WO2011102305A2 - Procédé de production d'un acide l-aminé utilisant une bactérie de la famille des enterobacteriaceae ayant une adénylate cyclase mutante - Google Patents

Procédé de production d'un acide l-aminé utilisant une bactérie de la famille des enterobacteriaceae ayant une adénylate cyclase mutante Download PDF

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WO2011102305A2
WO2011102305A2 PCT/JP2011/052967 JP2011052967W WO2011102305A2 WO 2011102305 A2 WO2011102305 A2 WO 2011102305A2 JP 2011052967 W JP2011052967 W JP 2011052967W WO 2011102305 A2 WO2011102305 A2 WO 2011102305A2
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amino acid
coli
gene
bacterium
strain
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WO2011102305A3 (fr
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Natalia Sergeevne Eremina
Natalia Viktorovna Stoynova
Tatyana Abramovna Yampolskaya
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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
    • 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
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y406/00—Phosphorus-oxygen lyases (4.6)
    • C12Y406/01—Phosphorus-oxygen lyases (4.6.1)
    • C12Y406/01001—Aodenylate cyclase (4.6.1.1)

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 a mutant adenylate cyclase.
  • 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.
  • Adenylate cyclase deficient ⁇ cya) mutants of E. coli K-12 were found to be resistant to fosmidomycin, a specific inhibitor of the non-mevalonate pathway, just like to fosfomycin.
  • E. coli glpT mutants were resistant to fosfomycin and also to fosmidomycin. This fact shows that fosmidomycin was transported inside via the glycerol-3-phosphate transporter, GlpT.
  • DNA micro-array analysis showed that the transcription of glpT and other genes concerning glycerol utilization were highly dependent on the presence of cAMP (Sakamoto Y, et al., Biosci Biotechnol
  • aspects of the present invention include enhancing the productivity of L-amino acid-producing strains and providing a method for producing non-aromatic or aromatic L-amino acids 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-arginine, L
  • Enterobacteriaceae family having an increased ability to produce 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.
  • 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-
  • mutant adenylate cyclase selected from the group consisting of:
  • (B) a variant of protein (A), which has an activity of adenylate cyclase.
  • Enterobacteriaceae family which contains the DNA as described above.
  • It is a further aspect of the present invention to provide a method for producing L-amino acid comprising cultivating the bacterium as described above in a culture medium containing glycerol or ethanol, and collecting the L-amino acid from the culture medium.
  • 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.
  • the cyaA gene (synonyms: ECK3800, b3806) encodes the Cya protein, adenylate cyclase (synonym B3806).
  • the cyaA gene (nucleotides from 3,989,176 to 3,991,722; GenBank accession no. NC_000913.2; gi: 49175990) is located between the gene hemC, oriented in the opposite direction, and the gene cyaY, oriented in the opposite direction, on the chromosome of E. coli K-12.
  • the nucleotide sequence of the cyaA gene and the amino acid sequence of Cya encoded by the cyaA gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • adenylate cyclase activity means the activity of catalyzing the synthesis of cyclic AMP (cAMP) by an intramolecular transfer of the adenylyl group of ATP to the 3 '-hydroxy group, releasing pyrophosphate.
  • cAMP cyclic AMP
  • the adenylate cyclase activity assay can be carried out, for example, using the method described by Yang J.K. and Epstein W. (J Biol Chem.; 258(6):3750-8(1983)).
  • the adenylate cyclase in which L-lysine at a position corresponding to position 432 in SEQ ID NO: 2 is replaced with another L-amino acid residue may be referred to as "the mutant adenylate cyclase"
  • a DNA coding for the mutant adenylate cyclase may be referred to as "the mutant cyaAgene” or "mutant adenylate cyclase gene”
  • an adenylate cyclase without the substitution may be referred to as "a wild-type adenylate cyclase”.
  • a position of an amino acid residue may change. For example, if an amino acid residue is inserted at an N-terminus portion, the amino acid residue inherently locates at the position 432 becomes position 433. In such a case, the amino acid residue corresponding to the original position 432 is designated as the amino acid residue at the position 432 in the present invention.
  • Another L-amino acid which can substitute for L-lysine at the position corresponding to position 432 is not particularly limited so long as the amino acid is other than L-lysine, and a bacterium containing the mutant adenylate cyclase having the mutation has a productivity of L-amino acid higher than that of a bacterium which does not contain the mutant adenylate cyclase.
  • An example of the other amino acid is glutamine.
  • the cyaA gene is not limited to the gene shown in SEQ ID No: 1, but may include genes homologous to SEQ ID No: 1. Therefore, the protein variant encoded by the cyaA gene may have a homology of not less than 80%, or not less than 90%, or not less than 95 %, or not less than 98%, or even not less than 99% with respect to the entire amino acid sequence shown in SEQ ID NO. 2, as long as the activity of the Cya protein is maintained.
  • the phrase "protein variant" as used in the present invention means proteins which have changes in the sequences, whether they are deletions, insertions, additions, or substitutions of amino acids.
  • the number of changes in the variant proteins depends on the position in the three dimensional structure of the protein or the type of amino acid residues. It may be 1 to 30, 1 to 15, or even 1 to 5 in SEQ ID NO: 2. These changes in the variants can occur in regions of the protein which are not critical for the three dimensional structure of the protein. This is because some amino acids have high homology to one another so the three dimensional structure is not affected by such a change.
  • 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 term "homology” can mean "identity”.
  • the substitution, deletion, insertion or addition of one or several amino acid residues can be conservative mutation(s) so that the activity is maintained.
  • the representative conservative mutation is a conservative substitution.
  • 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 of Asn, Glu or Gin for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp or Arg for Gin, substitution of Asn, Gin, Lys or Asp for Glu, substitution of Pro for Gly, substitution of Asn, Lys, Gin, Arg or Tyr for His, substitution of Leu, Met, Val or Phe for He, substitution of He, Met, Val or Phe for Leu, substitution of 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, Ser or Ala for Trp, substitution of His, Phe or Trp for Tyr, and substitution of Met, He
  • the cyaA gene may be a variant which hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 1, or a probe which can be prepared from the nucleotide sequence, provided that it encodes a functional Cya protein.
  • Stringent conditions include those under which a specific hybrid, for example, a hybrid having homology of not less than 80%, not less than 90%, not less than 95%), not less than 97%, not less than 98%, or even 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 are exemplified by washing one time or more, preferably two or three times at a salt concentration of 1 xSSC, 0.1% SDS, or 0.1 SSC, 0.1% SDS at 60°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. For example, the recommended duration of washing for the HybondTM N+ nylon membrane (Amersham) under stringent conditions is 15 minutes. Preferably, 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.
  • 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).
  • the bacterium of the present invention is an L-amino acid-producing bacterium of the Enter obacteriaceae family, wherein the bacterium has mutant adenylate cyclase.
  • the bacterium according to the present invention can be obtained by introduction in the cell of the bacterium a DNA fragment encoding mutant adenylate cyclase according to the present invention.
  • L-amino acid-producing bacterium can mean 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 can also mean 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 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, for example, not less than 0.5 g/L, or 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 examples.
  • the Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc. Specifically, those classified into the Enterobacteriaceae according to the taxonomy used by the NCBI (National Center for Biotechnology Information) database
  • 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, for example, 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 present invention.
  • a bacterium belonging to the genus Pantoea means that the bacterium is classified as 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)).
  • a bacterium belonging to either Enterobacter or Pantoea can be used so long as it is classified as the family Enterobacteriaceae.
  • bacteria which are able to produce either an aromatic or a non-aromatic L-amino acid may be used.
  • the bacterium of the present invention can be obtained by modifying a bacterium which inherently has the ability to produce L-amino acids so that the bacterium contains the mutant adenylate cyclase.
  • the bacterium of present invention can be obtained by imparting the ability to produce L-amino acids to a bacterium which already contains the mutant adenylate cyclase .
  • the bacterium can contain a wild-type adenylate cyclase as long as the bacterium contains the mutant adenylate cyclase. However, it is preferable that the bacteirum does not contain the wild-type adenylate cyclase.
  • a gene encoding the mutant adenylate cyclase can be prepared by modifying a wild-type cyaA gene so that the L- amino acid residue corresponding to position 432 in SEQ ID NO: 2 is replaced by another amino acid residue, and then a bacterium of the Enterobacteriaceae family can be transformed with a DNA containing the mutant gene to cause recombination of a corresponding gene on the genome with the mutant gene to substitute the mutant gene for the objective gene on the genome.
  • Examples of such gene substitution using homologous recombination include methods of using a linear DNA such as the method called Red-driven integration (Datsenko, K.A, and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 97:6640-6645 (2000)), and the method utilizing the Red-driven integration in combination with an excisive system derived from ⁇ phage (Cho, E.H., Gumport, R.I., Gardner, J.F., J. Bacteriol., 184:5200-5203 (2002)) (refer to WO2005/010175), a method of using a plasmid containing a temperature-sensitive replication origin (U.S. Patent No. 6,303,383, Japanese Patent Laid-open No. 05-007491), and so forth.
  • Red-driven integration Datsenko, K.A, and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 97:6640-6645
  • site-specific mutagenesis based on gene substitution using homologous recombination can also be performed by using a plasmid which is not able to replicate in a host.
  • glycerol or ethanol in the bacterium of this invention, glycerol or ethanol
  • expression of the glpR gene may be attenuated, or expression of the glycerol metabolism genes (EP 1715055 A) such as glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, tpiA, gldA, dhaK, dhaL, dhaM, dhaR, fsa, and talC genes may be enhanced.
  • the glycerol metabolism genes such as glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, tpiA, gldA, dhaK, dhaL, dhaM,
  • a gene which encodes a mutant glycerol kinase resistant to inhibition by fructose- 1 ,6-bisphosphate may be preferably enhanced (Pettigrew, D. W., Liu, W. Z., Holmes, C, Meadow, N. D., and Roseman, S., J. Bacteriol. 178, 10, 2846-52 (1996), Honisch, C. et.al., Genome Reseasch, 14: 2495-2502 (2004), WO2008/081959 and WO2008/107277). Furthermore, activities of glycerol dehydrogenase and
  • dihydroxyacetone kinase may be enhanced (WO2008/102861).
  • the gene encoding alcohol dehydrogenase (adhE) is modified to expressunder the control of a non-native promoter which functions under aerobic cultivation conditions. (WO2008/010565)
  • a bacterial strain used for producing an L-amino acid is modified so that expression of the adhE gene is controlled by a non-native promoter, i.e., a promoter that does not control the expression of the adhE gene in a wild-type strain.
  • a non-native promoter i.e., a promoter that does not control the expression of the adhE gene in a wild-type strain.
  • Such modification can be achieved by replacing the native promoter of the adhE gene on the choromosome with a non-native promoter which functions under an aerobic cultivation condition so that the adhE gene is operably linked with the non- native promoter.
  • a non-native promoter which functions under aerobic cultivation conditions any promoter which can express the adhE gene above a certain level under aerobic cultivation conditions may be used.
  • the activity of alcohol dehydrogenase in the cell free extract measured according to the method by Clark and Cronan can be, for example, 1.5 units or more, 5 units or more, or 10 units or more, per mg of protein.
  • Aerobic cultivation conditions can be those usually used for cultivation of bacteria in which oxygen is supplied by methods such as shaking, aeration and agitation.
  • any promoter which is known to express a gene under aerobic cultivation conditions can be used.
  • the P tac promoter, the lac promoter, the trp promoter, the trc promoter, the PR, or the PL promoters of lambda phage are all known to be strong promoters which function under aerobic cultivation conditions, and are preferably used.
  • a wild-type alcohol dehydrogenase may be subject to metal catalyzed oxidation. Although such a wild-type alcohol dehydrogenase can be used, a mutant alcohol dehydrogenase which is resistant to aerobic inactivation is preferable in the present invention.
  • the phrase "mutant alcohol dehydrogenase which is resistant to aerobic inactivation" means that the mutant alcohol dehydrogenase maintains its activity under aerobic conditions, or the activity is reduced by a negligible amount compared to the wild-type alcohol dehydrogenase.
  • mutant alcohol dehydrogenase which is resistant to aerobic inactivation can be used in the following:
  • 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. coli NRRL-21593 (U.S. Patent No. 5,939,307), E. coli FERM BP- 3756 (U.S. Patent No. 5,474,918), E.
  • E. coli TDH-6/pVIC40 VKPM B-3996
  • E.S. Patent No. 5, 175, 107, U.S. Patent No. 5,705,371 E. coli 472T23/pYN7 (ATCC 98081) (U.S. Patent
  • the strain TDH-6 is deficient in the thrC gene, as well as being sucrose- assimilative, and the ilvA 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 B-3996 contains the plasmid pVIC40 which was obtained by inserting a thrA*BC operon which includes a mutant thrA gene into a RSF1010- derived vector.
  • This mutant thrA gene encodes aspartokinase homoserine
  • dehydrogenase I which has substantially desensitized feedback inhibition by threonine.
  • the strain B-3996 was deposited on November 19, 1987 in the Ail-Union Scientific Center of Antibiotics ( Russian, 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) (GNU genetika, 1 Dorozhny proezd, 1, Moscow 117545, Russian Federation) on April 7, 1987 under the accession number B-3996.
  • 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 iso leucine, and a temperature-sensitive lambda-phage CI 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:
  • mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine;
  • the thrA gene which encodes aspartokinase homoserine dehydrogenase I of Escherichia coli has been elucidated (nucleotide positions 337 to 2799, GenBank accession no.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 no.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 no.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 function as a single threonine operon.
  • the attenuator region which affects the transcription is desirably removed from the operon (WO2005/049808,
  • 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 present 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 ORF1 (ybiF gene, nucleotide positions 764 to 1651, GenBank accession number AAA218541, gi:440181) and is located between the pexB and ompX genes.
  • the unit expressing a protein encoded by the ORF1 has been designated the rhtA gene (rht: resistance to homoserine and threonine).
  • the asd gene of E. coli has already been elucidated (nucleotide positions 3572511 to 3571408, GenBank accession no. 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 no. 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, a-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 AJ11442 (FERM BP-1543, NRRL B-12185; see U.S. Patent No. 4,346,170) and Escherichia coli VL61 1. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is desensitized.
  • the strain WC196 may be used as an L-lysine producing bacterium of
  • Escherichia coli This bacterial strain was bred from the W3110 strain, which was derived from Escherichia coli K-12, by replacing the wild type lysC gene on the chromosome of the W31 10 strain with a mutant lysC gene encoding a mutant aspartokinase III desensitized to feedback inhibition by L-lysine in which threonine at position 352 had been replaced with isoleucine, and conferring AEC resistance to the resulting strain (U.S. patent No. 5,661 ,012).
  • the resulting strain was designated Escherichia coli AJ13069 strain and was deposited at the National Institute of
  • 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 (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (U.S. Patent No.
  • the parent strains may have increased 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
  • 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 JM15 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 W3110 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
  • JP11155571 A2 E. coli W3110 with increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene (WOO 127307 A 1), and the like.
  • 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-hydroxyleucine, 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.
  • leucine for example, the strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121)
  • leucine analogs including ⁇ -2- thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A)
  • 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 which is free 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). L-histidine-producing bacteria
  • 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, RU2119536); 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) (EP1085087); E. coli AI80/pFM201 (U,S. Patent No. 6,258,554) and the like.
  • E. coli strain 24 VKPM B-5945, RU2003677
  • E. coli strain 80 VKPM B-7270, RU2119536
  • 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 into ATP
  • 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 (EP1016710A), E. coli 80 strain imparted with sulfaguanidine, DL-l,2,4-triazole-3 -alanine, and streptomycin- resistance (VKPM B-7270, Russian Patent No. 21 19536), and so forth.
  • JP 56-005099 A E. coli strains introduced with rht, a gene for an amino acid-export
  • EP1016710A E. coli 80 strain imparted with sulfaguanidine, DL-l,2,4-triazole-3 -alanine, and streptomycin- resistance
  • 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 ilvA 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 PI which was grown on wild-type E. coli K12 (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 which are deficient in a- ketoglutarate dehydrogenase activity, or strains in which one or more genes encoding an L-glutamic acid biosynthetic enzyme are enhanced.
  • the genes involved in L-glutamic acid biosynthesis include genes encoding glutamate
  • dehydrogenase gdhA
  • glutamine synthetase glnA
  • glutamate synthetase gltAB
  • isocitrate dehydrogenase icdA
  • acnA, acnB citrate synthase
  • ppc phosphoenolpyruvate carboxylase
  • pyc pyruvate carboxylase
  • pyc pyruvate dehydrogenase
  • aceEF IpdA
  • pyruvate kinase pykA, pykF
  • phosphoenolpyruvate synthase ppsA
  • end phosphoglyceromutase
  • phosphoglycerate kinase pgk
  • glyceraldehyde-3-phophate dehydrogenase g pA
  • tpiA triose phosphate isomerase
  • fbp fructose bisphosphate aldolase
  • pflcA phosphofructokinase
  • pflcB glucose phosphate isomerase
  • pgi glucose phosphate isomerase
  • 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.
  • strains which have been modified so that expression of the citrate synthetase gene and/or the phosphoenolpyruvate carboxylase gene are reduced, and/or/are deficient in a-ketoglutarate dehydrogenase activity 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.
  • Such enzymes include isocitrate lyase (aceA), -ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), acetolactate synthase (ilvT), formate acetyltransferase (pfl), lactate dehydrogenase (Idh), and glutamate decarboxylase (gadAB).
  • aceA isocitrate lyase
  • sucA -ketoglutarate dehydrogenase
  • pta phosphotransacetylase
  • ack acetate kinase
  • ack acetohydroxy acid synthase
  • ilvT acetolactate synthase
  • pfl lactate dehydrogenase
  • Idh lactate dehydrogenase
  • glutamate decarboxylase glutamate
  • E. coli AJ12628 (FERM BP-3854)
  • E. coli AJ12949 (FERM BP-4881)
  • E. coli W3110sucA::Km R is a strain obtained by disrupting the a-ketoglutarate dehydrogenase gene (hereinafter referred to as "sucA gene") of E. coli W3110. This strain is completely deficient in the a-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 the ⁇ -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. 6,1 10,714), and the like.
  • L-glutamic acid-producing bacteria examples include mutant strains belonging to the genus Pantoea which are deficient in the a-ketoglutarate
  • 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.
  • Pantoea ananatis AJ13356 is deficient in the ⁇ -ketoglutarate dehydrogenase activity as a result of disruption of the ocKGDH-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.
  • AJ13356 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::Tnl0, tyrR) (VKPM B-8197); E. coli HW1089 (ATCC 55371) harboring the mutant pheA34 gene (U.S. Patent No. 5,354,672); E. coli MWEClOl-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. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12
  • 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) which is deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Patent No. 5,756,345); E.
  • coli SV164 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine and a trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan (U.S. Patent No. 6,180,373); E. coli AGX17 (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
  • 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 SV164 which harbors desensitized anthranilate synthase and a transformant strain obtained by introducing into the E.
  • coli SV164 the plasmid pGH5 (WO 94/08031), which contains a mutant serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase.
  • 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 a 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
  • 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 ilvA gene and is able to produce L-proline (EP 1172433).
  • 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. Such 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. 2001112869), E. coli strain 382 (VKPM B- 7926) (EP1170358A1), an arginine-producing strain into which argA gene encoding N-acetylglutamate synthetase is introduced therein (EP1 170361A1), 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 (carAB).
  • argC N-acetylglutamyl phosphate reductase
  • argJ ornithine acetyl transferase
  • parent strains for deriving L-valine-producing bacteria of the present invention include bacteria belonging to the genus Escherichia such as H-81 (VKPM B- 8066), NRRL B-12287 and NRRL B-12288 (US patent No. 4,391 ,907), VKPM B-441 1 (US patent No. 5,658,766), VKPM B-7707 (European patent application EP1016710A2), or the like.
  • 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 ilvGMEDA operon (U.S. Patent No. 5,998,178). It is desirable to remove the region of the ilvGMEDA operon which is required for attenuation so that expression of the operon is not attenuated by L-valine that is produced. Furthermore, the ilvA 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 also include mutants having a mutation of amino-acyl t-RNA synthetase (U.S. Patent No. 5,658,766).
  • E. coli VL1970 which has a mutation in the ileS gene encoding isoleucine tRNA synthetase, can be used.
  • E. coli VL1970 has been deposited in the Russian National Collection of Industrial
  • VKPM Microorganisms
  • 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.
  • L-methionine-producing bacteria and parent strains for deriving L-methionine producing bacteria include, but are not limited to, L-threonine- auxotrophic mutant strain and norleucine-resistant mutant strain (JP 2000-139471 A). Furthermore, a methionine repressor-deficient strain and recombinant strains transformed with genes encoding proteins involved in L-methionine biosynthesis such as homoserine transsuccinylase and cystathionine ⁇ -synthase (JP 2000-139471 A) can also be used as parent strains.
  • the method of the present invention is a method for producing an L-amino acid comprising 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.
  • Glycerol and/or Ethanol can be used as a carbon source in the fermentation medium.
  • Glycerol refers to a substance of the nomenclatural name of propane- 1,2,3- triol. Crude glycerol refers to industrially produced glycerol which also contains impurities. Crude glycerol is industrially produced by hydrolyzing fats and oils with water at a high temperature and under high pressure, or by the esterification reaction for biodiesel fuel production. Biodiesel fuel includes aliphatic acid methyl esters produced from fats and oils and methanol produced by a transesterification. Crude glycerol is produced as a by-product of this reaction (refer to Fukuda, H., Kondo, A., and Noda, H., 2001 , J. Biosci.
  • the crude glycerol preferably contains ions from the alkali and the neutralization acid, such as sodium ions, potassium ions, chloride ions, and sulfate ions in an amount of, for example, 2 to 7%, 3 to 6%, or 4 to 5.8%, based on the weight of the crude glycerol.
  • ions from the alkali and the neutralization acid such as sodium ions, potassium ions, chloride ions, and sulfate ions in an amount of, for example, 2 to 7%, 3 to 6%, or 4 to 5.8%, based on the weight of the crude glycerol.
  • methanol may not be present, it can be present in an amount of 0.01% or less.
  • the crude glycerol may further contain trace amounts of metals, organic acids, phosphorus, aliphatic acids, and so forth.
  • organic acids include formic acid, acetic acid, and so forth, and although they may not be present, they are preferably present in an amount of 0.01% or less.
  • Trace metals required for growth of the microorganism are preferred, and examples include, for example, magnesium, iron, calcium, manganese, copper, zinc, and so forth.
  • Magnesium, iron, and calcium are preferably present in an amount of for example, 0.00001 to 0.1%, 0.0005 to 0.1%, 0.004 to 0.05%, or 0.007 to 0.01%, in terms of the total amount based on the weight of the crude glycerol.
  • Manganese, copper, and zinc are preferably present in an amount of, for example, 0.000005 to 0.01%, 0.000007 to 0.005%, or 0.00001 to 0.001%, in terms of the total amount.
  • the purity of the crude glycerol may be 10% or higher, 50% or higher, 70% or higher, or 80% or higher. As long as the impurities are within the aforementioned range, the purity of the glycerol may be 90% or higher.
  • Crude glycerol produced in the production of biodiesel fuel is preferred.
  • crude glycerol which enables production of more L-amino acid is preferred, compared with that when using an equal weight of reagent glycerol.
  • To produce more L-amino acid as compared with reagent glycerol means to increase the amino acid production amount by, for example, 5% or more, 10% or more, or 20% or more, as compared with when reagent glycerol is used as the carbon source.
  • "Reagent glycerol” means glycerol marketed as regent grade or having a purity equivalent to the purity of glycerol marketed as regent grade.
  • the glycerol has a purity of 99% by weight or higher, and pure glycerol is particularly preferred.
  • the expression "reagent glycerol in the same amount as crude glycerol” means that the reagent glycerol is the same weight as crude glycerol except for water, when the crude glycerol contains water.
  • Crude glycerol may be diluted with a solvent such as water.
  • a solvent such as water.
  • the aforementioned descriptions concerning the amount of glycerol and impurities can be applied to crude glycerol before dilution. That is, when a solution of crude glycerol in a solvent is used and the solvent is reduced so that it is 30% by weight or less, 20% by weight or less, or 10% by weight or less, if the content of glycerol and any impurities are within the aforementioned ranges, this crude glycerol corresponds to the "crude glycerol" used in the present invention.
  • Ethanol is a monohydric alcohol represented by the molecular formula C 2 H 5 OH, and may be used alone, or may be present as a mixture in the medium, such as the ethanol which is produced in ethanol fermentation in the medium etc..
  • Ethanol may be present in the medium at any concentration so long as the chosen bacterium can assimilate it as the carbon source. When it is used as the sole carbon source in the medium, it is present in an amount of 20% w/v or less, 10% w/v or less, or 2%> w/v or less. Furthermore, ethanol may be present in the medium at any concentration so long as it can be assimilated as the carbon source by the chosen bacterium. When it is used as the sole carbon source in the medium, it is desirably present in the medium in an amount of 0.001% w/v or more, 0.05% w/v or more, or 0.1%) w/v or more.
  • the feed medium when ethanol is used as the sole carbon source, it can be present in the medium in an amount of 10% w/v or less, 5% w/v or less, or 1% w/v or less.
  • concentration of ethanol can be measured by various methods, the enzymatic method is convenient and common (Swift R., 2003, Addiction, 98:73-80).
  • concentration of aliphatic acid can be measured by known methods such as gas chromatography and HPLC (TrAC Trends Anal. Chem., 2002, 21 :686-697; Lin J.T., Snyder L.R., and McKeon, T.A., 1998, J. Chromatogr. A., 808:43-49).
  • the medium may contain a mixture of ethanol.
  • concentrations of ethanol which are added may be any concentration so long as the chosen bacterium can assimilate them as the carbon source.
  • concentrations of ethanol which are added may be any concentration so long as the chosen bacterium can assimilate them as the carbon source.
  • concentrations of ethanol which are added may be any concentration so long as the chosen bacterium can assimilate them as the carbon source.
  • a mixture of ethanol when used as the sole carbon source in the medium, it can be present in an amount of 20% w/v or less, 10% w/v or less, or 2% w/v or less, in terms of the total concentration.
  • a mixture of ethanol may be present in the medium at any concentration so long as it can be assimilated as the carbon source by the bacterium.
  • a mixture of ethanol when used as the sole carbon source in the medium, it can be contained in the medium in an amount of 0.001 ) w/v or more, 0.05% w/v or more, or 0.1% w/v or more, in terms of the total concentration of ethanol .
  • other carbon sources may also be added to the medium, for example, such as saccharides such as glucose, fructose, sucrose, lactose, galactose, blackstrap molasses, and starch hydrolysate, polyhydric alcohols, and organic acids such as fumaric acid, citric acid, and succinic acid. Glucose, sucrose, and fructose are especially preferred.
  • the carbon source may be one kind of substance or a mixture of two or more kinds of substances. When other carbon sources are used, the ratio of ethanol, aliphatic acid, or a mixture of ethanol and glycerol in the carbon source can be 10% by weight or more, 30% by weight or more, or 50%» by weight or more.
  • 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 amino acid is produced using a bacterium.
  • a medium used for culture may be either a synthetic or natural medium, so long as the medium includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth.
  • Ethanol or glycerol is used as the carbon source.
  • 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.
  • minerals 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 by shaking and/or stirring 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
  • 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.
  • the collected L-amino acid may contain bacterial cells, medium components, moisture, and by-product metabolites of the bacterium in addition to the L-amino acid.
  • Purity of the collected L-amino acid can be 50% or higher, 85% or higher, or even 95% or higher (Japanese Patent No. 1214636, U.S. Patent Nos. 5,431,933, 4,956,471, 4,777,051, 4,946,654, 5,840,358, 6,238,714, U.S. Patent Published Application No. 2005/0025878).
  • Example 1 Preparation of the E. coli strain MG1655 cyaA K432Q Pi, tar ,-adhE * .
  • strain MG1655 P L-tac adhE* (WO2008010565) was maintained in prolonged exponential growth by daily passage into fresh minimal medium (M9) supplemented with 2% (v/v) ethanol. Each 100 hours of experiment, probes of culture were taken and growth rate on the same medium was measured.
  • the strain MG1655 PL-tacadhE* was derived from the strain MG1655.
  • the wild-type adhE gene is replaced with a mutant adhE gene which encodes a mutant alcohol dehydrogenase having Glu568Lys (E568K) mutation.
  • the mutant adhE gene is expressed by P L -tac promoter.
  • the strain MG1655 (ATCC47076, ATCC700926) can be available from the American Type Culture Collection (Address: 12301 Parklawn Drive, Rockville, Maryland 20852, P.O. Box 1549, Manassas, VA 20108, United States of America). At the 700 hours of experiment no further improvement of growth rate was observed. The cell suspension of this "fitness" culture was spread on the LB plates to form the individual colonies. The growth rate of seven independent clones was measured. It was shown that all these clones and "fitness" culture has the same growth rate on ethanol. One from these clones, Fit73, was chosen for further investigation.
  • chloramphenicol resistance marker (Cm R ) downstream cyaA K432Q gene was marked with chloramphenicol resistance marker (Cm R ) downstream cyaA K432Q gene as follows.
  • the DNA fragment carrying chloramphenicol resistance marker (Cm R ) encoded by the cat gene was integrated into the chromosome of strain Fit73 downstream cyaA K432Q gene by the method described by Datsenko K.A. and Wanner B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97, 6640-6645) which is also called “Red-mediated integration" and/or "Red-driven integration".
  • a DNA fragment containing a Cm R marker encoded by the cat gene was obtained by PCR using the pMWl 18-attL-Cm-attR plasmid (WO 05/010175) as a template, and primers PI (SEQ ID NO: 3) and P2 (SEQ ID NO: 4).
  • PCR was provided using the "Gene Amp PCR System 2700" amplificatory (Applied Biosystems).
  • the reaction mixture (total volume - 50 ⁇ ) consisted of 5 ⁇ of lOx PCR-buffer with 25 mM MgCl 2 ("Fermentas", Lithuania), 200 ⁇ each of dNTP, 25 pmol each of the exploited primers and 1 U of Taq-polymerase ("Fermentas", Lithuania).
  • the temperature profile was the following: initial DNA denaturation for 5 min at 95 °C, followed by 25 cycles of denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec, elongation at 72 °C for 40 sec; and the final elongation for 5 min at +72 °C.
  • the amplified DNA fragment was purified by agarose gel-electrophoresis, extracted using "GenElute Spin Columns" ("Sigma", USA) and precipitated by ethanol.
  • the obtained DNA fragment was used for electroporation and Red-mediated integration into the bacterial chromosome of the selected E. coli "fitness" strain Fit73.
  • the grown cells from 10 ml of the bacterial culture were washed 3 times by the ice-cold de-ionized water, followed by suspension in 100 ⁇ of the water. 10 ⁇ of DNA fragment (100 ng) dissolved in the de-ionized water was added to the cell suspension.
  • the electroporation was performed by "Bio-Rad” electroporator (USA) (No. 165-2098, version 2-89) according to the manufacturer's instructions.
  • the temperature profile follows: initial DNA denaturation for 5 min at 95 °C; then 30 cycles of denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec and elongation at 72 °C for 30 sec; the final elongation for 5 min at 72 °C.
  • a few Cm R colonies tested contained the desired 2076 bp DNA fragment, confirming the presence of Cm R marker DNA downstream cyaA K432Q gene.
  • One of the obtained strains was cured of the thermosensitive plasmid pKD46 by culturing at 37 °C.
  • mutant strain containing mutant cyaA K432Q gene was obtained.
  • the strain was named MG1655 cyaA K432Q P Lt ac-adhE*.
  • Example 2 The effect of mutation in the cyaA gene on L-threonine production.
  • the E. coli strain MG1655Atdh, rhtA*, cyaA K432Q P L - tac dhE* was obtained by transferring the DNA fragment from the chromosome of the E. coli strain MG1655 cyaA K432Q ?u ⁇ -adhE* to the E. coli strain MG1655 Atdh rhtA* P Ltac -adhE*
  • the strain MG1655 Atdh rhtA* P Lt ac-adhE* was obtained from the MG1655Atdh rhtA* strain.
  • the MG1655Atdh rhtA* strain corresponds to the MG1655 strain, but the tdh gene encoding threonine dehydrogenase is disrupted by the method of Datsenko and Wanner and a rhtA23 mutation is introduced therein, which imparts resistance to high concentrations of threonine in a minimal medium to the rhtA gene (Livshits, V.A., Zakataeva, N.P., Aleshin, V.V., Vitushkina, M.V., 2003, Res. Microbiol., 154:123-135).
  • MG1655Atdh, rhtA*, P L- ta C adhE* and MG1655Atdh, rhtA*, c ⁇ i e P L-tac adhE* were transformed with the plasmid pVIC40 (the plasmid pVIC40 is described in detail in U.S. Patent No. 5,705,371).
  • pVIC40 contains a thrA*BC operon including a mutant thrA gene which encodes aspartokinase homoserine dehydrogenase I which is substantially desensitized feedback inhibition by threonine.
  • pVIC40 can be obtained from the strain E.
  • VKPM B-3996 The strain E. coli TDH-6/pVIC40 was deposited at the Russian National Collection of Industrial Microorganisms (VKPM) (GNU genetika, 1 Dorozhny proezd, 1 , Moscow 1 17545, Russian Federation) on April 7, 1987 under the accession number VKPM B- 3996.
  • K 2 HP0 4 and CaC0 3 were sterilized separately.
  • the pH was adjusted to 7.0.
  • Example 3 The effect of mutation in the cyaA gene on on L-lysine production.
  • pCABD2 is a plasmid including the dapA gene coding for a dihydrodipicolinate synthase having a mutation which desensitizes feedback inhibition by L-lysine, the lysC gene coding for aspartokinase III having a mutation which desensitizes feedback inhibition by L-lysine, the dapB gene coding for a dihydrodipicolinate reductase, and the ddh gene coding for diaminopimelate dehydrogenase (U.S. Patent No. 6,040, 160).
  • the E. coli strain WC196 P L- ta C adhE* cat- cyaA K432Q (pCABD2) can be obtained by transferring DNA fragments from the chromosome of the E. coli MG1655 cyaA K432Q P Ltac -adhE* into the E. coli strain WC196 P L-tac adhE* (pCABD2) by PI transduction.
  • the E. coli strains WC 196 P L-tac adhE* cat- cyaA K432Q (pC ABD2) and WC 196 PL- t acadhE* (pCABD2) can be separately cultured in L-medium containing 20 mg/1 of streptomycin 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 hours by using a reciprocal shaker at the agitation speed of 115 rpm.
  • the amounts of L-lysine and residual ethanol in the medium can be measured by a known method (Biotech-analyzer AS210, manufactured by Sakura Seiki Co.). Then, the yield of L-lysine relative to consumed ethanol can be calculated for each of the strains.
  • pH is adjusted to 7.0 by KOH and the medium is autoclaved at 115°C for 10 min. Ethanol and MgS0 4 7H 2 0 are sterilized separately. CaC0 3 is dry-heat sterilized at 180°C for 2 hours and added to the medium at a final concentration of 30 g/1.
  • Example 4 The effect of mutation in the cyaA gene on L-histidine production.
  • strain 80 has been described in Russian patent 2119536 and deposited in the Russian National Collection of Industrial Microorganisms (VKPM)(GNII genetika, 1 Dorozhny proezd, 1, Moscow 117545, Russian Federation) on October 15, 1999 under accession number VKPM B-7270 and then converted to a deposit under the Budapest Treaty on July 12, 2004.
  • VKPM National Collection of Industrial Microorganisms
  • the E. coli strain 80 P L-tac adhE* cat- cyaA K432Q can be obtained by transferring DNA fragments from the chromosome of the E. coli MG1655 cyaA K4S2Q P ac-adhE* into the E. coli strain 80 P L - tac adhE* by PI transduction.
  • composition of the fermentation medium (pH 6.0) (g/1):
  • Ethanol, proline, betaine and CaC0 3 are sterilized separately. pH is adjusted to 6.0 before sterilization.
  • Example 5 The effect of mutation in the cyaA gene on on L-leucine production.
  • coli strain NS 1391 P L-tac adhE* cyaA K432Q can be obtained by transferring DNA fragments from the chromosome of the E. coli MG1655 Atdh rhtA* cy A K432Q P Uac - adhE* to the E. coli strain NS1391 P L-t a C adhE* (WO2008010565) by PI transduction.
  • the E. coli strains NS1391 P L-t a C adhE* cyaA K432Q and NS1391 P L-t a C adhE* can be separately cultured for 18-24 hours at 37°C on L-agar plates.
  • the strains can be 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% sucrose.
  • the fermentation medium can be inoculated with 0.21 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°C with shaking at 250 rpm.
  • composition of the fermentation medium (g/1) (pH 7.2):
  • Glucose, ethanol and CaC0 3 are sterilized separately.
  • Example 6 The effect of mutation in the cvaA gene on L-phenylalanine production.
  • the strain AJ12739 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (GNU genetika, 1 Dorozhny proezd, 1 , Moscow 117545, Russian Federation) on November 6, 2001 under accession number VKPM B- 8197 and then converted to a deposit under the Budapest Treaty on August 23, 2002.
  • VKPM National Collection of Industrial Microorganisms
  • the E. coli strain AJ 12739 P L - t a C adhE* cyaA K432Q can be obtained by transferring DNA fragments from the chromosome of the E. coli strain MG1655 cyaA K432Q Puac-adhE* to the strain E. co//.AJ12739 P L- ta C adhE* by PI transduction.
  • coli strains AJ12739 P L-tac adhE* cat- cyaA K432Q and AJ12739 P L- tac adhE* can be separately cultivated at 37°C for 18 hours in a nutrient broth, and 0.3 ml of the obtained cultures can each be inoculated into 3 ml of a fermentation medium in a 20 x 200 mm test tube and cultivated at 37 °C for 48 hours with a rotary shaker. After cultivation, the amount of phenylalanine which accumulates in the medium can be determined by TLC.
  • Sorbfil silica gel without fluorescent indicator (Stock Company Sorbpolymer, Krasnodar, Russia) can be used.
  • a solution (2%) of ninhydrin in acetone can be used as a visualizing reagent.
  • composition of the fermentation medium (g/1)
  • Ethanol and magnesium sulfate are sterilized separately.
  • CaC0 3 dry-heat sterilized at 180°C for 2 hours. pH is adjusted to 7.0.
  • Example 7 The effect of mutation in the cyaA gene on L-arginine production.
  • E. coli strain 382 P L - tac dhE* cyaA K432Q can be obtained by transferring DNA fragments from the chromosome of the E. coli strain MG1655 cyaA K432Q cat PL tac - adhE* into the strain E. coli 382 P L - t a C adhE* by PI transduction.
  • the E. coli strains 382 P L-tac adhE* cat- cyaA K432Q and 382 P L - ta cadhE* can be separately cultivated with shaking at 37 °C for 18 hours in 3 ml of nutrient broth, and 0.3 ml of the obtained cultures can be 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.
  • the amount of L-arginine which accumulates in the medium can be determined by paper chromatography using the following mobile phase:
  • a solution of ninhydrin (2%) in acetone can be used as a visualizing reagent.
  • a spot containing L-arginine can be cut out, L- arginine can be eluted with 0.5% water solution of CdCl 2 , and the amount of L- arginine can be estimated spectrophotometrically at 540 nm.
  • composition of the fermentation medium (g/l):
  • MgS0 4 -7H 2 0, ethanol and CaC0 3 are each sterilized separately.
  • Example 8 The effect of mutation in the cyaA gene on L-tryptophan production.
  • the strain SV164 has the trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan.
  • the plasmid pGH5 harbors a mutant serA gene encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine.
  • the strain SV164 (pGH5) is described in detail in U.S. Patent No. 6,180,373.
  • the E. coli strain SV164 P L-tac adhE* cyaA K432Q (pGH5) can be obtained by transferring DNA fragments from the chromosome of the E. coli strain MG1655 cyaA K432Q P Ltac -adhE* into the E. coli strain SV164 P L-tac adhE*(pGH5) by PI transduction.
  • the E. coli strains SV164 P L- ta C adhE* cat- cyaA K432Q (pGH5) and SV164 P L - t ac adhE* (pGH5) can be separately cultivated with shaking at 37 °C for 18 hours in a 3 ml of nutrient broth supplemented with 20 mg/1 of tetracycline (marker of pGH5 plasmid).
  • 0.3 ml of the obtained cultures can each 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 °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 6.
  • the fermentation medium components are set forth 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.
  • Solution A had a pH of 7.1, adjusted by NH 4 OH.
  • Example 9 The effect of mutation in the cyaA gene on L- glutamic acid production.
  • strain VL334thrC + has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (GNU genetika, 1 Dorozhny proezd, 1, Moscow 1 17545, Russian Federation) 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 National Collection of Industrial Microorganisms
  • the E. coli strain VL334thrC + PL- tac adhE* cyaA K432Q can be obtained by transferring DNA fragments from the chromosome of the E. coli strain MG1655 cyaA K432Q Puac-adhE* to the E. coli strain VL334thrC + P L- ta C adhE* by PI transduction.
  • the E. coli strains VL334thrC + P L-tac adhE* cyaA K432Q and VL334thrC + PL- t a c adhE* can be separately cultivated with shaking at 37 °C for 18 hours in a 3 ml of nutrient broth.
  • 0.3 ml of the obtained cultures can each be inoculated into 3 ml of a fermentation medium in 20 x 200 mm test tubes, and cultivated at 37 °C for 48 hours with a rotary shaker at 250 rpm.
  • composition of the fermentation medium (pH 7.2) (g/1):
  • Ethanol and CaC0 3 are sterilized separately.
  • Example 10 The effect of mutation in the cyaA gene on L-valine production To evaluate the effect of K432Q mutation in the cyaA gene on L-valine production, at first the L-valine producing strain E. coli H-81 PL-t ac adhE* was obtained by transferring DNA fragments from the chromosome of the E. coli strain
  • MG1655::P L - tac adhE* (US2009203090 (Al) to the valine-producing E. coli strain H-81 by PI transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY).
  • the H-81 strain was deposited at the Russian National Collection of Industrial Microorganisms (VKPM) (GNU genetika, 1 Dorozhny proezd, 1, Moscow 117545, Russian Federation) on January 30, 2001 under the accession number VKPM B-8066. Then the strain E. coli H-81 P L - tac dhE* cyaA K432Q was obtained by transferring DNA fragments from the chromosome of the E. coli strain MG1655 cyaA K432Q cat P Lt ac- adhE* into the strain E. coli H-81 P L-taC adhE* by PI transduction.
  • VKPM Russian National Collection of Industrial
  • the strains H-81 P L-tac adhE* cyaA K432Q and H-81 P L-tac adhE* were cultivated at 37 °C for 18 hours in a nutrient broth and 0.1 ml of each of the obtained cultures was inoculated into 2 ml of fermentation medium in a 20x200 mm test tube and cultivated at 32 °C for 72 hours with a rotary shaker. After cultivation for 24 hours in
  • H-81 P L -t ac adhE* cyaA K43 Q caused accumulation of a higher amount of L-valine, as compared with H-81 P L - ta cadhE*, both in the medium containing ethanol and in the medium containing glycerol.
  • CaC0 3 was dry-heat sterilized at 180 °C for 2 hours. The pH was adjusted to 7.0.

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Abstract

L'invention concerne un procédé de production d'acide L-aminé à partir d'éthanol ou de glycérol utilisant une bactérie de la famille des Enterobacteriaceae qui contient une adénylate cyclase mutante.
PCT/JP2011/052967 2010-02-18 2011-02-04 Procédé de production d'un acide l-aminé utilisant une bactérie de la famille des enterobacteriaceae ayant une adénylate cyclase mutante WO2011102305A2 (fr)

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WO2021122687A1 (fr) 2019-12-19 2021-06-24 Basf Se Augmentation du rendement spatio-temporel, de l'efficacité de conversion du carbone et de la flexibilité des substrat carbonés dans la production de produits chimiques fins
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WO2021122687A1 (fr) 2019-12-19 2021-06-24 Basf Se Augmentation du rendement spatio-temporel, de l'efficacité de conversion du carbone et de la flexibilité des substrat carbonés dans la production de produits chimiques fins
WO2023285585A2 (fr) 2021-07-16 2023-01-19 Biosyntia Aps Usines de cellules microbiennes produisant des composés de vitamine b

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