WO2008032757A1 - PROCÉDÉ DE FABRICATION D'UN ACIDE L-AMINO À L'AIDE D'UNE BACTÉRIE DE LA FAMILLE DES ENTEROBACTERIACEAE AVEC UNE EXPRESSION AMÉLIORÉE DE L'OPÉRON alsABC - Google Patents

PROCÉDÉ DE FABRICATION D'UN ACIDE L-AMINO À L'AIDE D'UNE BACTÉRIE DE LA FAMILLE DES ENTEROBACTERIACEAE AVEC UNE EXPRESSION AMÉLIORÉE DE L'OPÉRON alsABC Download PDF

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WO2008032757A1
WO2008032757A1 PCT/JP2007/067782 JP2007067782W WO2008032757A1 WO 2008032757 A1 WO2008032757 A1 WO 2008032757A1 JP 2007067782 W JP2007067782 W JP 2007067782W WO 2008032757 A1 WO2008032757 A1 WO 2008032757A1
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bacterium
gene
operon
amino acid
coli
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Konstantin Vyacheslavovich Rybak
Ekaterina Aleksandrovna Slivinskaya
Marina Evgenievna Sheremet'eva
Yury Ivanovich Kozlov
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Ajinomoto Co., Inc.
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/222—Phenylalanine
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    • 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
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/227—Tryptophan
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/24—Proline; Hydroxyproline; Histidine

Definitions

  • the present invention relates to a method for producing an L-amino acid such as L- threonine, L-lysine, L-leucine, L-histidine, L-cysteine, L-phenylalanine, L-arginine, L- tryptophan, L-glutamic acid, L-valine, L-proline and L-isoleucine by fermentation.
  • L-amino acid such as L- threonine, L-lysine, L-leucine, L-histidine, L-cysteine, L-phenylalanine, L-arginine, L- tryptophan, L-glutamic acid, L-valine, L-proline and L-isoleucine by fermentation.
  • 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.
  • Strains useful in production of L-threonine by fermentation are known, including strains with increased activities of enzymes involved in L-threonine biosynthesis (U.S. Patent Nos. 5,175,107; 5,661,012; 5,705,371; 5,939,307; EP 0219027), strains resistant to chemicals such as L-threonine and analogs thereof (WO 01/14525A1, EP 301572 A2, U.S. Patent No. 5,376,538), strains with target enzymes desensitized to feedback inhibition by the produced L- amino acid or its by-products (U.S. Patent Nos. 5,175,107; 5,661,012), and strains with inactivated threonine degradation enzymes (U.S. Patent Nos. 5,939,307; 6,297,031).
  • the known threonine-producing strain Escherichia coli VKPM B-3996 (U.S. Patent Nos. 5,175,107 and 5,705,371) is presently one of the best known threonine producers.
  • VKPM B-3996 strain To construct the VKPM B-3996 strain, several mutations and a plasmid, described below, were introduced into the parent strain E. coli K- 12 (VKPM B-7).
  • a mutant thrA gene (mutation thrA442) encodes aspartokinase homoserine dehydrogenase I, which is resistant to feedback inhibition by threonine.
  • a mutant HvA gene (mutation ilvA442) encodes threonine deaminase which has decreased activity, and results in a decreased rate of isoleucine biosynthesis and a leaky phenotype of isoleucine starvation.
  • transcription of the thrABC operon is not repressed by isoleucine; and therefore, this mutation results in very efficient threonine production.
  • Inactivation of the tdh gene encoding threonine dehydrogenase results in the prevention of threonine degradation.
  • the genetic determinant of saccharose assimilation (scrKYABR genes) was transferred to this strain.
  • the plasmid pVIC40 containing the mutant threonine operon thrA442BC was introduced into the intermediate strain TDH6.
  • the amount of L-threonine produced during fermentation of the strain can be up to 85 g/1.
  • L-amino acid producing strains By optimizing the main biosynthetic pathway of a desired compound, further improvement of L-amino acid producing strains can be accomplished via supplementation of the bacterium with increasing amounts of sugars as a carbon source, for example, glucose or arabinose. Despite the efficiency of glucose transport by PTS, access to the carbon source in a highly productive strain still may be insufficient.
  • ABC transport systems for import or export of nutrients and other substances across the cell membrane are widely diverse in nature.
  • a periplasmic component is the primary determinant of specificity of the transport complex as a whole (Chaudhuri BN, Ko J, Park C, Jones TA, Mowbray SL.;J MoI Biol. 1999 Mar 12;286(5):1519- 31).
  • Periplasmic binding protein-dependent transport systems are composed of a periplasmic substrate-binding protein, a set of two (sometimes one) very hydrophobic integral membrane proteins, and one (sometimes two) hydrophilic peripheral membrane protein that binds and hydrolyzes ATP. These systems are members of the superfamily of ABC transporters (Saurin W, Dassa E.; Protein Sci. 1994 Feb;3(2):325-44).
  • the AIsABC transporter belongs to the ATP-binding Cassette (ABC) Superfamily. It is responsible for the uptake of D-allose, an all-cis hexose that can be used by E. coli as the sole carbon source.
  • AIsB is a periplasmic protein that binds to D-allose with a K d of 0.33 ⁇ M, as determined by fluorescence spectroscopy. Based on sequence similarity, AIsA is the ATP-binding component, and AIsC is the membrane component of the ABC transporter. The AIsABC system also transports ribose at low affinity. Complementation analysis of ah mutants showed that the cloned ah genes could restore growth on ribose minimal media.
  • AIsR is a negative regulator of alsABC, and transcription o ⁇ alsR is regulated by allose (Kim C, Song S, Park C; J Bacteriol. 1997 Dec;179(24):7631-7).
  • the operon responsible for D-allose metabolism was localized at 92.8 min of the E. coli linkage map. It consists of six genes, ahRBACEK, which are inducible by D-allose and are under the control of the repressor gene alsR. This operon is also subject to catabolite repression. Three genes, alsB, als ⁇ , and ahC, appear to be necessary for transport of D-allose.
  • D-Allose-binding protein, encoded by alsB gene is a 32,779 Mr periplasmic protein that has an affinity for D-allose, with a K d of 0.33 ⁇ M.
  • the allose transport system includes an ATP- binding component (AIsA) and a transmembrane protein (AIsC).
  • AIsA is a 56,614 Mr protein that is encoded by the alsA gene which is downstream of alsB gene.
  • a 34,185 Mr protein is encoded by the ahC gene which is downstream of ah A gene.
  • AIsE a putative D-allulose-6- phosphate 3-epimerase
  • AIsK a putative D-allose kinase
  • the D-allose transporter is partially responsible for the low-affinity transport of D-ribose (Kim C, Song S, Park C; J Bacteriol. 1997 Dec;179(24):7631-7).
  • Objects of the present invention include enhancing the productivity of L-amino acid- producing strains and providing a method for producing L-amino acids using these strains.
  • the above objects were achieved by finding that enhancing the expression of the alsABC operon encoding the D-allose transporter can increase production of L-amino acids, such as L-threonine, L-lysine, L-leucine, L-histidine, L-cysteine, L-phenylalanine, L-arginine, L-tryptophan, L-glutamic acid, L-valine, L-proline and L-isoleucine, by fermentation using glucose as a carbon source.
  • the insufficient access to the carbon source was simulated by deleting the PTS transport system (ptsHI-crr) in the L-amino acid producing strain.
  • (C) a protein comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof; wherein said variants have the activity of the high-affinity D-allose transporter when said variants are combined together.
  • operon comprises: (A) a DNA comprising the nucleotide sequence of nucleotides 1 to 936 in SEQ ID NO: 1, or a DNA which is able to hybridize under stringent conditions to a sequence complementary to said sequence, or a probe prepared from said sequence;
  • thrB gene which codes for homoserine kinase
  • 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
  • L-amino acid is selected from the group consisting of L-lysine, L-cystein, L- leucine, L-histidine, L-phenylalanine, L-arginine, L-tryptophan, L-proline, and L-glutamic acid.
  • It is a further object of the present invention to provide a method for producing an L- amino acid comprising cultivating the bacterium described above in a culture medium which contains glucose, and isolating the L-amino acid from the culture medium.
  • L-amino acid is selected from the group consisting of L-threonine, L-lysine, L- cystein, L-leucine, L-histidine, L-phenylalanine, L-arginine, L-tryptophan, L-proline, and L- glutamic acid.
  • Figure 1 shows the relative positions of primers Pl and P2 on plasmid pMWl 18-attL- Cm-attR.
  • Figure 2 shows construction of a chromosomal DNA fragment which includes the inactivated ptsHI-crr operon.
  • Figure 3 shows substitution of the native promoter region of the alsABC operon in E. coli with the hybrid P L- tac promoter.
  • Figure 4 shows the influence of ⁇ . ⁇ alsABC on growth of the PTS " strain.
  • MG1655 means E. coli strain MGl 655;
  • MG ⁇ pts means E. coli strain MG1655 ⁇ ptsHI- crr;
  • MG ⁇ pts-P-alsABC means E. coli strain MGl 655 ⁇ ptsHI-crr V ⁇ alsABC.
  • Figure 5 shows the alignment of the primary sequences of the AIsB from Bacillus subtilis (BACSU, SEQ ID NO: 15), Bacillus cereus (BACCl, SEQ ID NO: 16), Yersinia pestis (YERPE, SEQ ID NO: 17), Yersinia pseudotuberculosis (YERPS, SEQ ID NO: 18), Escherichia coli (ECOLI, SEQ ED NO: 2), Symbiobacterium thermophilum (SYMTH, SEQ ID NO: 19).
  • the alignment was done by using the PIR Multiple Alignment program (http://pir.georgetown.edu).
  • the identical amino acids are marked by asterisk (*), similar amino acids are marked by colon (:).
  • Figure 6 (A) and (B) show the alignment of the primary sequences of the AIsA from Yersinia pestis (YERPE, SEQ ID NO: 20), Yersinia pseudotuberculosis (YERPS, SEQ ID NO: 21), Escherichia coli (ECOLI, SEQ ID NO: 4), Symbiobacterium thermophilum (SYMTH, SEQ ID NO: 22), Bacillus cereus (BACCl, SEQ ID NO: 23), Bacillus subtilis (BACSU, SEQ ID NO: 24).
  • the alignment was done by using the PIR Multiple Alignment program (http://pir.georgetown.edu).
  • the identical amino acids are marked by asterisk (*), similar amino acids are marked by colon (:).
  • Figure 7 shows the alignment of the primary sequences of the AIs C from Bacillus subtilis (BACSU, SEQ ID NO: 25), Bacillus cereus (BACCl, SEQ ID NO: 26), Yersinia pseudotuberculosis (YERPS, SEQ ID NO: 27), Yersinia pestis (YERPE, SEQ ID NO: 28), Escherichia coli (ECOLI, SEQ ID NO: 6), Symbiobacterium thermophilum (SYMTH, SEQ ID NO: 29).
  • the alignment was done by using the PIR Multiple Alignment program (http://pir.georgetown.edu).
  • the identical amino acids are marked by asterisk (*), similar amino acids are marked by colon (:).
  • L-amino acid-producing bacterium means a bacterium which has an ability to cause accumulation of an L-amino acid in a medium when the bacterium is cultured in the medium.
  • the L-amino acid-producing ability may be imparted or enhanced by breeding.
  • the phrase "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.
  • L-amino acids 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.
  • L-threonine L-lysine, L-cystein, L-leucine, L-histidine, L-phenylalanine, L-arginine, L-tryptophan, L-proline and L-glutamic acid 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, 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 into the genus Pantoea according to the classification known to a person skilled in the art of microbiology.
  • Some species of Enterobacter agglomerates 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)).
  • the bacterium of the present invention encompasses a strain of the Enter obacteriaceae family which has an ability to produce an L-amino acid and has been modified to enhance the expression of the alsABC operon.
  • the bacterium of the present invention encompasses a strain of the Enterobacteriaceae family which has an ability to produce an L- amino acid and has been transformed with a DNA fragment encoding the alsABC operon so that components of the D-allose transporter encoded by the DNA fragment are expressed.
  • activity of high-affinity D-allose transporter means an activity of transporting sugars, such as D-allose and glucose, into the cell.
  • the activity of the high- affinity D-allose transporter can be detected and measured by using membrane vesicles as described by Daruwalla et al (Biochem J., 200(3), 611-27 (1981)) or by complementation of high-affinity allose transport in an alsABC knockout strain (Horazdovsky, B. F. and Hogg, R.W., J. Bacteriol; 171(6):3053-9 (1989)).
  • the phrase "enhance the expression of the operon” means that the expression of the operon is increased compared to that of a non-modified strain, for example, a wild-type strain. Examples of such modifications include increasing the copy number of the operon(s) per cell, increasing the expression level of the operon(s), and so forth.
  • the quantity of the copy number of the operon is measured, for example, by Southern blotting using a probe based on the operon sequence, fluorescence in situ hybridization (FISH), and the like.
  • FISH fluorescence in situ hybridization
  • the level of operon expression can be measured by various known methods including Northern blotting, quantitative RT-PCR, and the like.
  • wild-type strains that can act as a control include, for example, Escherichia coli K- 12 or Pantoea ananatis FERM BP-6614 (WO2004099426, AU2004236516A1).
  • Pantoea ananatis FERM BP-6614 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on February 19, 1998 and received an accession number of FERM P- 16644.
  • the AIsABC transporter is encoded by the operon alsRBACEK.
  • alsB, alsA and alsC are necessary for allose metabolism.
  • alsABC operon means an operon which includes at least these three genes in the following order:
  • the alsB gene (synonyms - ECK4081 , B4088 , yjcX) encodes the D-allose transporter periplasmic subunit (synonyms - B4088 , YjcX).
  • the alsB gene (nucleotides complementary to nucleotides 4,309,130 to 4,310,065 in the sequence of GenBank accession NC_000913, gi: 49175990 ) is located between the rpiR and alsA genes on the chromosome of E. coli K- 12.
  • the alsA gene (synonyms - ECK4080 , b4087 , yjcW) encodes the fused D-allose transporter subunits of ABC superfamily: ATP-binding components (synonyms - B4087, YjcW).
  • the alsA gene (nucleotides complementary to nucleotides 4,307,471 to 4,309,003 in the sequence of GenBank accession NC_NC_000913.2 , gi: 16131913) is located between the alsB and alsC genes on the chromosome of E. coli K-12.
  • the alsC gene (synonyms - yjcV, ECK4079, b4086, JW4047) encodes the D-allose transporter subunit (synonym - B4086, YjcV).
  • the alsC gene (nucleotides complementary to nucleotides 4,306,512 to 4,307,492 in the sequence of GenBank accession NC_000913.2, gi: 49175990) is located between the alsA and alsE genes on the chromosome of E. coli K-12.
  • alsABC operons from the following microorganisms have also been elucidated : Shigella dysenteriae serotype 1, Shigella sonney, Erwinia carotovora subsp.
  • alsB, alsA, and alsC genes from Escherichia coli are represented by SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively.
  • the amino acid sequences encoded by the alsB, alsA, and alsC genes are presented by SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively.
  • glucokinase Upon being transported into the cell, glucose is phosphorylated by glucokinase, which is encoded by the glk gene. So, it is also desirable to modify the bacterium to have enhanced activity of gluco kinase.
  • the glk gene which encodes glucokinase of Escherichia coli has been elucidated (nucleotide numbers 2506481 to 2507446 in the sequence of GenBank accession NC_000913.1, gi:16127994). The glk gene is located between the b2387 and the b2389 ORFs on the chromosome of E. coli K-12.
  • xylose isomerase encoded by the xylA gene also efficiently catalyzes the conversion of D-glucose to D-fructose (Wovcha, M.G. et al, Appl Environ Microbiol. 45(4): 1402-4 (1983)). So, it is also desirable to modify the bacterium to have an enhanced activity of xylose isomerase.
  • the xylA gene which encodes xylose isomerase of Escherichia coli has been elucidated (nucleotide numbers 3728788 to 3727466 in the sequence of GenBank accession NC_000913.2, gi: 49175990). ThexylA gene is located between the xylB and xylF genes on the chromosome of E. coli K-12.
  • the alsABC, glk, and xylA genes can be obtained by PCR (polymerase chain reaction; refer to White, TJ. et al., Trends Genet., 5, 185 (1989)) utilizing primers prepared based on the known nucleotide sequences of the genes.
  • Genes coding for D-allose transporter from other microorganisms can be obtained in a similar manner.
  • the alsABC operon derived from Escherichia coli is exemplified by a DNA which encodes the following proteins:
  • (C) a protein comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof.
  • variant protein means a protein which has changes in the sequence, whether they are deletions, insertions, additions, or substitutions of amino acids, but still maintains the desired activity at a useful level, for example, useful for the enhanced production of an L-amino acid.
  • the number of changes in the variant protein depends on the position in the three dimensional structure of the protein or the type of amino acid .
  • the number of changes may be 1 to 30, preferably 1 to 15, and more preferably 1 to 5 for the proteins shown as SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO: 6. These changes in the variants can occur in regions of the protein which are not critical for the function of the protein.
  • the protein variants may have a homology of not less than 70 %, preferably not less than 80 %, and more preferably not less than 90 %, and most preferably not less than 95 % with respect to the entire amino acid sequences shown in any of SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 6 as long as the activity of D-allose transporter is maintained when combined with the corresponding components of the high-affinity D-allose transporter.
  • the components of the high-affinity D-allose transporter may be combined as follows: a variant of the protein shown in SEQ ID NO: 2 is combined with the proteins having the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 6, a variant of protein shown in SEQ ID NO: 4 is combined with the proteins having the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 6, and a variant of the protein shown in SEQ ID NO: 6 is combined with proteins having the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4 .
  • 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.
  • substitution, deletion, insertion, or addition of one or several amino acid residues should 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 GIu, GIn, Lys, His or Asp for Asn, substitution of Asn, GIu or GIn for Asp, substitution of Ser or Ala for Cys, substitution of Asn, GIu, Lys, His, Asp or Arg for GIn, substitution of Asn, GIn, Lys or Asp for GIu, substitution of Pro for GIy, substitution of Asn, Lys, GIn, Arg or Tyr for His, substitution of Leu, Met, VaI or Phe for He, substitution of He, Met, VaI or Phe for Leu, substitution of Asn, GIu, GIn, His or Arg for Lys, substitution of He, Leu, VaI or Phe for Met, substitution of Trp,
  • the DNAs which encode substantially the same proteins as components of D-allose transporter may be obtained, for example, by modifying the nucleotide sequences of DNAs encoding components of D-allose transporter (SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 respectively), for example, by means of the site-directed mutagenesis method so that one or more amino acid residues at a specified site are deleted, substituted, inserted, or added. DNAs modified as described above may be obtained by conventionally known mutation treatments.
  • Such treatments include hydroxylamine treatment of the DNA encoding proteins of present invention, or treatment of the bacterium containing the DNA with UV irradiation or a reagent such as N-methyl-N'-nitro-N-nitrosoguanidine or nitrous acid.
  • DNAs encoding substantially the same proteins as components of D-allose transporter can be obtained by expressing DNAs having a mutation as described above in an appropriate cell, and investigating the activity of the expressed product.
  • DNAs encoding substantially the same protein as components of D-allose transporter can also be obtained by isolating DNAs that are hybridizable with probes having nucleotide sequences which contain, for example, the nucleotide sequences shown in any of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 under stringent conditions, and encode proteins having the activities of components of D- allose transporter.
  • the "stringent conditions” referred to herein are conditions under which so- called specific hybrids are formed, and non-specific hybrids are not formed.
  • stringent conditions can be exemplified by conditions under which DNAs having high homology, for example, DNAs having homology of not less than 50%, preferably not less than 60%, more preferably not less than 70%, further preferably not less than 80%, and still more preferably not less than 90%, and most preferably not less than 95% are able to hybridize with each other, but DNAs having homology lower than the above are not able to hybridize with each other.
  • stringent conditions may be exemplified by conditions under which DNA is able to hybridize at a salt concentration equivalent to ordinary washing conditions in Southern hybridization, i.e., 1 x SSC, 0.1% SDS, preferably 0.1 x SSC, 0.1% SDS, at 60°C.
  • Duration of washing depends on the type of membrane used for blotting and, as a rule, what is recommended by the manufacturer. For example, recommended duration of washing, for example, for the HybondTM N+ nylon membrane (Amersham), under stringent conditions is approximately 15 minutes. Preferably, washing is performed 2 to 3 times.
  • Probes may be prepared by PCR using primers based on the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and DNA fragments containing the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 as templates.
  • the hybridization conditions for washing include, for example, 50 0 C, 2 x SSC and 0.1% SDS.
  • substitution, deletion, insertion, or addition of nucleotides as described above also may include a mutation which naturally occurs (mutant or variant), for example, due to variety in the species or genus of bacterium, which contains the components of the D-allose transporter.
  • Transformation of a bacterium with DNA encoding a protein means introduction of the DNA into a bacterium, for example, by conventional methods. Transformation of this DNA will result in an increase in expression of the gene encoding the protein of the present invention, and will enhance the activity of the protein in the bacterial cell. Methods of transformation include any known methods that have hitherto been reported. For example, a method of treating recipient cells with calcium chloride so as to increase permeability of the cells to DNA has been reported for Escherichia coli K- 12 (Mandel, M. and Higa, A., J. MoI. Biol, 53, 159 (1970)) and may be used.
  • Methods of enhancing gene expression include increasing the gene copy number. Introducing a gene into a vector that is able to function in a bacterium of the Enter obacteriaceae family increases the copy number of the gene.
  • low copy vectors are used. Examples of low-copy vectors include but are not limited to pSClOl, pMWl 18, pMWl 19, and the like.
  • the term "low copy vector" typically indicates vectors which have up to 5 copies per cell.
  • Increasing the copy number of the alsABC operon can also be achieved by introducing multiple copies of the alsABC operon into the chromosomal DNA of the bacterium.
  • homologous recombination is carried out using a sequence whose multiple copies exist as targets in the chromosomal DNA.
  • Sequences having multiple copies in the chromosomal DNA include, but are not limited to repetitive DNA, or inverted repeats existing at the end of a transposable element.
  • Introduction of multiple copies of the gene into a bacterial chromosome can be also achieved by Mu integration, or the like. For example, one act of Mu integration allows introduction of up to 3 copies of the gene into a bacterial chromosome.
  • Enhancing gene expression may also be achieved by placing the DNA of the present invention under the control of a potent promoter.
  • a potent promoter for example, the P tac promoter, the lac promoter, the trp promoter, the trc promoter, the P R , or the P L promoters of lambda phage are all known to be potent promoters.
  • the use of a potent promoter can be combined with increasing the gene copy number.
  • the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter to increase the transcription level of a gene located downstream of the promoter.
  • substitution of several nucleotides in the spacer between ribosome binding site (RBS) and the start codon, especially the sequences immediately upstream of the start codon profoundly affect the mRNA translatability. For example, a 20-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon (Gold et al, Annu. Rev. Microbiol., 35, 365-403, 1981; Hui et al, EMBO J., 3, 623-629, 1984).
  • the rhtA23 mutation is an A-for-G substitution at the - 1 position relative to the ATG start codon (ABSTRACTS of 17 th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457). Therefore, it may be suggested that the rhtA23 mutation enhances the rhtA gene expression and, as a consequence, increases the resistance to threonine, homoserine and some other substances transported out of cells.
  • Methods for preparation of plasmid DNA include, but are not limited to digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer and the like, or other 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 can be obtained by the introduction of the aforementioned DNAs into a bacterium which inherently has the ability to produce L-amino acids.
  • the bacterium of the present invention can be obtained by imparting an ability to produce L-amino acids to a bacterium which already contains the DNAs.
  • bacteria which are able to produce L-amino acids may be used.
  • the bacterium of the present invention can be obtained by enhancing expression of the alsABC genes 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 enhanced expression of the alsABC genes.
  • 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)
  • 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 1149911 A) 5 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 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 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 (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 (EP0593792B) 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 th
  • 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 tt&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, WO2003/097839).
  • a mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine, as well as, the thrB and thrC genes can be obtained as one operon from the 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 ORFl (ybiF gene, nucleotide positions 764 to 1651, GenBank accession number AAA218541, gi:440181) and is located between thepexB and ompX genes.
  • the unit expressing a protein encoded by the ORPl 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 NC_0O0913.1, 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 W3110, which was derived from Escherichia coli K- 12.
  • the resulting strain was designated Escherichia coli AJ13069 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. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on September 29, 1995, and received an accession number of FERM BP-5252 (U.S. Patent No. 5,827,698).
  • 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 QysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (U.S. Patent No.
  • 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 (pntAE) (U.S. Patent No. 5,830,716), the ybjE gene (WO2005/073390), or combinations thereof.
  • cyo energy efficiency
  • pntAE 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).
  • L-cysteine-producing bacteria examples 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 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 (JPl 1155571 A2); E. coli W3110 with increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene (WOO 127307Al), and the like.
  • E. coli JM 15 which is transformed with different cysE alleles coding for feedback-resistant serine acetyltransferases
  • 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.
  • 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-hydroxyleucine, 4-aza
  • 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, RU2119536); E. coli NRRL B-12116 - B 12121 (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.
  • genes include genes encoding ATP phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase (MsI), phosphoribosyl-ATP pyrophosphohydrolase (MsIE), phosphoribosylformimino-5- aminoimidazole carboxamide ribotide isomerase (MsA), amidotransferase (MsH), histidinol phosphate aminotransferase (MsQ, histidinol phosphatase (MsB), histidinol dehydrogenase (MsD), and so forth.
  • hisG phosphoribosyltransferase
  • MsI phosphoribosyl AMP cyclo
  • 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 (EP 1016710A), E. coli 80 strain imparted with sulfaguanidine, DL-l,2,4-triazole-3-alanine, and streptomycin-resistance (VKPM B-7270, Russian Patent No. 2119536), and so forth.
  • JP 56-005099 A E. coli strains introduced with rht, a gene for an amino acid-export
  • EP 1016710A 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 EschericMa, 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 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 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 (ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase ⁇ aceEF, ipdA), pyruvate kinase (pykA,pykF), phosphoenolpyruvate synthase (ppsA), eno
  • 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.
  • Such enzymes include isocitrate lyase (aceA), ⁇ -ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pt ⁇ ), acetate kinase (ack), acetohydroxy acid synthase (UvG), acetolactate synthase (HvI), formate acetyltransferase (pfl), lactate dehydrogenase (Idh), and glutamate decarboxylase (gadAB).
  • aceA isocitrate lyase
  • sucA ⁇ -ketoglutarate dehydrogenase
  • pt ⁇ phosphotransacetylase
  • ack acetate kinase
  • UvG acetohydroxy acid synthase
  • HvI acetolactate synthase
  • pfl lactate dehydrogenase
  • Idh lactate dehydrogenase
  • glutamate decarboxylase
  • E. coli AJ12949 (FERM BP-4881)
  • 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 11, 1999 and received an accession number of FERM BP-6615.
  • Pantoea ananatis AJl 3356 is deficient in ⁇ -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 AJl 3356.
  • 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.
  • L-phenylalanine-producing bacteria examples 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. co/JNRRL B-12141, NRRL B- 12145, NRRL B-12146 and NRRL B-12147 (U.S. Patent No.
  • E. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used (EP 488424 Bl).
  • L-phenylalanine producing bacteria belonging to the genus Escherichia with an enhanced activity of the protein encoded by theyedA 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) are 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 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 theyedA gene or the yddG gene may also be used (U.S. patent applications 2003/0148473 Al and 2003/0157667 Al).
  • Examples of 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 SVl 64 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 UvA 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).
  • Examples of bacteria belonging to the genus Escherichia, which have an activity to produce L-proline include the following E. coli strains: NRRL B- 12403 and NRRL B- 12404 (GB Patent 2075056), VKPM B-8012 (Russian patent application 2000124295), plasmid mutants described in DE Patent 3127361, plasmid mutants described by Bloom F.R. et al (The 15 th Miami winter symposium, 1983, p. 34), and the like.
  • 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) (EPl 170358A1), an arginine-producing strain into which argA gene encoding N-acetylglutamate synthetase is introduced therein (EPl 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 ⁇ car AB).
  • 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 the L-valine that is produced. Furthermore, the HvA 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.
  • E. coli VL1970 which has a mutation in the UeS gene encoding isoleucine tRNA synthetase, can be used.
  • E. coli VLl 970 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) ( Russian, 117545 Moscow, 1 Dorozhny Proezd, 1) on June 24, 1988 under accession number VKPM B-4411.
  • 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).
  • Oxaloacetate serves as a substrate for the reaction which results in synthesis of Thr and Lys.
  • OAA results from a reaction of PEP with phosphoenol pyrvate carboxlase (PEPC) functioning as a catalyst. Therefore, elevation of the PEPC concentration in a cell can be very important for fermentative production of these amino acids.
  • glucose is internalized by the glucose-phosphontransferase (GIc-PTS) system. This system consumes PEP, and proteins in the PTS are encoded by ptsG and ptsHIcrr.
  • GIc-PTS glucose-phosphontransferase
  • proteins in the PTS are encoded by ptsG and ptsHIcrr.
  • one molecule of PEP and one molecule of pyruvate (Pyr) are generated from one molecule of glucose.
  • Increasing the ratio of PEP/Pyr even more by increasing expression of the alsABC operon in a threonine-producing strain, a lysine-producing strain, a histidine-producing strain, a phenylalanine-producing strain, an arginine-producing strain, a tryptophan-producing strain and/or a glutamic acid-producing strain should further increase the corresponding amino acid production. Because four molecules of PEP are generated from two molecules of glucose, the ratio of PEP/Pyr is expected to be greatly improved. Due to the increased expression of the alsABC operon, removal of the expression control glc-PTS is expected.
  • 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 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.
  • 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 the E. coli strain having a disrupted PTS transport system.
  • the ptsHI-crr operon was deleted in the chosen strain 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 Cm R marker encoded by the cat gene was obtained by PCR, using primers Pl (SEQ ID NO: 7) and P2(SEQ ID NO: 8) 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 5' end of the ptsHI-crr operon and a region complementary to the 24-nt attL region.
  • Primer P2 contains both a region complementary to the 36-nt region located at the 3' end of the ptsHI-crr operon and a region complementary to the 24-nt attR region.
  • Conditions for PCR were as follows: denaturation for 3 min at 95°C; profile for two first cycles: 1 min at 95°C, 30 sec at 50 0 C, 40 sec at 72°C; profile for the last 25 cycles: 30 sec at 95 0 C, 30 sec at 54°C, 40 sec at 72°C; final step: 5 min at 72°C.
  • a 1699-bp PCR product (Fig. 2) was obtained and purified in agarose gel and was used for electroporation of the E. coli strain MG1655 (ATCC 700926), which contains the plasmid pKD46, the replication of which is temperature-sensitive.
  • 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.
  • J02459 contains genes of the ⁇ Red homologous recombination system ( ⁇ , ⁇ , exo genes) under the control of the arabinose-inducible P araB promoter.
  • the plasmid pKD46 is necessary for integration of the PCR product into the chromosome of strain MG1655.
  • MG1655 can be obtained from American Type Culture Collection. (P.O. Box 1549 Manassas, VA 20108, U.S.A.).
  • Electrocompetent cells were prepared as follows: E. coli MG1655/pKD46 was grown overnight at 30 °C in LB medium containing ampicillin (100 mg/1), and the culture was diluted 100 times with 5 ml of SOB medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) containing ampicillin and D-allose (1 mM). The cells were grown with aeration at 30°C to an OD 6 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 without the ptsHI-crr operon and having the Cm resistance gene were verified by PCR.
  • Locus-specific primers P3 (SEQ ID NO: 9) and P4 (SEQ ID NO: 10) 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 0 C, 1 min at 72°C; final step: 7 min at 72°C.
  • the PCR product obtained in the reaction using the parental ptsHI-crr + strain MG 1655 as a template was ⁇ 3.0 kbp in length.
  • the PCR product obtained in the reaction using the cells of the mutant strain as a template was ⁇ 2.0 kbp in length (Fig.2).
  • the mutant strain was named MG 1655 ⁇ ptsHI-cr ⁇ xat.
  • the Cm resistance gene ⁇ cat gene was deleted from the chromosome of the E. coli MG 1655 ⁇ ptsHI-crr::cat strain using the int-xis system.
  • E. coli strain MG 1655 ⁇ ptsHI-cr ⁇ xat was transformed with plasmid pMWts-Int/Xis (WO 05/010175). Transformant clones were selected on LB-medium containing 100 ⁇ g/ml of ampicillin. Plates were incubated overnight at 30°C.
  • Transformant clones were cured from the cat gene by spreading the separate colonies at 37°C (at this temperature repressor Cits is partially inactivated and transcription of the int/xis genes is derepressed) followed by selection of Cm Ap variants. Elimination of the cat gene from the chromosome of the strain was verified by PCR. Locus-specific primers P3 (SEQ ID NO: 9) and P4 (SEQ ID NO: 10) were used in PCR for the verification. Conditions for PCR verification were as described above. The PCR product obtained in reaction using cells without cat gene as a template was ⁇ 0.3 kbp in length. Thus, the strain with the inactivated ptsHI-crr operon and missing the cat gene was obtained. This strain was named MG 1655 ⁇ ptsHI-crr.
  • Example 2 Replacement of the native promoter region of the alsABC operon in E. coli with hybrid Pr-tan promoter.
  • the hybrid P L- ta c promoter was obtained by PCR using the chromosomal DNA of E. coli strain B-3996P L-tac xylE (PCT application WO2006043730) as the template, and primers P5 (SEQ ID NO: 11 and P6(SEQ ID NO: 12). PCR was conducted as described in Example 1.
  • 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 E. coli MG 1655 ⁇ ptsHI-crr /pKD46 as described in Example 1.
  • Colonies which grew within 24 h were tested for the presence of a Cm R marker instead of the alsABC operon native promoter region by PCR using primers P7 (SEQ ID NO: 13) and P8 (SEQ ID NO: 14). For this purpose, a freshly isolated colony was suspended in 20 ⁇ l water and then l ⁇ l of this suspension was used for PCR. PCR conditions were as described in Example 1. A few tested Cm R colonies contained the desired ⁇ 2.1 kb DNA fragment, confirming the presence of the hybrid P L - tac promoter and Cm R marker DNA instead of -0.3 kb alsABC operon native promoter region (see Figure 3). One of the obtained strains was cured from the thermosensitive plasmid pKD46 by culturing at 37 0 C and named E. coli MG1655 ⁇ ptsHI-crr ? L . tac alsABC.
  • Example 3 Effect of enhancing the alsABC operon expression in the strain having a disrupted PTS transport system on L-threonine production.
  • Locus-specific primers P3 (SEQ ID NO: 9) and P4 (SEQ ID NO: 10) were used in PCR for the verification. Conditions for PCR verification were as described above.
  • the PCR product obtained in the reaction using the parental ptsHI-crr + B-3996 strain as the template was ⁇ 3.0 kbp in length.
  • the PCR product obtained in the reaction using the mutant strain B-3996 ⁇ ptsHI-crr::cat as the template was ⁇ 2.0 kbp in length (Fig.2).
  • the Cm resistance gene ⁇ cat gene was deleted from the chromosome of the E. coli B- 3996 ⁇ ptsHI-cr ⁇ xat strain using the int-xis system.
  • E. coli strain B-3996 ⁇ ptsHI-cr ⁇ xat was transformed with plasmid pMWts-Int/Xis (WO 2005 010175).
  • Transformant clones were selected on the LB-medium containing 100 ⁇ g/ml of ampicillin. Plates were incubated overnight at 30°C.
  • Transformant clones were cured from the cat gene by spreading the separate colonies at 37°C (at this temperature repressor Cits is partially inactivated and transcription of the int/xis genes is derepressed) followed by selection of Cm s Ap R variants. Elimination of the cat gene from the chromosome of the strain was verified by PCR. Locus-specific primers P3 (SEQ ID NO: 9) and P4 (SEQ ID NO: 10) were used in PCR for the verification. Conditions for PCR verification were as described above. The PCR product obtained in reaction using cells without the cat gene as a template was ⁇ 0.3 kbp in length. Thus, the threonine-producing strain with the inactivated ptsHI-crr operon and missing the cat gene was obtained. This strain was named B-3996 ⁇ ptsHI-crr.
  • the native promoter of the alsABC operon was replaced with a hybrid P L-tac promoter.
  • DNA fragments from the chromosome of the above-described E. coli MG1655 ⁇ ptsHI-crr V ⁇ alsABC were transferred to the E. coli strain B-3996 ⁇ ptsHI-crr by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the E. coli strain B-3996- ⁇ ptsHI-crr ? L . tSLC alsABC.
  • coli strain B-3996- ⁇ ptsHI-crr V ⁇ - ⁇ alsABC were verified by PCR.
  • Locus-specific primers P7 (SEQ ID NO: 13) and P8 (SEQ ID NO: 14) were used in PCR for the verification.
  • Conditions for PCR verification were as described above.
  • the PCR product obtained in the reaction with the strain B-3996- ⁇ ptsHI-crr V L - X&C CIISABC as the template was ⁇ 2.1 kbp in length.
  • E. coli strains B-3996, B-3996- ⁇ ptsHI-crr and B-3996- ⁇ ptsHI-crr V L . ⁇ c alsABC were each cultivated at 37 0 C for 18 hours in a nutrient broth, and 0.3 ml of each of the obtained cultures was inoculated into 3 ml of fermentation medium having the following composition in a 20x200 mm test tube and cultivated at 32 0 C for 72 hours with a rotary shaker.
  • composition of the fermentation medium (g/1) was as follows:
  • strain AJ14442 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.
  • Both E. coli strains, AJl 1442 and AJl can each be cultured in L- medium at 37°C, and 0.3 ml of each of the obtained cultures 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 the consumed glucose for each of the strains.
  • the 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 coding for the allose transporter from the chromosome of the above-described E. coli MG1655- ⁇ ptsHI-crr P h - tac CilsABC strain 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 JM 15(ydeD)-P L-tac ⁇ / ⁇ 5C.
  • E. coli strain JM15(ydeD) is a derivative of E. coli strain JM15 (US Patent No. 6,218,168) which can be transformed with DNA having the ydeD gene, which codes for a membrane protein, and is not involved in a biosynthetic pathway of any L-amino acid (U.S. Patent No. 5,972,663).
  • the strain JM15 (CGSC# 5042) can be obtained from The Coli Genetic Stock Collection at the E. coli Genetic Resource Center, MCD Biology Department, Yale University (http://cgsc.biology.yale.edu/).
  • Example 6 Production of L-leucine by E. coli 57- Pi ⁇ lsABC
  • DNA fragments coding for the allose transporter from the chromosome of the above-described E. coli MG1655- ⁇ ptsHI-crr Y ⁇ alsABC strain 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 51-? ⁇ c alsABC.
  • the strain 57 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 1 17545 Moscow, 1 Dorozhny proezd, 1) on May 19, 1997 under accession number VKPM B-7386.
  • Both E. coli strains, 57 and 51-V ⁇ . ⁇ alsABC can each be 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) is as follows:
  • Glucose and CaCO 3 are sterilized separately.
  • DNA fragments coding for the allose transporter from the chromosome of the above-described E. coli MG1655- ⁇ ptsHI-crr strain 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.
  • strain 80 is described in Russian patent 2119536, and was deposited in the Russian National Collection of Industrial Microorganisms (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on October 15, 1999 under accession number VKPM B-7270 and then converted to a deposit under the Budapest Treaty on July 12, 2004.
  • composition of the fermentation medium (g/1) is as follows (pH 6.0):
  • Glucose, proline, betaine and CaCO 3 are sterilized separately.
  • the pH is adjusted to 6.0 before sterilization.
  • Example 8 Production of L-glutamate by E. coli strain VL334thrC + -Pr-t 3£ ⁇ /,s'./4i?C
  • DNA fragments coding for the allose transporter from the chromosome of the above-described E. coli MG1655- ⁇ ptsHI-crr P L . tac alsABC strain can be transferred to the E. coli L-glutamate- producing strain VL334thrC + (EP 1172433) by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab.
  • Both strains, VL334thrC + and VL334thrC + - ? L . ⁇ c alsABC can each be 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 (I 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.
  • Example 9 Production of L-phenylalanine by E. coli strain AJl2139-P_] : t 3 c 1 aIsABC
  • DNA fragments coding for the allose transporter from the chromosome of the above-described E. coli MG1655- ⁇ ptsHI-crr ? L . t ⁇ c alsABC strain can be transferred to the 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 the strain AJ12739- ?L.
  • Both strains, AJ12739- V L - ⁇ aIsABC and AJ12739 can each be cultivated at 37°C for 18 hours in a nutrient broth, and 0.3 ml of each of the obtained cultures can each be inoculated into 3 ml of a fermentation medium in a 20x200-mm test tube and cultivated at 37°C for 48 hours with shaking on a rotary shaker. After cultivation, 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° for 2 hours. The pH is adjusted to 7.0.
  • Example 10 Production of L- tryptophan by E. coli strain SVl 64 ( ⁇ GH5)-PJ .. ⁇ aIsABC
  • DNA fragments coding for the allose transporter from the chromosome of the above-described E. coli MG1655- ⁇ ptsHI-crr P ⁇ .t ⁇ C alsABC strain 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)-P L- taC alsABC.
  • the strain SV 164 has the trpE allele encoding anthranilate synthase which is not subject to feedback inhibition by tryptophan.
  • the plasmid pGH5 harbors a mutant serA gene encoding phosphoglycerate dehydrogenase which is not subject to feedback inhibition by serine.
  • the strain SV164 (pGH5) is described in detail in US patent No. 6,180,373 and European patent 0662143.
  • Both strains, SV164(pGH5)-P L - taC a/ ⁇ 45C and SV164(pGH5) can each be cultivated with shaking at 32°C for 18 hours in 3 ml of nutrient broth supplemented with tetracycline (10 mg/1, marker of pGH5 plasmid).
  • the obtained cultures (0.3 ml each) can each be inoculated into 3 ml of a fermentation medium containing tetracycline (10 mg/1) in 20 x 200-mm test tubes, and cultivated at 32°C for 72 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 G), as shown, to avoid adverse interactions during sterilization.
  • Group A had pH of 7.1, adjusted by NH 4 OH. Each group was sterilized separately.
  • DNA fragments coding for the allose transporter from the chromosome of the above-described E. coli MG1655- ⁇ ptsHI-crr P]_. tac alsABC strain 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 -Pi- t ⁇ alsABC.
  • the strain 702ilvA has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (USD, 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
  • DNA fragments coding for the allose transporter from the chromosome of the above-described E. coli MG1655- ⁇ ptsHI-crr Pi ⁇ ⁇ alsABC strain can be transferred to the arginine-producing E. coli strain 382 by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain 382 -Y L - ⁇ aIsABC.
  • 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 strains, 382-P ⁇ lsABC and 382 can each be cultivated with shaking at 37 0 C for 18 hours in 3 ml of nutrient broth, and 0.3 ml of each 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.
  • 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/1) is as follows:
  • Glucose and magnesium sulfate are sterilized separately.
  • CaCO 3 is dry-heat sterilized at 180°C for 2 hours. The pH is adjusted to 7.0.

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Abstract

L'invention concerne un procédé de fabrication d'un acide L-amino, par exemple les L-thréonine, L-lysine, L-leucine, L-histidine, L-cystéine, L-phénylalanine, L-arginine, L-tryptophane, acide L-glutamique, L-valine, L-proline et L-isoleucine, par fermentation de glucose à l'aide d'une bactérie de la famille des entérobactériaceæ, la bactérie ayant été modifiée pour augmenter l'activité du transporteur d'alose d'affinité élevée codé par l'opéron alsABC.
PCT/JP2007/067782 2006-09-13 2007-09-06 PROCÉDÉ DE FABRICATION D'UN ACIDE L-AMINO À L'AIDE D'UNE BACTÉRIE DE LA FAMILLE DES ENTEROBACTERIACEAE AVEC UNE EXPRESSION AMÉLIORÉE DE L'OPÉRON alsABC WO2008032757A1 (fr)

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US7833761B2 (en) 2007-09-04 2010-11-16 Ajinomoto Co., Inc. Amino acid producing microorganism and a method for producing an amino acid
US8679798B2 (en) 2007-12-21 2014-03-25 Ajinomoto Co., Inc. Method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family
CN110734887A (zh) * 2018-07-18 2020-01-31 中国科学院微生物研究所 产n-乙酰谷氨酸的基因工程菌及其构建方法与应用
WO2020204179A1 (fr) * 2019-04-05 2020-10-08 Ajinomoto Co., Inc. Procédé de production d'acides l-aminés

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WO2006068273A1 (fr) * 2004-12-23 2006-06-29 Ajinomoto Co., Inc. Méthode de synthèse de l-acides aminés impliquant des bactéries de la famille enterobacteriaceae

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