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WO2009022755A1 - Procédé de production d'acide l-amino au moyen d'une bactérie de la famille enterobacteriaceae avec une expression atténuée du gène chac - Google Patents

Procédé de production d'acide l-amino au moyen d'une bactérie de la famille enterobacteriaceae avec une expression atténuée du gène chac Download PDF

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WO2009022755A1
WO2009022755A1 PCT/JP2008/064772 JP2008064772W WO2009022755A1 WO 2009022755 A1 WO2009022755 A1 WO 2009022755A1 JP 2008064772 W JP2008064772 W JP 2008064772W WO 2009022755 A1 WO2009022755 A1 WO 2009022755A1
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gene
amino acid
coli
bacterium
strain
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Konstantin Vyacheslavovich Rybak
Elvira Borisovna Voroshilova
Mikhail Markovich Gusyatiner
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Ajinomoto Co Inc
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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
    • 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
    • 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
    • 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

Definitions

  • the present invention relates to the microbiological industry, and specifically to a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family which has been modified to attenuate expression of the chaC gene.
  • 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 way to enhance L-amino acid production yields is to attenuate expression of a gene or several genes which are involved in degradation of the target L-amino acid, diverting the precursors of the target L-amino acid from the L-amino acid biosynthetic pathway, or redistribution of carbon, nitrogen, and phosphate fluxes, and genes coding for toxins etc.
  • the cha operon consists of 2 genes, chaB and chaC, found at -27 minutes on the Escherichia coli chromosome.
  • Objects of the present invention are to enhance the productivity of L-amino acid- producing strains and to provide a method for producing an L-amino acid using these strains.
  • L-amino acids such as L-threonine, L-lysine, L- cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L- alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine, L- phenylalanine, L-tyrosine, and L-tryptophan.
  • L-amino acids such as L-threonine, L-lysine, L- cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L- alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L
  • the present invention provides a bacterium of the Enterobacteriaceae family having an increased ability to produce L-amino acids, such as L-threonine, L-lysine, L-cysteine, L- methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L- asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine, L- phenylalanine, L-tyrosine, and L-tryptophan.
  • L-amino acids such as L-threonine, L-lysine, L-cysteine, L- methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L- asparagine, L-as
  • L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.
  • aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L- tyrosine, and L-tryptophan.
  • non-aromatic L-amino acid is selected from the group consisting of L-threonine, L- lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L- arginine.
  • L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.
  • aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L- tyrosine, and L-tryptophan.
  • non-aromatic L-amino acid is selected from the group consisting of L-threonine, L- lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L- arginine.
  • Figure 1 shows the relative positions of primers Pl and P2/P5 on plasmid pMWl 18-attL-Cm- attR which is used as a template for PCR amplification of the cat gene.
  • Figure 2 shows the construction of the chromosomal DNA fragment containing the inactivated chaC gene or inactivated chaBC operon.
  • the bacterium of the present invention is an L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified to attenuate expression of the chaC gene.
  • L-amino acid-producing bacterium means a bacterium which has an ability to produce and excrete an L-amino acid into a medium, when the bacterium is cultured in the medium.
  • L-amino acid-producing bacterium as used herein also means a bacterium which is able to produce and cause accumulation of an L-amino acid in a culture medium in an amount larger than a wild-type or parental strain of the bacterium, for example, E. coli, such as E. coli K- 12, and preferably means that the microorganism is able to cause accumulation in a medium of an amount not less than 0.5 g/L, more preferably not less than 1.0 g/L, of the target L-amino acid.
  • L-amino acid includes 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.
  • aromatic L-amino acid includes L-phenylalanine, L-tyrosine, and L- tryptophan.
  • non-aromatic L-amino acid includes L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L- asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine.
  • L- threonine L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L-phenylalanine, L- tryptophan, L-proline, and L-arginine are particularly preferred.
  • a bacterium belonging to the genus Escherichia means that the bacterium is classified into the genus Escherichia according to the classification known to a person skilled in the art of microbiology.
  • Examples of a bacterium belonging to the genus Escherichia as used in the present invention include, but are not limited to, Escherichia coli (E. coli).
  • the bacterium belonging to the genus Escherichia that can be used in the present invention is not particularly limited; however, e.g., bacteria described by Neidhardt, F. C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D. C, 1208, Table 1) are encompassed by the bacterium of the present invention.
  • a bacterium belonging to the genus Pantoea means that the bacterium is classified into the genus Pantoea according to the classification known to a person skilled in the art of microbiology.
  • Some species of Enterobacter agglomerans have been recently re-classified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like, based on the nucleotide sequence analysis of 16S rRNA, etc. (Int. J. Syst. Bacteriol., 43, 162-173 (1993)).
  • bacterium has been modified to attenuate expression of the chaC gene means that the bacterium has been modified in such a way that the modified bacterium contains a reduced amount of the ChaC protein as compared with an unmodified bacterium, or the modified bacterium is unable to synthesize the ChaC protein.
  • the phrase "bacterium has been modified to attenuate expression of the chaC gene” may also mean that the bacterium has been modified in such a way that the modified gene encodes a mutant ChaC protein with a decreased activity.
  • the expression of the chaC gene can be attenuated by inactivating the chaC gene.
  • the chaC gene can also be attenuated by inactivating the chaB gene because the chaB gene and the chaC gene constitue an operon in this order. Furthermore, the expression of the chaC gene can also be attenuated by inactivating the the chaBC operon.
  • the presence or absence of the chaC gene, chaB gene or the chaBC operon in the chromosome of a bacterium can be detected by well-known methods, including PCR, Southern blotting, and the like.
  • the level of gene expression can be estimated 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 encoded by the chaC gene or the chaB gene can be measured by well-known methods, including SDS-PAGE followed by immunoblotting assay (Western blotting analysis), and the like.
  • activation of the chaC gene, chaB gene or the chaBC operon means that the modified gene or operon encodes a completely inactive protein(s). It is also possible that the modified DNA region is unable to naturally express the gene or operon due to the deletion of a part of or the entire gene or operon, the shifting of the reading frame of the gene or operon, the introduction of missense/nonsense mutation(s), or the modification of an adjacent region of the gene or operon, including sequences controlling gene expression, such as promoter(s), enhancer(s), attenuator(s), ribosome-binding site(s), etc..
  • the chaC gene encodes the ChaC protein (synonym - B1218).
  • the chaC gene of E. coli (nucleotides 1,271,709 to 1,272,425 in the GenBank accession number NC 000913.2; gi:49175990) is located between the gene chaB and the ORF ychN on the chromosome of E. coli strain K- 12.
  • the nucleotide sequence of the chaC gene and the amino acid sequence of ChaC encoded by the chaC gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the chaB gene encodes the ChaB protein (synonym - B 1217).
  • the chaB gene of E. coli (nucleotides 1,271,342 to 1,271,572 in the GenBank accession number NC 000913.2; gi:49175990) is located between the chaA gene and the chaC gene on the chromosome of E. coli strain K- 12.
  • the nucleotide sequence of the chaB gene and the amino acid sequence of ChaB encoded by the chaB gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the chaC gene or the chaB gene is not limited to the gene shown in SEQ ID No: 1 or SEQ ID No: 3, but may include genes homologous to SEQ ID No: 1 or SEQ ID No: 3 which encodes a variant protein of the ChaC or ChaB.
  • the phrase "variant protein" as used in the present invention means a protein which has changes in the sequence, whether they are deletions, insertions, additions, or substitutions of amino acids, but still maintains the activity of the product as the ChaC or ChaB protein. 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.
  • the variant protein encoded by the chaC gene or the chaB gene may be one which has a homology of not less than 80%, preferably not less than 90%, and most preferably not less than 95%, with respect to the entire amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: 4, as long as the activity of the ChaC or ChaB protein prior to inactivation of the chaC gene, the chaC gene or the chaBC operon is maintained.
  • Homology between two amino acid sequences can be determined using the well-known methods, for example, the computer program BLAST 2.0, which calculates three parameters: score, identity and similarity.
  • the chaC gene ,or the chaB gene may be a variant which hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or a probe which can be prepared from the nucleotide sequence under stringent conditions, provided that it encodes a functional ChaC/ChaB protein prior to inactivation.
  • Stringent conditions include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, preferably not less than 70%, more preferably not less than 80%, still more preferably not less than 90%, and most preferably not less than 95%, is formed and a nonspecific hybrid, for example, a hybrid having homology lower than the above, is not formed.
  • stringent conditions are exemplified by washing one time or more, preferably two or three times at a salt concentration of 1 X SSC, 0.1% SDS, preferably 0.1 X SSC, 0.1% SDS at 60 0 C.
  • Duration of washing depends on the type of membrane used for blotting and, as a rule, should be what is recommended by the manufacturer.
  • the recommended duration of washing for the HybondTM N+ nylon membrane (Amersham) under stringent conditions is 15 minutes.
  • washing may be performed 2 to 3 times.
  • the length of the probe may be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp.
  • Expression of the chaC gene can be attenuated by introducing a mutation into the gene on the chromosome so that intracellular activity of the protein encoded by the gene is decreased as compared with an unmodified strain.
  • a mutation on the gene can be replacement of one base or more to cause an amino acid substitution in the protein encoded by the gene (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, or deletion of a part of the gene or the entire gene (Qiu, Z. and Goodman, M.F., J. Biol. Chem., 272, 861 1-8617 (1997); Kwon, D. H. et al, J. Antimicrob.
  • Expression of the chaC gene can also be attenuated by modifying an expression regulating sequence such as the promoter, the Shine- Dalgarno (SD) sequence, etc. (WO95/34672, Carrier, T.A. and Keasling, J.D., Biotechnol Prog 15, 58-64 (1999)) of the chaBC operon.
  • SD Shine- Dalgarno
  • the following methods may be employed to introduce a mutation by gene recombination.
  • a mutant gene encoding a mutant protein having a decreased activity is prepared, and a bacterium to be modified is transformed with a DNA fragment containing the mutant gene. Then the native gene on the chromosome is replaced with the mutant gene by homologous recombination, and the resulting strain is selected.
  • Such gene replacement using homologous recombination can be conducted by the method employing a linear DNA, which is known as "Red-driven integration" (Datsenko, K. A. and Wanner, B.L., Proc. Natl. Acad. Sci.
  • Expression of the gene can also be attenuated by insertion of a transposon or an IS factor into the coding region of the gene (U.S. Patent No. 5,175,107), or by conventional methods, such as mutagenesis treatment using UV irradiation or nitrosoguanidine (N-methyl-N'-nitro-N- nitrosoguanidine).
  • Inactivation of the gene or operon can also be performed by conventional methods, such as a mutagenesis 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 and Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 6640- 45), also called "Red-driven integration".
  • Methods for preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer, and the like may be ordinary methods well known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E. F., and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).
  • L-amino acid-producing bacteria As a bacterium of the present invention which is modified to attenuate expression of the chaC gene, bacteria which are able to produce either an aromatic or a non-aromatic L-amino acids may be used.
  • the bacterium of the present invention can be obtained by attenuating expression of the chaC gene in a bacterium which inherently has the ability to produce L-amino acids.
  • the bacterium of present invention can be obtained by imparting the ability to produce L-amino acids to a bacterium already having the attenuated expression of the chaC gene.
  • Examples of parent strains for deriving the L-threonine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli TDH-6/pVIC40 (VKPM B-3996) (U.S. Patent No. 5, 175, 107, U.S. Patent No. 5,705,371), E. coli 472T23/pYN7 (ATCC 98081) (U.S. Patent No.5,631, 157), E. co// NRRL-21593 (U.S. Patent No. 5,939,307), E. coli FERM BP-3756 (U.S. Patent No. 5,474,918), E.
  • E. coli FERM BP- 3519 and FERM BP-3520 U.S. Patent No. 5,376,538, E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978)), E. coli VL643 and VL2055 (EP 114991 1 A), and the like.
  • the strain TDH-6 is deficient in the thrC gene, as well as being sucrose-assimilative, and the HvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene, which imparts resistance to high concentrations of threonine or homoserine.
  • the strain VKPM B-3996 contains the plasmid pVIC40 which was obtained by inserting a thrA*BC operon which includes a mutant thrA gene into a RSFlOlO-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which is substantially desensitized to feedback inhibition by threonine.
  • the strain B-3996 was deposited on November 19, 1987 in the Ail-Union Scientific Center of Antibiotics (USD, 117105 Moscow, Nagatinskaya Street 3 -A) under the accession number RIA 1867. The strain was also deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (USD, 117545 Moscow, 1 Dorozhny proezd, 1) on April 7, 1987 under the accession number VKPM B-3996.
  • VKPM Russian National Collection of Industrial Microorganisms
  • E. coli VKPM B-5318 (EP 0593792B) may also be used as a parent strain for deriving L- threonine-producing bacteria of the present invention.
  • the strain B-5318 is prototrophic with regard to isoleucine, and a temperature-sensitive lambda-phage Cl repressor and PR promoter replaces the regulatory region of the threonine operon in plasmid pVIC40.
  • the strain VKPM B- 5318 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) on May 3, 1990 under accession number of VKPM B-5318.
  • the bacterium of the present invention is additionally modified to enhance expression of one or more of the following genes: the mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine; the thrB gene which codes for homoserine kinase; the thrC gene which codes for threonine synthase; the rhtA gene which codes for a putative transmembrane protein; the asd gene which codes for aspartate- ⁇ -semialdehyde dehydrogenase; and the aspC gene which codes for aspartate aminotransferase (aspartate transaminase);
  • the mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine
  • the thrB gene which codes for homoserine kinase
  • the thrC gene which codes for thre
  • the thrA gene which encodes aspartokinase homoserine dehydrogenase I of Escherichia coli has been elucidated (nucleotide positions 337 to 2799, GenBank accession NC 000913.2, gi: 49175990).
  • the thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K- 12.
  • the thrB gene which encodes homoserine kinase of Escherichia coli has been elucidated (nucleotide positions 2801 to 3733, GenBank accession NC 000913.2, gi: 49175990).
  • the thrB gene is located between the thrA and thrC genes on the chromosome of E. coli K- 12.
  • the thrC gene which encodes threonine synthase of Escherichia coli has been elucidated (nucleotide positions 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 on the chromosome of E. coli K- 12. All three genes functions as a single threonine operon.
  • the attenuator region which affects the transcription is desirably removed from the operon (WO2005/049808, WO2003/097839).
  • a mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine, as well as, the thrB and thrC genes can be obtained as one operon from well-known plasmid pVIC40 which is presented in the threonine producing E. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S. Patent No. 5,705,371.
  • the rhtA gene exists at 18 min on the E. coli chromosome close to the glnHPQ operon, which encodes components of the glutamine transport system.
  • the rhtA gene is identical to ORFl ⁇ ybiF gene, nucleotide positions 764 to 1651, GenBank accession number AAA218541, gi:440181) and located between the pexB and ompX genes.
  • the unit expressing a protein encoded by the ORFl has been designated the rhtA gene (rht: resistance to homoserine and threonine).
  • the asd gene of E. coli has already been elucidated (nucleotide positions 357251 1 to 3571408, GenBank accession NC 000913.1, gi:16131307), and can be obtained by PCR (polymerase chain reaction; refer to White, T.J. et al., Trends Genet., 5, 185 (1989)) utilizing primers prepared based on the nucleotide sequence of the gene.
  • the asd genes of other microorganisms can be obtained in a similar manner.
  • the aspC gene of E. coli has already been elucidated (nucleotide positions 983742 to 984932, GenBank accession NC 000913.1, gi:16128895), and can be obtained by PCR.
  • the aspC genes of other microorganisms can be obtained in a similar manner.
  • L-lysine-producing bacteria belonging to the genus Escherichia include mutants having resistance to an L-lysine analogue.
  • the L-lysine analogue inhibits growth of bacteria belonging to the genus Escherichia, but this inhibition is fully or partially desensitized when L-lysine coexists in a medium.
  • Examples of the L-lysine analogue include, but are not limited to, oxalysine, lysine hydroxamate, S-(2-aminoethyl)-L-cysteine (AEC), ⁇ -methyllysine, ⁇ -chlorocaprolactam and so forth.
  • Mutants having resistance to these lysine analogues can be obtained by subjecting bacteria belonging to the genus Escherichia to a conventional artificial mutagenesis treatment.
  • bacterial strains useful for producing L-lysine include Escherichia coli AJl 1442 (FERM BP-1543, NRRL B-12185; see U.S. Patent No. 4,346,170) and Escherichia coli VL611. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is desensitized.
  • the strain WC 196 may be used as an L-lysine producing bacterium of Escherichia coli. This bacterial strain was bred by conferring AEC resistance to the strain W31 10, which was derived from Escherichia coli K- 12. The resulting strain was designated Escherichia coli AJ 13069 strain and was deposited at the National Institute of Bioscience and Human- Technology, Agency of Industrial Science and Technology (currently National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on December 6, 1994 and received an accession number of FERM P- 14690.
  • Examples of parent strains for deriving L-lysine-producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-lysine biosynthetic enzyme are enhanced.
  • genes include, but are not limited to, genes encoding dihydrodipicolinate synthase ⁇ dap A), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (U.S. Patent No. 6,040,160), phosphoenolpyrvate carboxylase (ppc), aspartate semialdehyde dehydrogenease (asd), and aspartase (aspA) (EP 1253195 A).
  • the parent strains may have an increased level of expression of the gene involved in energy efficiency (cyo) (EP 1170376 A), the gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (U.S. Patent No. 5,830,716), the ybjE gene (WO2005/073390), or combinations thereof.
  • cyo energy efficiency
  • pntAB nicotinamide nucleotide transhydrogenase
  • ybjE gene WO2005/073390
  • Examples of parent strains for deriving L-lysine-producing bacteria of the present invention also include strains having decreased or eliminated activity of an enzyme that catalyzes a reaction for generating a compound other than L-lysine by branching off from the biosynthetic pathway of L-lysine.
  • Examples of the enzymes that catalyze a reaction for generating a compound other than L-lysine by branching off from the biosynthetic pathway of L-lysine include homoserine dehydrogenase, lysine decarboxylase (U.S. Patent No. 5,827,698), and the malic enzyme (WO2005/010175).
  • parent strains for deriving L-cysteine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JM 15 which is transformed with different cysE alleles coding for feedback-resistant serine acetyltransferases (U.S. Patent No. 6,218,168, Russian patent application 2003121601); E. coli W31 10 having over-expressed genes which encode proteins suitable for secreting substances toxic for cells (U.S. Patent No. 5,972,663); E. coli strains having lowered cysteine desulfohydrase activity (JPl 1155571A2); E. coli W31 10 with increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene (WO0127307A1), and the like.
  • E. coli JM 15 which is transformed with different cysE alleles coding for feedback-resistant serine acetyltrans
  • parent strains for deriving L-leucine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strains resistant to leucine (for example, the strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121)) or leucine analogs including ⁇ -2-thienylalanine, 3 -hydroxy leucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A); E. coli strains obtained by the gene engineering method described in WO96/06926; E. coli H-9068 (JP 8-70879 A), and the like.
  • E. coli strains resistant to leucine for example, the strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121)
  • leucine analogs including ⁇ -2-thienylalanine, 3 -hydroxy leu
  • the bacterium of the present invention may be improved by enhancing the expression of one or more genes involved in L-leucine biosynthesis.
  • genes of the leuABCD operon which are preferably represented by a mutant leuA gene coding for isopropylmalate synthase freed from feedback inhibition by L-leucine (US Patent 6,403,342).
  • the bacterium of the present invention may be improved by enhancing the expression of one or more genes coding for proteins which excrete L-amino acid from the bacterial cell. Examples of such genes include the b2682 and b2683 genes (ygaZH genes) (EP 1239041 A2).
  • Examples of parent strains for deriving L-histidine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 24 (VKPM B-5945, RU2003677); E. coli strain 80 (VKPM B-7270, RU21 19536); E. coli NRRL B-12116 - B12121 (U.S. Patent No. 4,388,405); E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Patent No. 6,344,347); E. coli H-9341 (FERM BP-6674) (EP 1085087); E. coli AI80/pFM201 (U 5 S. Patent No. 6,258,554) and the like.
  • E. coli AI80/pFM201 U 5 S. Patent No. 6,258,554 and the like.
  • Examples of parent strains for deriving L-histidine-producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-histidine biosynthetic enzyme are enhanced.
  • examples of such genes include genes encoding ATP phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase (hisl), phosphoribosyl- ATP pyrophosphohydrolase (hisIE), phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (hisA), amidotransferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD), and so forth.
  • L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are inhibited by L-histidine, and therefore an L-histidine-producing ability can also be efficiently enhanced by introducing a mutation conferring resistance to the feedback inhibition into ATP phosphoribosyltransferase ( Russian Patent Nos. 2003677 and 21 19536).
  • strains having an L-histidine-producing ability include E. coli FERM-P 5038 and 5048 which have been introduced with a vector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP 56-005099 A), E. coli strains introduced with rht, a gene for an amino acid-export (EPl 01671 OA), E. coli 80 strain imparted with sulfaguanidine, DL- 1,2,4- triazole-3 -alanine, and streptomycin-resistance (VKPM B-7270, Russian Patent No. 2119536), and so forth.
  • L-glutamic acid-producing bacteria include E. coli FERM-P 5038 and 5048 which have been introduced with a vector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP 56-005099 A), E. coli strains introduced with rht, a gene for an amino acid-export (EPl 01
  • Examples of parent strains for deriving L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli VL334thrC + (EP 1172433).
  • E. coli VL334 (VKPM B-1641) is an L-isoleucine and L- threonine auxotrophic strain having mutations in thrC and HvA genes (U.S. Patent No. 4,278,765).
  • a wild-type allele of the thrC gene was transferred by the method of general transduction using a bacteriophage Pl grown on the wild-type E. coli strain Kl 2 (VKPM B-7) cells.
  • an L-isoleucine auxotrophic strain VL334thrC + (VKPM B-8961), which is able to produce L-glutamic acid, was obtained.
  • parent strains for deriving the L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains in which expression of one or more genes encoding an L-glutamic acid biosynthetic enzyme are enhanced.
  • genes include genes encoding glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), phosphoenolpyruvate carboxylase ippc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, ipdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), en
  • strains modified so that expression of the citrate synthetase gene, the phosphoenolpyruvate carboxylase gene, and/or the glutamate dehydrogenase gene is/are enhanced include those disclosed in EP1078989A, EP955368A, and EP952221A.
  • Examples of parent strains for deriving the L-glutamic acid-producing bacteria of the present invention also include strains having decreased or eliminated activity of an enzyme that catalyzes synthesis of a compound other than L-glutamic acid by branching off from an L- glutamic acid biosynthesis pathway.
  • genes include genes encoding isocitrate lyase (ace A), ⁇ -ketoglutarate dehydrogenase (sue A), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (HvG), acetolactate synthase (UvI), formate acetyltransferase (pfl), lactate dehydrogenase (Idh), and glutamate decarboxylase (gadAB).
  • E. coli W3110sucA::Km R is a strain obtained by disrupting the ⁇ -ketoglutarate dehydrogenase gene (hereinafter referred to as "sucA gene") of E. coli W31 10. This strain is completely deficient in ⁇ -ketoglutarate dehydrogenase.
  • L-glutamic acid-producing bacterium examples include those which belong to the genus Escherichia and have resistance to an aspartic acid antimetabolite. These strains can also be deficient in ⁇ -ketoglutarate dehydrogenase activity and include, for example, E. coli AJ13199 (FERM BP-5807) (U.S. Patent No. 5,908,768), FFRM P-12379, which additionally has a low L-glutamic acid decomposing ability (U.S. Patent No. 5,393,671); AJ13138 (FERM BP- 5565) (U.S. Patent No. 6,110,714), and the like.
  • L-glutamic acid-producing bacteria examples include mutant strains belonging to the genus Pantoea which are deficient in ⁇ -ketoglutarate dehydrogenase activity or have a decreased ⁇ -ketoglutarate dehydrogenase activity, and can be obtained as described above.
  • Such strains include Pantoea ananatis AJ13356. (U.S. Patent No. 6,331,419).
  • Pantoea ananatis AJ13356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on February 19, 1998 under an accession number of FERM P- 16645. It was then converted to an international deposit under the provisions of Budapest Treaty on January 1 1, 1999 and received an accession number of FERM BP-6615.
  • Pantoea ananatis AJ13356 is deficient in the ⁇ - ketoglutarate dehydrogenase activity as a result of disruption of the ⁇ KGDH-El subunit gene (sucA).
  • the above strain was identified as Enterobacter agglomerans when it was isolated and deposited as the Enterobacter agglomerans AJ13356.
  • it was recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth.
  • AJ 13356 was deposited at the aforementioned depository as Enterobacter agglomerans, for the purposes of this specification, they are described as Pantoea ananatis.
  • parent strains for deriving L-phenylalanine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli AJ12739 (tyrA::TnlO, tyrR) (VKPM B-8197); E. coli HW1089 (ATCC 55371) harboring the mutant pheA34 gene (U.S. Patent No. 5,354,672); E. coli MWEC101-b (KR8903681); E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (U.S. Patent No. 4,407,952). Also, as a parent strain, E.
  • L-phenylalanine producing bacteria belonging to the genus Escherichia with an enhanced activity of the protein encoded by Xh ⁇ yedA gene or the yddG gene may also be used (U.S. patent applications 2003/0148473 Al and 2003/0157667 Al).
  • parent strains for deriving the L-tryptophan-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Patent No. 5,756,345); E.
  • coli SV 164 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase not subject to feedback inhibition by serine and a trpE allele encoding anthranilate synthase not subject to feedback inhibition by tryptophan (U.S. Patent No. 6,180,373); E. coli AGXl 7 (pGX44) (NRRL B- 12263) and AGX6(pGX50)aroP (NRRL B- 12264) deficient in the enzyme tryptophanase (U.S. Patent No. 4,371,614); E.
  • coli AGX17/pGX50,pACKG4-pps in which a phosphoenolpyruvate- producing ability is enhanced (WO9708333, U.S. Patent No. 6,319,696), and the like may be used.
  • L-tryptophan-producing bacteria belonging to the genus Escherichia with an enhanced activity of the identified protein encoded by and the yedA gene or the yddG gene may also be used (U.S. patent applications 2003/0148473 Al and 2003/0157667 Al).
  • parent strains for deriving the L-tryptophan-producing bacteria of the present invention also include strains in which one or more activities of the enzymes selected from anthranilate synthase, phosphoglycerate dehydrogenase, and tryptophan synthase are enhanced.
  • the anthranilate synthase and phosphoglycerate dehydrogenase are both subject to feedback inhibition by L-tryptophan and L-serine, so that a mutation desensitizing the feedback inhibition may be introduced into these enzymes.
  • Specific examples of strains having such a mutation include a E. coli SV 164 which harbors desensitized anthranilate synthase and a transformant strain obtained by introducing into the E. coli SVl 64 the plasmid pGH5 (WO 94/08031), which contains a mutant serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase.
  • Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention also include strains into which the tryptophan operon which contains a gene encoding desensitized anthranilate synthase has been introduced (JP 57-71397 A, JP 62-244382 A, U.S. Patent No. 4,371,614).
  • L-tryptophan-producing ability may be imparted by enhancing expression of a gene which encodes tryptophan synthase, among tryptophan operons (trpBA).
  • the tryptophan synthase consists of ⁇ and ⁇ subunits which are encoded by the trpA and trpB genes, respectively.
  • L-tryptophan-producing ability may be improved by enhancing expression of the isocitrate lyase-malate synthase operon (WO2005/103275).
  • Examples of parent strains for deriving L-proline-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli 702ilvA (VKPM B-8012) which is deficient in the HvA gene and is able to produce L- proline (EP 1 172433).
  • the bacterium of the present invention may be improved by enhancing the expression of one or more genes involved in L-proline biosynthesis. Examples of such genes for L-proline producing bacteria which are preferred include the proB gene coding for glutamate kinase of which feedback inhibition by L-proline is desensitized (DE Patent 3127361).
  • the bacterium of the present invention may be improved by enhancing the expression of one or more genes coding for proteins excreting L-amino acid from bacterial cell.
  • genes are exemplified by b2682 and b2683 genes (ygaZH genes) (EP1239041 A2).
  • parent strains for deriving L-arginine-producing bacteria of the present invention 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 ( Russian Patent Application No. 2001 1 12869), E. coli strain 382 (VKPM B-7926) (EPl 170358A1), an arginine-producing strain into which argA gene encoding N-acetylglutamate synthetase is introduced therein (EPl 170361 Al), and the like.
  • Examples of parent strains for deriving L-arginine producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-arginine biosynthetic enzyme are enhanced.
  • Examples of such genes include genes encoding N- acetylglutamyl phosphate reductase (argC), ornithine acetyl transferase (argJ), N- acetylglutamate kinase (argB), acetylornithine transaminase (argD), ornithine carbamoyl transferase (argF), argininosuccinic acid synthetase (argG), argininosuccinic acid lyase (argH), and carbamoyl phosphate synthetase ⁇ car AE).
  • argC N- acetylglutamyl phosphate reductase
  • argJ ornithine acety
  • Example of parent strains for deriving L-valine-producing bacteria of the present invention include, but are not limited to, strains which have been modified to overexpress the HvGMEDA operon (U.S. Patent No. 5,998,178). It is desirable to remove the region of the HvGMEDA operon which is required for attenuation so that expression of the operon is not attenuated by L-valine that is produced. Furthermore, the UvA gene in the operon is desirably disrupted so that threonine deaminase activity is decreased.
  • Examples of parent strains for deriving L-valine-producing bacteria of the present invention include also include mutants having a mutation of amino-acyl t-RNA synthetase (U.S. Patent No. 5,658,766).
  • E. coli VL 1970 which has a mutation in the HeS gene encoding isoleucine tRNA synthetase, can be used.
  • E. coli VLl 970 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 1 17545 Moscow, 1 Dorozhny Proezd, 1) on June 24, 1988 under accession number VKPM B-441 1.
  • mutants requiring lipoic acid for growth and/or lacking H + -ATPase can also be used as parent strains (WO96/06926).
  • parent strains for deriving L-isoleucine producing bacteria of the present invention include, but are not limited to, mutants having resistance to 6-dimethylaminopurine (JP 5-304969 A), mutants having resistance to an isoleucine analogue such as thiaisoleucine and isoleucine hydroxamate, and mutants additionally having resistance to DL-ethionine and/or arginine hydroxamate (JP 5-130882 A).
  • recombinant strains transformed with genes encoding proteins involved in L-isoleucine biosynthesis can also be used as parent strains (JP 2-458 A, FR 0356739, and U.S. Patent No. 5,998,178).
  • the method of the present invention is a method for producing an L-amino acid by cultivating the bacterium of the present invention in a culture medium to produce and excrete the L-amino acid into the medium, and collecting the L-amino acid from the medium.
  • the cultivation, collection, and purification of an L-amino acid 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 bacterium.
  • the medium used for culture may be either synthetic or natural, so long as it includes a carbon source, a nitrogen source, minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth.
  • the carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the mode of assimilation of the chosen microorganism, alcohol, including ethanol and glycerol, may be used.
  • As the nitrogen source various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean- hydrolysate, and digested fermentative microorganism can be used.
  • potassium monophosphate magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used.
  • vitamins thiamine, yeast extract, and the like, can be used.
  • the cultivation is preferably performed under aerobic conditions, such as a shaking culture, and a stirring culture with aeration, at a temperature of 20 to 40 °C, preferably 30 to 38 °C.
  • the pH of the culture is usually between 5 and 9, preferably between 6.5 and 7.2.
  • the pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5-day cultivation leads to accumulation of the target L-amino acid in the liquid medium.
  • solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and then the L-amino acid can be collected and purified by ion-exchange, concentration, and/or crystallization methods.
  • Example 1 Construction of a strain with an inactivated chaC gene
  • a strain in which the chaC gene has been deleted was constructed by the method initially developed by Datsenko, K. A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) called "Red-driven integration".
  • the DNA fragment containing the chloramphenicol-resistant (Cm R ) marker encoded by the cat gene was obtained by PCR, using primers Pl (SEQ ID NO: 5) and P2 (SEQ ID NO: 6) and plasmid pMWl 18-attL-Cm-attR as a template (WO 05/010175).
  • Primer Pl contains both a region complementary to the 36-nt region located at the 3' end of the chaC gene and a region complementary to the attL region.
  • Primer P2 contains both a region complementary to the 35-nt region located at the 5' end of the chaC gene and a region complementary to the attR region.
  • Conditions for PCR were as follows: denaturation step for 3 min at 95 °C; profile for two first cycles: 1 min at 95 °C, 30 sec at 50 °C, 40 sec at 72 °C; profile for the last 25 cycles: 30 sec at 95 °C, 30 sec at 54 °C, 40 sec at 72 °C; final step: 5 min at 72 0 C.
  • a 1699-bp PCR product (Fig. 1) was obtained and purified in agarose gel and was used for electroporation of the E. coli strain MGl 655 (ATCC 700926), which contains the plasmid pKD46 having a temperature-sensitive replication control region.
  • the plasmid pKD46 (Datsenko, K. A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 2000, 97:12:6640-45) includes a 2,154 nucleotide DNA fragment of phage ⁇ (nucleotide positions 31088 to 33241, GenBank accession no.
  • the plasmid pKD46 is necessary for integration of the PCR product into the chromosome of strain MGl 655.
  • the strain MGl 655 can be obtained from American Type Culture Collection. (P.O. Box 1549 Manassas, VA 20108, United States of America).
  • the mutants having the chaC gene deleted and marked with the Cm resistance gene were verified by PCR.
  • Locus-specific primers P3 (SEQ ID NO: 5) and P4 (SEQ ID NO: 6) were used in PCR for the verification.
  • Conditions for PCR verification were as follows: denaturation step for 3 min at 94 °C; profile for 30 cycles: 30 sec at 94 °C, 30 sec at 54 °C, 1 min at 72 °C; final step: 7 min at 72 °C.
  • the PCR product obtained in the reaction with the cells of parental chaC + strain MG 1655 as a template was ⁇ 0.9 kbp in length.
  • the PCR product obtained in the reaction with the cells of the mutant strain as the template was -1.8 kbp in length (Fig.2).
  • the mutant strain was named MGl 655 ⁇ chaC::cat.
  • Example 2 Construction of a strain with an inactivated chaBC operon
  • a strain having deletion of the chaBC operon was constructed by the "Red-driven integration".
  • the DNA fragment containing the Cm R marker encoded by the cat gene was obtained by PCR, using primers Pl (SEQ ID NO: 5) and P5 (SEQ ID NO: 9) and plasmid pMWl 18-attL-Cm-attR as a template.
  • Primer P5 contains both a region complementary to the 35-nt region located at the 5' end of the chaB gene and a region complementary to the attR region.
  • Conditions for PCR were as follows: denaturation step for 3 min at 95 °C; profile for two first cycles: 1 min at 95° C, 30 sec at 50 °C, 40 sec at 72 0 C; profile for the last 25 cycles: 30 sec at 95 °C, 30 sec at 54°C, 40 sec at 72 0 C; final step: 5 min at 72 °C.
  • a 1699-bp PCR product (Fig. 1) was obtained and purified in agarose gel and was used for electroporation of the E. coli strain MG 1655, which contains the plasmid pKD46 having a temperature-sensitive replication control region.
  • Electrocompetent cells were prepared as follows: E. coli MG1655/pKD46 was grown overnight at 30 0 C in LB medium containing ampicillin (100 mg/1), and the culture was diluted 100 times with 5 ml of SOB medium containing ampicillin and L-arabinose (1 mM). The cells were grown with aeration at 30 °C to an OD ⁇ oo of «0.6 and then were made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H 2 O. Electroporation was performed using 70 ⁇ l of cells and »100 ng of the PCR product.
  • the mutants having the chaBC operon deleted and marked with the Cm R gene were verified by PCR.
  • Locus-specific primers P6 (SEQ ID NO: 10) and P4 (SEQ ID NO: 8) were used in PCR for the verification.
  • Conditions for PCR verification were as follows: denaturation step for 3 min at 94 °C; profile for 30 cycles: 30 sec at 94 °C, 30 sec at 54 °C, 1 min at 72 °C; final step: 7 min at 72 °C.
  • the PCR product obtained in the reaction with the cells of parental chaC + strain MGl 655 as a template was ⁇ 1.3 kbp in length.
  • the PCR product obtained in the reaction with the cells of the mutant strain as the template was ⁇ 1.9 kbp in length (Fig.2).
  • the mutant strain was named MG1655 ⁇ chaBC::cat.
  • Both E. coli strains, B-3996 and B-3996- ⁇ chaC were grown for 18-24 hours at 37 0 C on L-agar plates.
  • the strains were grown on a rotary shaker (250 rpm) at 32 °C for 18 hours in 20x200-mm test tubes containing 2 ml of L-broth supplemented with 4% glucose.
  • the fermentation medium was inoculated with 0.2 ml (10%) of seed material.
  • the fermentation was performed in 2 ml of minimal medium for fermentation in 20x200-mm test tubes. Cells were grown for 65 hours at 32 °C with shaking at 250 rpm.
  • B-3996- ⁇ chaC produced a higher amount of L-threonine, as compared with B-3996.
  • L- threonine can also be produced by fermentation using the B-3996- ⁇ chaBC strain in the same manner.
  • composition of the fermentation medium (g/1) was as follows:
  • Glucose and magnesium sulfate were sterilized separately.
  • CaCO 3 was sterilized by dry-heat at 180 °C for 2 hours.
  • the pH was adjusted to 7.0.
  • the antibiotic was introduced into the medium after sterilization.
  • DNA fragments from the chromosome of the above-described E. coli strain MG 1655 ⁇ chaC::cat or MG 1655 ⁇ chaBC::cat can be transferred to the lysine-producing E. coli strain AJl 1442 by Pl transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain AJl 1442- ⁇ chaC/ AJl 1442- ⁇ chaBC strain.
  • the strain AJ l 1442 was deposited at the National Institute of Bioscience and Human- Technology, Agency of Industrial Science and Technology (currently National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on May 1, 1981 and received an accession number of FERM P-5084. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on October 29, 1987, and received an accession number of FERM BP- 1543.
  • the pCABD2 plasmid includes the dap A gene encoding dihydrodipicolinate synthase having a mutation which desensitizes feedback inhibition by L-lysine, the lysC gene encoding aspartokinase III having a mutation which desensitizes feedback inhibition by L-lysine, the dapB gene encoding dihydrodipicolinate reductase, and the ddh gene encoding diaminopimelate dehydrogenase (US Patent 6,040,160).
  • E. coli strains AJl 1442, AJl 1442- ⁇ chaC and AJl 1442- ⁇ chaBC, can be separately cultured in L-medium containing streptomycin (20 mg/1) at 37 °C, and 0.3 ml of the obtained culture can be inoculated into 20 ml of the fermentation medium containing the required drugs in a 500-ml flask.
  • the cultivation can be carried out at 37 °C for 16 h by using a reciprocal shaker at the agitation speed of 115 rpm.
  • the amounts of L-lysine and residual glucose in the medium can be measured by a known method (Biotech-analyzer AS210 manufactured by Sakura Seiki Co.). Then, the yield of L-lysine can be calculated relative to consumed glucose for each of the strains.
  • composition of the fermentation medium (g/1) is as follows:
  • the pH is adjusted to 7.0 by KOH and the medium is autoclaved at 115 °C for 10 min. Glucose and MgSO 4 7H 2 O are sterilized separately. CaCO 3 is dry-heat sterilized at 180 °C for 2 hours and added to the medium for a final concentration of 30 g/1.
  • DNA fragments from the chromosome of the above-described E. coli MG 1655 ⁇ chaC::cat or MG1655 ⁇ chaBC::cat can be transferred to the E. coli L-cysteine-producing strain JM15(ydeD) by Pl transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain JM15(ydeD)- ⁇ chaC/ JM15(ydeD)- ⁇ chaBC.
  • E. coli JM15(ydeD) is a derivative of E. coli JM 15 (US Patent 6,218,168), which has been transformed with DNA having the ydeD gene encoding a membrane protein, and is not involved in a biosynthetic pathway of any L-amino acid (US Patent No. 5,972,663).
  • DNA fragments from the chromosome of the above-described E. coli strain MG 1655 ⁇ chaC::cat or MG1655 ⁇ chaBC::cat can be transferred to the E. coli L-leucine-producing strain 57 (VKPM B-7386, US Patent No. 6,124,121) by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain 57- ⁇ chaC/57- ⁇ chaBC.
  • the strain 57 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1 ) on May 19, 1997 under the accession number VKPM B-7386.
  • E. coli strains, 57, 57- ⁇ chaC and 57- ⁇ chaBC can be separately cultured for 18-24 hours at 37 0 C on L-agar plates.
  • the strains can be grown on a rotary shaker (250 rpm) at 32 0 C for 18 hours in 20x200-mm test tubes containing 2 ml of L-broth supplemented with 4% sucrose.
  • the fermentation medium can be inoculated with 0.2 ml of seed material (10%).
  • the fermentation can be performed in 2 ml of a minimal fermentation medium in 20x200-mm test tubes.
  • Cells can be grown for 48-72 hours at 32 0 C with shaking at 250 rpm.
  • composition of the fermentation medium (g/1) (pH 7.2) is as follows:
  • DNA fragments from the chromosome of the above-described E. coli MG 1655 ⁇ chaC::cat or MG1655 ⁇ chaBC::cat can be transferred to the histidine-producing E. coli strain 80 by Pl transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 80- ⁇ chaC/80- ⁇ chaBC.
  • the strain 80 has been described in Russian patent 2119536 and deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (USD, 1 17545 Moscow, 1 Dorozhny proezd, 1) on October 15, 1999 under accession no. VKPM B-7270.
  • VKPM National Collection of Industrial Microorganisms
  • composition of the fermentation medium (g/1) (pH 6.0) is as follows:
  • Glucose, proline, betaine and CaCO 3 are sterilized separately.
  • the pH is adjusted to 6.0 before sterilization.
  • Example 8 Production of L-glutamic acid by E. coli VL334thrC + - ⁇ chaC/£. coli VL334thrC + - ⁇ chaBC
  • DNA fragments from the chromosome of the above-described E. coli strain MG 1655 ⁇ chaC::cat or MG 1655 ⁇ chaBC::cat can be transferred to the E. coli L-glutamic acid-producing strain VL334thrC + (EP 1172433) by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab.
  • strain VL334thrC + - ⁇ chaC/ VL334thrC + - ⁇ chaBC has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 1 17545 Moscow, 1 Dorozhny proezd, 1) on December 6, 2004 under the accession number VKPM B-8961 and then converted to a deposit under the Budapest Treaty on December 8, 2004.
  • VKPM Russian National Collection of Industrial Microorganisms
  • E. coli strains VL334thrC + , VL334thrC + - ⁇ chaC and VL334thrC + - ⁇ chaBC, can be separately grown for 18-24 hours at 37 0 C on L-agar plates. Then, one loop of the cells can be transferred into test tubes containing 2ml of fermentation medium.
  • the fermentation medium contains glucose (60g/l), ammonium sulfate (25 g/1), KH 2 PO 4 (2g/l), MgSO 4 (1 g/1), thiamine (0.1 mg/ml), L-isoleucine (70 ⁇ g/ml), and CaCO 3 (25 g/1).
  • the pH is adjusted to 7.2. Glucose and CaCO 3 are sterilized separately.
  • DNA fragments from the chromosome of the above-described E. coli MGl 655 ⁇ chaC::cat or MG1655 ⁇ chaBC::cat can be transferred to the L-phenylalanine-producing E. coli strain AJ 12739 by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain AJ12739- ⁇ chaC/ AJ12739- ⁇ chaBC.
  • the strain AJ12739 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (USD, 1 17545 Moscow, 1 Dorozhny proezd, 1) on November 6, 2001 under accession no. VKPM B-8197 and then converted to a deposit under the Budapest Treaty on August 23, 2002.
  • VKPM Russian National Collection of Industrial Microorganisms
  • E. coli strains can be separately cultivated at 37 °C for 18 hours in a nutrient broth, and 0.3 ml of the obtained culture can be each inoculated into 3 ml of a fermentation medium in a 20x200-mm test tube and cultivated at 37 0 C for 48 hours with shaking on a rotary shaker.
  • the amount of phenylalanine which accumulates in the medium can be determined by TLC.
  • the 10xl5-cm TLC plates coated with 0.11 -mm layers of Sorbfil silica gel containing no fluorescent indicator (Stock Company Sorbpolymer, Krasnodar, Russia) can be used.
  • a solution of ninhydrin (2%) in acetone can be used as a visualizing reagent.
  • composition of the fermentation medium (g/1) is as follows:
  • Glucose and magnesium sulfate are sterilized separately.
  • CaCO 3 is dry-heat sterilized at 180 0 C for 2 hours. The pH is adjusted to 7.0.
  • DNA fragments from the chromosome of the above-described E. coli strain MGl 655 ⁇ chaC::cat or MG1655 ⁇ chaBC::cat can be transferred to the tryptophan-producing E. coli strain SV 164 (pGH5) by Pl transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain SV164(pGH5)- ⁇ chaC/ SV164(pGH5)- ⁇ chaBC.
  • the strain SV 164 has the trpE allele encoding anthranilate synthase not subject to feedback inhibition by tryptophan.
  • the plasmid pGH5 harbors a mutant serA gene encoding phosphoglycerate dehydrogenase not subject to feedback inhibition by serine.
  • the strain SV 164 (pGH5) was described in detail in US patent No. 6,180,373 or European patent No. 0662143. E.
  • coli strains SV164(pGH5), SV164(pGH5)- ⁇ chaC and SV164(pGH5)- ⁇ chaBC, can be separately cultivated with shaking at 37 °C for 18 hours in 3 ml of nutrient broth supplemented with tetracycline (20 mg/1, marker of pGH5 plasmid).
  • the obtained cultures (0.3 ml each) can be inoculated into 3 ml of a fermentation medium containing tetracycline (20 mg/1) in 20 x 200-mm test tubes, and cultivated at 37 0 C for 48 hours with a rotary shaker at 250 rpm.
  • the amount of tryptophan which accumulates in the medium can be determined by TLC as described in Example 9.
  • the fermentation medium components are listed in Table 2, but should be sterilized in separate groups (A, B, C, D, E, F, and H), as shown, to avoid adverse interactions during sterilization.
  • Group A had pH 7.1 adjusted by NH 4 OH. Each group is sterilized separately, chilled and then mixed together.
  • Example 11 Production of L-proline by E. coli 702ilvA- ⁇ chaC/E. coli 702ilvA- ⁇ chaBC
  • E. coli 702ilvA- ⁇ chaC DNA fragments from the chromosome of the above-described E. coli strain MG 1655 ⁇ chaC::cat or MG1655 ⁇ chaBC::cat can be transferred to the proline-producing E. coli strain 702ilvA by Pl transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain 702ilvA- ⁇ chaC/702ilvA- ⁇ chaBC.
  • strain 702ilvA has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on July 18, 2000 under accession number VKPM B-8012 and then converted to a deposit under the Budapest Treaty on May 18, 2001.
  • VKPM Russian National Collection of Industrial Microorganisms
  • E. coli strains, 702ilvA, 702ilvA- ⁇ chaC and 702ilvA- ⁇ chaBC can be separately grown for 18-24 hours at 37 °C on L-agar plates. Then, these strains can be cultivated under the same conditions as in Example 8.
  • the strain 382 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (USD, 117545 Moscow, 1 Dorozhny proezd, 1) on April 10, 2000 under accession number VKPM B-7926 and then converted to a deposit under the Budapest Treaty on May 18, 2001.
  • VKPM Russian National Collection of Industrial Microorganisms
  • Both E. coli strains, 382 and 382- ⁇ chaC were separately cultivated with shaking at 37 °C for 18 hours in 3 ml of nutrient broth, and 0.3 ml of the obtained cultures were inoculated into 2 ml of a fermentation medium in 20 x 200-mm test tubes and cultivated at 32 °C for 48 hours on a rotary shaker.
  • composition of the fermentation medium (g/1) was as follows:
  • Glucose 48.0 (NH 4 ) 2 SO 4 35.0 KH 2 PO 4 2.0 MgSO 4 -7H 2 O 1.0 Thiamine HCl 0.0002 Yeast extract 1.0 L-isoleucine 0.1 CaCO 3 5.0
  • Glucose and magnesium sulfate were sterilized separately.
  • CaCO 3 was dry-heat sterilized at 180 0 C for 2 hours. The pH was adjusted to 7.0.
  • L-amino acid of a bacterium of the Enterobacteriaceae family can be enhanced.

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Abstract

La présente invention concerne un procédé de production d'un acide L-amino au moyen d'une bactérie de la famille Enterobacteriaceae, en particulier une bactérie appartenant au genre Escherichia ou Pantoea, qui a été modifiée pour atténuer l'expression du gène chaC.
PCT/JP2008/064772 2007-08-14 2008-08-13 Procédé de production d'acide l-amino au moyen d'une bactérie de la famille enterobacteriaceae avec une expression atténuée du gène chac Ceased WO2009022755A1 (fr)

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RU2007131006/13A RU2392327C2 (ru) 2007-08-14 2007-08-14 СПОСОБ ПОЛУЧЕНИЯ L-ТРЕОНИНА ИЛИ L-АРГИНИНА С ИСПОЛЬЗОВАНИЕМ БАКТЕРИИ, ПРИНАДЛЕЖАЩЕЙ К РОДУ Escherichia, В КОТОРОЙ ИНАКТИВИРОВАН ГЕН chaC ИЛИ ОПЕРОН chaBC
RU2007131006 2007-08-14
US5370408P 2008-05-16 2008-05-16
US61/053,704 2008-05-16

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WO2006123764A1 (fr) * 2005-05-16 2006-11-23 Ajinomoto Co., Inc. Methodes de production d'un l-amino-acide en utilisant une bacterie de la famille des enterobacteriacees avec l'expression attenuee du gene kefb

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SU1694643A1 (ru) * 1987-11-26 1991-11-30 Всесоюзный научно-исследовательский институт генетики и селекции промышленных микроорганизмов Штамм бактерий ЕSснеRIснIа coLI - продуцент L-треонина
JPH07155184A (ja) * 1993-12-08 1995-06-20 Ajinomoto Co Inc 発酵法によるｌ−リジンの製造法
RU2229513C2 (ru) * 2001-11-23 2004-05-27 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" Способ получения l-аминокислот, штамм escherichia coli - продуцент l-аминокислоты (варианты)

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Publication number Priority date Publication date Assignee Title
WO2006123764A1 (fr) * 2005-05-16 2006-11-23 Ajinomoto Co., Inc. Methodes de production d'un l-amino-acide en utilisant une bacterie de la famille des enterobacteriacees avec l'expression attenuee du gene kefb

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OSHIMA T ET AL: "A 718-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 12.7-28.0 min region on the linkage map (supplement).", DNA RESEARCH : AN INTERNATIONAL JOURNAL FOR RAPID PUBLICATION OF REPORTS ON GENES AND GENOMES 30 JUN 1996, vol. 3, no. 3, 30 June 1996 (1996-06-30), pages 211 - 223, XP002504919, ISSN: 1340-2838 *

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