HUE025593T2 - A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE ENTEROBACTERIACEAE FAMILY WITH ATTENUATED EXPRESSION OF THE sfmACDFH-fimZ CLUSTER OR THE fimZ GENE - Google Patents

Abstract

FIELD: chemistry. ^ SUBSTANCE: invention relates to biotechnology and represents a method for obtaining L-threonine using bacterium belonging to genus Escherichia, which is modified in such a way that gene selected from the group consisting of gene cluster sfmACDFH-fimZ and gene fimZ is inactivated. ^ EFFECT: obtaining L-threonine with a high degree of efficiency. ^ 4 cl, 5 dwg, 3 tbl, 24 ex

Description

Description

Technical Field [0001] The present invention relates to the microbiological industry, and specifically to a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family which has been modified to attenuate expression of the fimZ gene:, by inactivation of the gene.

Background Art [0002] Conventionally, 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.

[0003] Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganisms with recombinant DNA (see, for example, US patent No. 4,278,765). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes of the feedback inhibition by the resulting L-amino acid (see, for example, WO 95/16042 or US patent Nos. 4,346,170; 5,661,012 and 6,040,160).

[0004] Another way to enhance L-amino acid production yields is to attenuate expression of a gene or several genes encoding protein(s) involved in degradation of the target L-amino acid, involved in diverting the precursors of the target L-amino acid from the L-amino acid biosynthetic pathway, involved in the redistribution of carbon, nitrogen, and phosphate fluxes, and genes encoding toxins etc..

[0005] EP 0 796 912 describes a process for producing L-lysine comprising cultivating in a liquid medium a microorganism belonging to the genus Escherichia in which L-lysine decarboxylase activity is decreased or abolished.

[0006] US 2004/0229320 describes a process for producing an L-amino acid such as L-threonine comprising the step of cultivating a bacterium belonging to the genus Escherichia which has been modified to inactivate the mlc gene.

[0007] WO 03/054207 describes a fermentative process for preparing an L-amino acid using coryneform bacteria in which at least the gene coding for a small integral C4-dicarboxylate membrane transport protein and/or the gene coding for the 2-oxoglutarate/malate translocator are attenuated.

[0008] The sfmA gene encodes the SfmA protein, which is a putative fimbrial-like protein. The sfmC gene encodes the SfmC protein, which is a putative shaperone. The sfmD gene encodes the SfmD protein, which is a putative outer membrane protein. The sfmH gene encodes the SfmH protein, which is a putative protein involved fimbrial assembly. The sfmF gene encodes the SfmF protein, which is a putative fimbrial-like protein. The fimZ gene encodes the FimZ protein, which is a putative transcriptional regulator (http://ecocyc.org).

[0009] But currently, there have been no reports of attenuating expression of the sfmACDFH-fimZ cluster or the fimZ gene for the purpose of producing L-amino acids.

Summary of the Invention [0010] Objects of the present invention include enhancing the productivity of L-amino acid-producing strains and providing a method for producing an L-amino acid using these strains.

[0011] The above objects were achieved by finding that attenuating expression of the fimZ gene can enhance production of 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-pheny-lalanine, L-tyrosine, and L-tryptophan.

[0012] The present application describes a bacterium of the 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-histi-dine, 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.

[0013] It is an object of the present invention to provide a method using an L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified to attenuate expression of the fimZ gene by inactivation of the gene.

[0014] It is a further object of the present invention to provide a method using the bacterium as described above, wherein the bacterium belongs to the genus Escherichia.

[0015] It is a further object of the present invention to provide a method using the bacterium as described above, wherein the bacterium belongs to the genus Pantoea.

[0016] It is a further object of the present invention to provide a method using the bacterium as described above, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L- amino acid.

[0017] It is a further object of the present invention to provide a method using the bacterium as described above, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.

[0018] It is a further object of the present invention to provide a method using the bacterium as described above, wherein said 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.

[0019] It is a further object of the present invention to provide a method for producing an L-amino acid comprising: cultivating the bacterium as described above in a medium to produce and excrete said L-amino acid into the medium, and collecting said L-amino acid from the medium.

[0020] It is a further object of the present invention to provide the method as described above, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

[0021] It is a further object of the present invention to provide the method as described above, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.

[0022] It is a further object of the present invention to provide the method as described above, wherein said nonaromatic 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.

[0023] The present invention is described in detail below.

Detailed Description of the Preferred Embodiments 1. Bacterium for use in a method of the present invention [0024] The bacterium for use in a method of the present invention is an L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified to attenuate expression of the fimZ gene by inactivation.

[0025] In the present invention, "L-amino acid-producing bacterium" means a bacterium which has an ability to produce and excrete an L-amino acid into a medium, when the bacterium is cultured in the medium.

[0026] The term "L-amino acid-producing bacterium" as used herein also means a bacterium which is able to produce and cause accumulation of an L-amino acid in a culture medium in an amount larger than a wild-type or parental strain of the bacterium, for example, E. coli, such as E. coli K-12, and preferably means that the bacterium is able to cause accumulation in the medium of an amount not less than 0.5 g/L, more preferably not less than 1.0 g/L, of the target L-amino acid. The term "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.

[0027] The term "aromatic L-amino acid" includes L-phenylalanine, L-tyrosine, and L-tryptophan. The term "non-aro-matic L-amino acid" includes L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine. L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L-phenylalanine, L-tryptophan, L-proline, and L-arginine are particularly preferred.

[0028] . 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 (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) can be used. A bacterium belonging to the genus Escherichia or Pantoea is preferred.

[0029] The phrase "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).

[0030] 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.

[0031] The phrase "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)).

[0032] The phrase "bacterium has been modified to attenuate expression of the fimZ gene" by "inactivation of the gene" means that the modified gene encodes a completely non-functional protein. It is also possible that the modified DNA region is unable to naturally express the gene due to the deletion of a part of the gene or of the gene entirely, the shifting of the reading frame of the gene, the introduction of missense/nonsense mutation(s), or the modification of an adjacent region of the gene, including sequences controlling gene expression, such as promoters, enhancers, attenuators, ribosome-binding sites, etc.. The presence or absence of the fimZ gene in the chromosome of a bacterium can be detected by well-known methods, including PCR, Southern blotting, and the like. In addition, the the level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene using various known methods including Northern blotting, quantitative RT-PCR, and the like.

[0033] The amount of the proteins encoded by the fimZ genes can be measured by well-known methods, including SDS-PAGE followed by immunoblotting assay (Western blotting analysis) and the like.

[0034] The sfmACDFH-fimZ cluster includes the sfmACDFH operon and the fimZ gene.

[0035] The sfmACDFH operon includes genes in the following order.

[0036] The sfmA gene (synonym -b0530) encodes a putative fimbrial-like protein (synonyms-SfmA, B0530). The sfmA gene of £. coli (nucleotide positions 557,402 to557,977; GenBank accession no. NC_000913.2; gi:49175990) is located between the folD and sfmC genes on the chromosome of E. coli K-12. The nucleotide sequence of the sfmA gene and the amino acid sequence of SfmA encoded by the sfmA gene are shown in SEQ ID NO: 1 andSEQ ID NO: 2, respectively.

[0037] The sfmC gene (synonym - b0531) encodes a putative shaperon (synonyms-SfmC, B0531). The sfmC gene of E. coli (nucleotide positions 558,197 to 558,889; GenBank accession no. NC_000913.2; gi:49175990) is located between the sfmA and sfmD genes on the chromosome ofE. coli K-12. The nucleotide sequence of the sfmC gene and the amino acid sequence of SfmC encoded by the sfmC gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0038] The sfmD gene (synonym -b0532) encodes a putative outer membrane protein with export function (synonyms-Sfm D, B0532). The sfmD gene ofE. coli( nucleotide positions 558,920 to 561,523; GenBank accession no. NC_000913.2; gi:49175990) is located between the sfmC and sfmHgenes on the chromosome of E. coli K-12. The nucleotide sequence of the sfmD gene and the amino acid sequence of SfmD encoded by the sfmD gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively [0039] The sfmH gene (synonym - b0533) encodes a protein involved in fimbrial assembly (synonyms-SfmH, B0533). The sfmH gene of E. coli (nucleotides 561,565 to 562,542; GenBank accession no. NC_000913.2; gi:49175990) is located between the sfmD and sfmF genes on the chromosome ofE. coli K-12. The nucleotide sequence of the sfmH gene and the amino acid sequence of SfmH encoded by the sfmH gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0040] The sfmF gene (synonyms - b0534, ybcG) encodes a putative fimbrial-like protein (synonyms - SfmF, B0534, YbcG,). The sfmF gene ofE. coli (nucleotides 562,553 to 563,068; GenBank accession no. NC_000913.2; gi:49175990) is located between the sfmH and fimZ genes on the chromosome of £. coli K-12. The nucleotide sequence of the sfmF gene and the amino acid sequence of SfmF encoded by the sfmF gene are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0041] The fimZ gene (synonyms - b0535, ybcG) encodes a transcriptional regulator(synonyms - FimZ, B0535, YbcA). The fimZ gene of £. coli (nucleotides complementary to nucleotides 563,071 to 563,703 GenBank accession no. NC_000913.2; gi:49175990) is located between the sfmF and argil genes on the chromosome of £. coli K-12. The nucleotide sequence of the fimZ gene and the amino acid sequence of FimZ encoded by the fimZ gene are shown in SEQ ID NO: 1 and SEQ ID NO: 12, respectively.

[0042] Since there may be some differences in DNA sequences between the genera or strains of the Enterobacte-riaceae family, the fimZ gene to be inactivated on the chromosome is not limited to the gene shown in SEQ ID NO 11, but may include genes homologous to SEQ ID NO: 11 which encode variant proteins. The phrase "variant protein" as used in the present invention means a protein which has changes in the sequence, whether they are deletions, insertions, additions, or substitutions of amino acids, but still maintains the activity of the protein. 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 residues. It may be 1 to 30, preferably 1 to 15, and more preferably 1 to 5 in SEQ ID NO:12. These changes in the variants are conservative mutations that preserve the function of the protein. In other words, these changes in the variants can occur in regions of the protein which are not critical for the function of the protein. This is because some amino acids have high homology to one another so the three dimensional structure or activity is not affected by such a change. A conservative mutation is a mutation wherein substitution takes place mutually among Phe, Trp, Tyr, if the substitution site is an aromatic amino acid; among Leu, Ile, Val, if the substitution site is a hydrophobic amino acid; between Gin, Asn, if it is a polar amino acid; among Lys, Arg, His, if it is a basic amino acid; between Asp, Glu, if it is an acidic amino acid; and between Ser, Thr, if it is an amino acid having a hydroxyl group. Typical conservative mutations are conservative substitutions. Specific examples of substitutions that are considered to be conservative include: substitution of Ala with Ser or Thr; substitution of Arg with Gin, His, or Lys; substitution of Asn with Glu, Gin, Lys, His, or Asp; substitution of Asp with Asn, Glu, or Gin; substitution of Cys with Ser or Ala; substitution of Gin with Asn, Glu, Lys, His, Asp, or Arg; substitution of Glu with Gly, Asn, Gin, Lys, or Asp; substitution of Gly with Pro; substitution of His with Asn, Lys, Gin, Arg, or Tyr; substitution of lie with Leu, Met, Val, or Phe; substitution of Leu with Ile, Met, Val, or Phe; substitution of Lys with Asn, Glu, Gin, His, or Arg; substitution of Met with lie, Leu, Val, or Phe; substitution of Phe with Trp, Tyr, Met, lie, or Leu; substitution of Ser with Thror Ala; substitution of Thr with Ser or Ala; substitution of Trp with Phe or Tyr; substitution of Tyr with His, Phe, or Trp; and substitution of Val with Met, lie, or Leu. Substitutions, deletions, insertions, additions, or inversions and the like of the amino acids described above include naturally occurred mutations (mutant or variant) depending on differences in species, or individual differences of microorganisms that retain the ybdA gene. Such a gene can be obtained by modifying the nucleotide sequences shown in SEQ ID NO:11 using, for example, site-directed mutagenesis, so that the site-specific amino acid residue in the protein encoded includes substitutions, deletions, insertions, or additions.

[0043] Moreover, the protein variants encoded by the fimZgene may have a homology of not less than 80%, preferably not less than 90%, and most preferably not less than 95%, with respect to the entire amino acid sequences shown in SEQ ID NO. 12 as long as the native activity of FimZ proteins prior to inactivation are maintained.

[0044] Homology between two amino acid sequences can be determined using well-known methods, for example, the computer program BLAST 2.0, which calculates three parameters: score, identity and similarity.

[0045] Moreover, the fimZ gene may be variants which hybridize under stringent conditions with the nucleotide sequences shown in SEQ ID NO:11 or probes which can be prepared from the nucleotide sequences, provided that functional FimZ proteins is encoded. "Stringent conditions" include those under which a specific hybrid, for example, a hybrid having homology of 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% is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above, is not formed. For example, stringent conditions are exemplified by washing one time or more, preferably two or three times at a salt concentration of 1XSSC, 0.1% SDS, preferably 0.1 XSSC, 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 Hybond™ 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 usually varies from 100 bp to 1 kbp.

[0046] Inactivation of the gene can be performed by conventional methods, such as mutagenesis with UV irradiation or nitrosoguanidine (N-methyl-N’-nitro-N-nitrosoguanidine) treatment, site-directed mutagenesis, gene disruption using homologous recombination, or/and insertion-deletion mutagenesis (Yu, D. étal., Proc. Natl. Acad. Sei. USA, 2000,97:12: 5978-83 and Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sei. USA, 2000, 97:12: 6640-45) also called "Red-driven integration".

[0047] Functional properties are not known for the proteins encoded by the fimZ gene. The presence or absence of the fimZ gene in the chromosome of a bacterium can be detected by well-known methods, including PCR, Southern blotting, and the like. In addition, the level of gene expression can be estimated by measuring the amount of mRNA transcribed from the fimZ gene using various well-known methods, including Northern blotting, quantitative RT-PCR, and the like. The amount of the protein encoded by the fimZ gene can be measured by well-known methods, including SDS-PAGE followed by immunoblotting assay (Western blotting analysis) and the like.

[0048] Methods for preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer, and the like may be ordinary methods well-known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989). L-amino acid-producing bacteria [0049] As a bacterium for use in a method of the present invention which is modified to attenuate expression of the fimz gene by inactivation, bacteria which are able to produce either aromatic or non-aromatic L-amino acids may be used.

[0050] The bacterium for use in a method of the present invention can be obtained by attenuating expression of the fimZ gene in a bacterium for use in a method which inherently has the ability to produce L-amino acids. Alternatively, the bacterium of present invention can be obtained by imparting the ability to produce L-amino acids to a bacterium already having the attenuated expression of the sfmACDHF-fimZ cluster or the fimZ gene. L-threonine-producina bacteria

[0051] Examples of parent strains for deriving the L-threonine-producing bacteria for use in a method 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), £ coli NRRL-21593 (U.S. Patent No. 5,939,307), £. coli FERM BP-3756 (U.S. Patent No. 5,474,918), £. coli FERM BP-3519 and FERM BP-3520 (U.S. Patent No. 5,376,538), £. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14,947-956 (1978)), £. coli VL643 and VL2055 (EP 1149911 A), and the like.

[0052] 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 All-Union Scientific Center of Antibiotics (Nagatinskaya Street 3-A, 117105 Moscow, Russian Federation) under the accession number RIA 1867. The strain was also deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow 1, Dorozhny proezd. 1) on April 7, 1987 under the accession number VKPM B-3996.

[0053] £. coli VKPM B-5318 (EP 0593792B) may also be used as a parent strain for deriving L-threonine-producing bacteria for use in a method of the present invention. The strain B-5318 is prototrophic with regard to isoleucine, and a temperature-sensitive lambda-phage C 1 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.

[0054] Preferably, the bacterium for use in a method 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-ß-semialdehyde dehydrogenase; and the aspC gene which codes for aspartate aminotransferase (aspartate transaminase); [0055] 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 £. 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 £ coli K-12. The thrC gene which encodes threonine synthase of Escherichia coli has been elucidated (nucleotide positions 3734 to 5020, GenBank accession NC_000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaXopen reading frame on the chromosome of £. coli K-12. All three genes functions as a single threonine operon. To enhance expression of the threonine operon, the attenuator region which affects the transcription is desirably removed from the operon (W02005/049808, W02003/097839).

[0056] 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 the well-known plasmid pVIC40 which is presented in the threonine producing £. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S. Patent No. 5,705,371.

[0057] The rhtA gene exists at 18 min on the £. coli chromosome close to the ginHPQ 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 thepexßand ompXgenes. The unit expressing a protein encoded by the ORF1 has been designated the rhtA gene (rht: resistance to homoserine and threonine). Also, it was revealed that the rhtA23 mutation is an A-for-G substitution at position -1 with respect to the ATG start codon (ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A).

[0058] The asd gene of £. coli has already been elucidated (nucleotide positions 3572511 to 3571408, GenBank accession NC_000913.1, gi:16131307), and can be obtained by PCR (polymerase chain reaction; refer to White, T.J. et al., Trends Genet., 5, 185 (1989)) utilizing primers prepared based on the nucleotide sequence of the gene. The asd genes of other microorganisms can be obtained in a similar manner.

[0059] Also, the aspC gene of £. 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-Ivsine-producinq bacteria [0060] Examples of 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), γ-methyllysine, a-chlorocapro-lactam and so forth. Mutantshaving resistance to these lysine analogues can be obtained by subjecting bacteria belonging to the genus Escherichia to a conventional artificial mutagenesis treatment. Specific examples of bacterial strains useful for producing L-lysine include Escherichia coli M11442 (FERM BP-1543, NRRLB-12185;seeU.S. Patent No. 4,346,170) and Escherichia coli VL611. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is desensitized.

[0061] The strain WC196 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 strain and was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Tsukuba Central 6,1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on December 6, 1994 and received an accession number of FERM P-14690. 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).

[0062] Examples of parent strains for deriving L-lysine-producing bacteria for use in a method of the present invention also include strains in which expression of one or more genes encoding an L-lysine biosynthetic enzyme are enhanced. Examples of such genes include, but are not limited to, genes encoding dihydrodipicolinate synthase (dapA), aspartokinase (/ysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (U.S. Patent No. 6,040,160), phosphoenolpyrvate carboxylase (ppc), aspartate semialdehyde dehydroge-nease (asd), and aspartase (aspA) (EP 1253195 A). In addition, the parent strains may have an increased level of expression of the gene involved in energy efficiency (cyo) (EP 1170376 A), the gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (U.S. Patent No. 5,830,716), the ybjE gene (W02005/073390), or combinations thereof.

[0063] Examples of parent strains for deriving L-lysine-producing bacteria for use in a method 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 ofFfrom the biosynthetic pathway of L-lysine include homoserine dehydrogenase, lysine decarboxylase (U.S. Patent No. 5,827,698), and the malic enzyme (W02005/010175). L-cvsteine-producina bacteria [0064] Examples of parent strains for deriving L-cysteine-producing bacteria for use in a method 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. co//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 (JP11155571A2); E. co//W3110 with increased activity of a positive transcriptional regulatorfor cysteine regulon encoded by the cysB gene (W00127307A1), and the like. L-leucine-producinq bacteria [0065] Examples of parent strains forderiving L-leucine-producing bacteria for use in a method 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 β-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.

[0066] The bacterium for use in a method of the present invention may be improved by enhancing the expression of one or more genes involved in L-leucine biosynthesis. Examples include 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 No. 6,403,342). In addition, the bacterium for use in a method 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-producinq bacteria [0067] Examples of parent strains for deriving L-histidine-producing bacteria for use in a method 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 coliH-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Patent No. 6,344,347); E co//H-9341 (FERM BP-6674) (EP1085087); E. coli AI80/pFM201 (U.S. Patent No. 6,258,554) and the like.

[0068] Examples of parent strains for deriving L-histidine-producing bacteria for use in a method 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 (hislE), phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (hisA), amidotransferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD), and so forth.

[0069] It is known that the L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are inhibited by L-histidine, and therefore an L-histidine-producing ability can also be efficiently enhanced by introducing a mutation conferring resistance to the feedback inhibition into ATP phosphoribosyltransferase (Russian Patent Nos. 2003677 and 2119536).

[0070] Specific examples of 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-1,2,4-triazole-3-alanine, and streptomycin-resistance (VKPM B-7270, Russian Patent No. 2119536), and so forth. L-qlutamic acid-producing bacteria [0071] Examples of parent strains for deriving L-glutamic acid-producing bacteria for use in a method 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 P1 grown on the wild-type E coli strain K12 (VKPM B-7) cells. As a result, an L-isoleucine auxotrophic strain VL334thrC+ (VKPM B-8961), which is able to produce L-glutamic acid, was obtained.

[0072] Examples of parent strains for deriving the L-glutamic acid-producing bacteria for use in a method 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. Examples of such 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), enolase (eno), phos-phoglyceromutase (pgmA, pgml), phosphoglycerate kinase (pgk), glyceraldehyde-3-phophate dehydrogenase {gapA), triose phosphate isomerase {tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), and glucose phosphate isomerase (pgi).

[0073] Examples of strains modified so that expression of the citrate synthetase gene, the phosphoenolpyruvate carboxylase gene, and/áorthe glutamate dehydrogenase gene is/are enhanced include those disclosed in EP1078989A, EP955368A, and EP952221A.

[0074] Examples of 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.

[0075] Exam pies of parent strains for deriving the L-glutamic acid-producing bacteria for use in a method 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. Examples of such enzymes include isocitrate lyase (aceA), α-ketoglutarate dehydrogenase {sucA), phosphotransacetylase (pfa), acetate kinase (ac/c), acetohydroxy acid synthase (//vG), acetolactate synthase {ilvl), formate acetyltransferase (pfl), lactate dehydrogenase {Idh), and glutamate decarboxylase {gadAB). Bacteria belonging to the genus Escherichia deficient in a-ketogl-utarate dehydrogenase activity or having a reduced α-ketoglutarate dehydrogenase activity and methods for obtaining them are described in U.S. Patent Nos. 5,378,616 and 5,573,945. Specifically, these strains include the following: E. coli W3110sucA::Kmr E coli AJ 12624 (FERM BP-3853) E coli AJ 12628 (FERM BP-3854) E coli AJ 12949 (FERM BP-4881) £ coli W3110sucA::KmR is a strain obtained by disrupting the a-ketoglutarate dehydrogenase gene (hereinafter referred to as "sucA gene") of £. co//W3110. This strain is completely deficient in the a-ketoglutarate dehydrogenase.

[0076] Other examples of L-glutamic acid-producing bacterium include those which belong to the genus Escherichia and have resistance to an aspartic acid antimetabolite. These strains can also be deficient in a-ketoglutarate dehydrogenase activity and include, for exam pie, £. coli AJ 13199 (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.

[0077] Examples of L-glutamic acid-producing bacteria, include mutant strains belonging to the genus Pantoea which are deficient in a-ketoglutarate dehydrogenase activity or have decreased a-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-Chôme, 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 AJ13356 is deficient in the a-ketoglutarate dehydrogenase activity as a result of disruption of the aKGDH-E1 subunit gene (sucA). The above strain was identified as Enterobacter agglomerans when it was isolated and deposited as the Euterobacter agglomerans AJ13356. However, it was recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth. Although AJ13356 was deposited at the aforementioned depository as Enterobacter agglomerans, for the purposes of this specification, they are described as Pantoea ananatis. L-phenvialanine-producing bacteria [0078] Examples of parent strains for deriving L-phenylalanine-producing bacteria for use in a method of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as£. coliAJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197); £. coli HW1089 (ATCC 55371) harboring the mutant pheA34 gene (U.S. Patent No. 5,354,672); £. coli MWEC101-b (KR8903681); £. 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, £ co//K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), £. co//K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659), £ coli K-M [W3110 (tyrA)/pPHATerm] (FERM BP-12662) and £ coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used (EP 488424 B1). Furthermore, L-phenylalanine producing bacteria belonging to the genus Escherichia with an enhanced activity of the protein encoded by the yedA gene or the yddG gene may also be used (U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1). L-tryptophan-producinq bacteria [0079] Examples of parent strains for deriving the L-tryptophan-producing bacteria for use in a method of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as £. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Patent No. 5,756,345); £ coliSV164 (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); £ 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); £ coli AGX17/pGX50,pACKG4-pps in which a phosphoenolpyruvate-producing ability is enhanced (WO9708333, U.S. Patent No. 6,319,696), and the like may be used. L-tryptophan-producing bacteria belonging to the genus Escherichia with an enhanced activity of the identified protein encoded by and the yedA gene or the yddG gene may also be used (U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1).

[0080] Examples of parent strains for deriving the L-tryptophan-producing bacteria for use in a method 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 feed back 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 £ coli SV164 which harbors desensitized anthranilate synthase and a transformant strain obtained by introducing into the £ co//SV164 the plasmid pGH5 (WO 94/08031), which contains a mutant serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase.

[0081] Examples of parent strains for deriving the L-tryptophan-producing bacteria for use in a method 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). Moreover, L-tryptophan-producing ability may be imparted by enhancing expression of a gene which encodes tryptophan synthase, among tryptophan opérons (trpBA). The tryptophan synthase consists of a and ß subunits which are encoded by the trpA and trpB genes, respectively. In addition, L-tryptophan-producing ability may be improved by enhancing expression of the isocitrate lyase-malate synthase operon (W02005/103275). L-proline-producinq bacteria [0082] Exam pies of parent strains for deriving L-proline-producing bacteria for use in a method of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli702ilvA (VKPM B-8012) which is deficient in the UvA gene and is able to produce L-proline (EP 1172433). The bacterium for use in a method 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 praß gene coding for glutamate kinase of which feedback inhibition by L-proline is desensitized (DE Patent 3127361). In addition, the bacterium for use in a method 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).

[0083] Examples of bacteria belonging to the genus Escherichia, which have an activity to produce L-proline include the following £. 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 15th Miami winter symposium, 1983, p.34), and the like. L-arqinine-producinq bacteria [0084] Exam pies of parent strains forderiving L-arginine-producing bacteria for use in a method of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as £. coli strain 237 (VKPM B-7925) (U.S. Patent Application 2002/058315 A1) 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 (EP1170361A1 ), and the like.

[0085] Examples of parent strains for deriving L-arginine producing bacteria for use in a method 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 (argß), acetylornithine transaminase (argD), ornithine carbamoyl transferase (argF), argininosuccinic acid synthetase (argG), argininosuccinic acid lyase (argH), and carbamoyl phosphate synthetase {carAB). L-valine-producina bacteria [0086] Example of parent strains for deriving L-valine-producing bacteria for use in a method 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 the L-valine that is produced. Furthermore, the UvA gene in the operon is desirably disrupted so that threonine deaminase activity is decreased.

[0087] Examples of parent strains for deriving L-valine-producing bacteria for use in a method of the present invention include also include mutants having a mutation of amino-acyl t-RNA synthetase (U.S. Patent No. 5,658,766). For example, E. coliVL1970, which has a mutation in the ileS gene encoding isoleucine tRNA synthetase, can be used. £. coliVL1970 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 113545 Moscow, 1 Dorozhny Proezd, 1) on June 24, 1988 under accession number VKPM B-4411.

[0088] Furthermore, mutants requiring lipoic acid for growth and/or lacking H+-ATPase can also be used as parent strains (WO96/06926). L-isoleucine-producina bacteria [0089] Examples of parent strains for deriving L-isoleucine producing bacteria for use in a method 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). In addition, recombinant strains transformed with genes encoding proteins involved in L-isoleucine biosynthesis, such as threonine deaminase and acetohy- droxate synthase, can also be used as parent strains (JP 2-458 A, FR 0356739, and U.S. Patent No. 5,998,178). 2. Method of the present invention [0090] The method of the present invention is a method for producing an L-amino acid by cultivating the bacterium for use in a method 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.

[0091] In the present invention, 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.

[0092] 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. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used. As vitamins, thiamine, yeast extract, and the like, can be used.

[0093] 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.

[0094] After cultivation, 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.

Brief Description of Drawings [0095]

Figure 1 shows the construction of the pMW118-attL-Cm-attR plasmid, which is used as a template for PCR. Figure 2 shows the relative positions of primers P17 and P 18 on plasmid pACYC184, which is used for PCR amplification of the caf gene.

Figure 3 shows the relative positions of primers P21 and P22 on plasmid pMW118-attL-Cm-attR, which is used for PCR amplification of the cat gene.

Figure 4 shows the construction of the chromosomal DNA fragment containing the inactivated sfmACDHF-fimZ cluster.

Figure 5 shows the construction of the chromosomal DNA fragment containing the inactivated fimZ gene. Examples [0096] The present invention will be more concretely explained below with reference to the following non-limiting Examples.

Example 1. Preparation of the PCR template and helper plasmids [0097] The PCR template plasmid pMW118-attL-Cm-attR and the helper plasmid pMW-intxis-ts were prepared as follows:

(1) pMW118-attL-Cm-attR

The pMW118-attL-Cm-attR plasmid was constructed on the basis of pMW118-attL-Tc-attR that was obtained by ligation of the following four DNA fragments: 1) the Bgl\\-EcoR\ fragment (114 bp) carrying attL (SEQ ID NO:13) which was obtained by PCR amplification of the corresponding region of the E. coli W3350 (contained λ prophage) chromosome using oligonucleotides P1 and P2 (SEQ ID NOS: 14 and 15) as primers (these primers contained the subsidiary recognition sites for

BglII and EcoR\ endonucleases); 2) the Pst\-Hind\\\ fragment (182 bp) carrying attR (SEQ ID NO: 16) which was obtained by PCR amplification of the corresponding region of the E. co//W3350 (contained λ prophage) chromosome using the oligonucleotides P3 and P4 (SEQ ID NOS: 17 and 18) as primers (these primers contained the subsidiary recognition sites for Pst\ and Hind III endonucleases); 3) the large Sg/ll-H/ndlll fragment (3916 bp) of pMW118-ter_rrnS. The plasmid pMW118-ter_rrnß was obtained by ligation of the following three DNA fragments: • the large DNA fragment (2359 bp) carrying the Aat\\-EcoR\ fragment of pMW118 that was obtained in the following way: pMW118 was digested with EcoR\ restriction endonuclease, treated with Klenow fragment of DNA polymerase I, and then digested with Aafll restriction endonuclease; • the small Aat\\-Bgl\ I fragment (1194 bp) of pUC19 carrying the bla gene for ampicillin resistance (ApR) was obtained by PCR amplification of the corresponding region of the pUC19 plasmid using oligonucleotides P5 and P6 (SEQ ID NOS: 19 and 20) as primers (these primers contained the subsidiary recognition sites for Aafll and BglU endonucleases); • the small Sg/ll-Psflpol fragment (363 bp) of the transcription terminator ter_rrnB was obtained by PCR amplification of the corresponding region of the £. coli MG1655 chromosome using oligonucleotides P7 and P8 (SEQ ID NOS: 21 and 22) as primers (these primers contained the subsidiary recognition sites for BglII and Pst\ endonucleases); 4) the small EcoR\-Pst\ fragment (1388 bp) (SEQ ID NO:23) of pML-Tc-ter_thrL bearing the tetracycline resistance gene and the ter_thrL transcription terminator; the pML-Tc-ter_fbrL plasmid was obtained in two steps: • the pML-ter_fbr/_ plasmid was obtained by digesting the pML-MCS plasmid (Mashko, S.V. et al., Bi-otekhnologiya (in Russian), 2001, no. 5,3-20) with the Xbal and BamHI restriction endonucleases, followed by ligation of the large fragment (3342 bp) with the Xba\-BamH\ fragment (68 bp) carrying terminatorter_fbrL obtained by PCR amplification of the corresponding region of the E. coli MG1655 chromosome using oligonucleotides P9 and P10 (SEQ ID NOS: 24 and 25) as primers (these primers contained the subsidiary recognition sites for the Xbal and BamH I endonucleases); • the pML-Tc-ter_fbr£. plasmid was obtained by digesting the pML-ter_thrL plasmid with the Kpn I and Xbal restriction endonucleases followed by treatment with Klenow fragment of DNA polymerase I and ligation with the small EcoR\-Van9l\ fragment (1317 bp) of pBR322 bearing the tetracycline resistance gene (pBR322 was digested with EcoRI and Van9lI restriction endonucleases and then treated with Klenow fragment of DNA polymerase I).

The above £. coll W3350 is a derivative of wild-type strain £. coli K-12. The £. coli MG1655 (ATCC 700926) is a wild-type strain and can be obtained from American Type Culture Collection (P.O. Box 1549 Manassas, VA 20108, United States of America). The plasmids pMW118 and pUC19 are commercially available. The ßg/ll-£coRlfragment carrying attL and the Bgl\\-Pst\ fragment of the transcription terminator ier rrnB can be obtained from other strains of £ coli in the same manner as described above.

The pMW118-attL-Cm-attR plasmid was constructed by ligation of the large BamH\-Xba\ fragment (4413 bp) of pMW118-attL-Tc-attR and the artificial DNA Bgl\\-Xba\ fragment (1162 bp) containing the PA2 promoter (the early promoter of the phage T7), the cat gene for chloramphenicol resistance (CmR), the ter_thrL transcription terminator, and attR. The artificial DNA fragment (SEQ ID NO:26) was obtained as follows: 1. The pML-MCS plasmid was digested with the Kpn I and Xbal restriction endonucleases and ligated with the small Kpn\-Xba\fragment (120 bp), which included the PA2 promoter (the early promoter of phage T7) obtained by PCR amplification of the corresponding DNA region of phage T7 using oligonucleotides P11 and P12 (SEQ ID NOS: 27 and 28, respectively) as primers (these primers contained the subsidiary recognition sites for Kpn I and Xbal endonucleases). As a result, the pML-PA2-MCS plasmid was obtained. The complete nucleotide sequence of phage T7 has been reported (J. Mol. Biol., 166: 477-535 (1983). 2. The Xbal site was deleted from pML-PA2-MCS. As a result, the pML-PA2-MCS(Xbah) plasmid was obtained. 3. The small Bgl\\-Hind\\\ fragment (928 bp) of pML-PA2-MCS(Xbah) containing the PA2 promoter (the early promoter of the phage T7) and the cat gene for chloramphenicol resistance (CmR) was ligated with the small /-//ndlll-/-//ncilll fragment (234 bp) of pMW118-attL-Tc-attR containing the ter_thrL transcription terminator and attR. 4. The required artificial DNA fragment (1156 bp) was obtained by PCR amplification of the ligation reaction mixture using oligonucleotides P9 and P4 (SEQ ID NOS: 24 and 18) as primers (these primers contained the subsidiary recognition sites for Hind\\\ and XbaI endonucleases). (2) pMW-intxis-ts [0098] Recombinant plasmid pMW-intxis-ts containing the cl repressor gene and the int-xis genes of phage λ under control of promoter PR was constructed on the basis of vector pMWP|aclacl-ts. To construct the pMWP|aclacl-ts variant, the Aafll-EcoRV fragment of the pMWP|aclacl plasmid (Skorokhodova, A. Yu. et al., Biotekhnologiya (in Russian), 2004, no. 5,3-21) was substituted with the Aafll-EcoRV fragment of the pMAN997 plasmid (Tanaka, K. et al., J. Bacteriol., 2001, 183(22): 6538-6542, WO99/03988) bearing the par and ori loci and the repAis gene (a temperature sensitive-replication origin) of the pSC101 replicon. The plasmid pMAN997 was constructed by exchanging the Vspl-Hindlll fragments of pMAN031 (J. Bacteriol., 162, 1196 (1985)) and pUC19.

[0099] Two DNA fragments were amplified using phage λ DNA ("Fermentas") as a template. The first one contained the DNA sequence from 37,168 to 38,046, the cl repressor gene, promoters PRM and PR, and the leader sequence of the cro gene. This fragment was PCR-amplified using oligonucleotides P13 and P14 (SEQ ID NOS: 29 and 30) as primers. The second DNA fragment containing the x/'s-/hf genes of phage λ and the DNA sequence from 27801 to 29100 was PCR-amplified using oligonucleotides P15 and P16 (SEQ ID NOS: 31 and 32) as primers. All primers contained the corresponding restriction sites.

[0100] The first PCR-amplified fragment carrying the cl repressor was digested with restriction endonuclease C/al, treated with Klenow fragment of DNA polymerase I, and then digested with restriction endonuclease EcoRI. The second PCR-amplified fragment was digested with restriction endonucleases EcoRI and Psfl. The pMWP|aclacl-ts plasmid was digested with the BglU endonuclease, treated with Klenow fragment of DNA polymerase I, and digested with the Psfl restriction endonuclease. The vector fragment of pMWPIaclacl-ts was eluted from agarose gel and ligated with the above-mentioned digested PCR-amplified fragments to obtain the pMW-intxis-ts recombinant plasmid.

Example 2 (Reference). Construction of a strain with the inactivated sfmACDHF-fimZ cluster 1. Deletion of the sfmACDHF-fimZ cluster [0101] A strain with the sfinACDHF-fimZ duster deieted was constructed by the method initially developed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sei. USA, 2000, 97(12): 6640-6645) called "Red-driven integration". According to this procedure, the PCR primers P17 (SEQ ID NO: 33) and P18 (SEQ ID NO:34), which are complementary to both the region adjacent to the sfmACDHF-fimZ cluster and the gene conferring antibiotic resistance in the template plasmid, were constructed. The plasmid pACYC184 (NBL Gene Sciences Ltd., UK) (GenBank/EMBL accession numberX06403) was used as a template in the PCR reaction. Conditions for PCR were as follows: dénaturation step: 3 min at 95°C; profile for two first cycles: 1 min at 95°C, 30 sec at 50°C, 40 sec at 72°C; profile for the last 25 cycles: 30 sec at 95°C, 30 sec at 54°C, 40 sec at 72°C; final step: 5 min at 72°C.

[0102] A 1152-bp PCR product (Fig. 2) was obtained and purified in agarose gel and was used for electroporation of E coli MG 1655 (ATCC 700926), which contains the pKD46 plasmid having temperature-sensitive replication. The pKD46 plasmid (Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sei. USA, 2000, 97(12):6640-6645) includes a 2,154-bp DNA fragment of phage λ (nucleotide positions 31088 to 33241, GenBank accession no. J02459), and contains genes of the λ Red homologous recombination system (γ, β, exo genes) under the control of the arabinose-inducible ParaB promoter. The plasmid pKD46 is necessary for integration of the PCR product into the chromosome of strain MG1655. The strain MG1655 can be obtained from American Type Culture Collection. (P.O. Box 1549 Manassas, VA 20108, U.S.A.).

[0103] Electrocompetent cells were prepared as follows: E. coli MG1655/pKD46 was grown overnight at 30 °C in LB medium containing ampicillin (100 mg/l), 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 L-arabinose (1 mM). The cells were grown with aeration at 30°C to an OD600 of «0.6 and then were made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H20. Electroporation was performed using 70 μΙ of cells and «100 ng of the PCR product. Cells after electroporation were incubated with 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C for 2.5 hours and then were plated onto L-agar containing chloramphenicol (30 μg/ml) and grown at 37°C to select CmR recombinants. Then, to eliminate the pKD46 plasmid, two passages on L-agar with Cm at 42°C were performed and the colonies were tested for sensitivity to ampicillin.

2. Verification of the sfmACDHF-fimZ cluster deletion by PCR

[0104] The mutants having the sfmACDHF-fimZ cluster deleted and marked with the Cm resistance gene were verified by PCR. Locus-specific primers P19 (SEQ ID NO:35) and P20 (SEQ ID NO:36) were used in PCR for the verification. Conditions for PCR verification were as follows: dénaturation step: 3 min at 94°C; profile for 30 cycles: 30 sec at 94°C, 30 sec at 54°C, 1 min at 72°C; final step: 7 min at 72°C. The PCR product obtained in the reaction with the parental sfmACDHF-fimZ+ MG1655 strain as the template was 6528 bp in length. The PCR product obtained in the reaction with the mutant strain as the template was 1306 bp in length (Fig.4). The mutant strain was named MG1655 AsfmACDHF-fimZ::cat.

Example 3 (Referenced. Production of L-threonine by E. coli strain B-3996-AsfmACDHF-fimZ

[0105] To test the effect of inactivation of the sfmACDHF-fimZ cluster on threonine production, DNA fragments from the chromosome of the above-described E. coli MG1655 AsfmACDHF-fimZ::cat were transferred to the threonine-producing E. coli strain VKPM B-3996 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain B-3996-AsfmACDHF-fimZ.

[0106] Both E. coli strains, B-3996 and B-3996-AsfmACDHF-fimZ, were grown for 18-24 hours at 37°C on L-agar plates. To obtain a seed culture, the strains were grown on a rotary shaker (250 rpm) at 32°C for 18 hours in 20x200-mm test tubes containing 2 ml of L-broth supplemented with 4% glucose. Then, the fermentation medium was inoculated with 0.21 ml (10%) of seed material. The fermentation was performed in 2 ml of minimal medium for fermentation in 20x200-mm test tubes. Cells were grown for 65 hours at 32°C with shaking at 250 rpm.

[0107] After cultivation, the amount of L-threonine which had accumulated in the medium, was determined by paper chromatography using the following mobile phase: butanol - acetic acid - water = 4 : 1 : 1 (v/v). A solution of ninhydrin (2%) in acetone was used as a visualizing reagent. A spot containing L-threonine was cut out, L-threonine was eluted with 0.5 % water solution of CdCI2, and the amount of L-threonine was estimated spectrophotometrically at 540 nm. The results of eight independent test tube fermentations are shown in Table 1. As follows from Table 1, B-3996-AsfmACDHF-fimZ caused accumulation of a higher amount of L-threonine, as compared with B-3996.

[0108] The composition of the fermentation medium (g/l) was as follows:

[0109] Glucose and magnesium sulfate were sterilized separately. CaC03 was sterilized by dry-heat at 180°C for 2 hours. The pH was adjusted to 7.0. The antibiotic was introduced into the medium after sterilization.

Example 4 (Reference’). Production of L-Ivsine bv E. coli AJ11442-AsfmACDHF-fimZ

[0110] To test the effect of inactivation of the sfmACDHF-fimZ cluster on lysine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 AsfmACDHF-fimZ::cat can be transferred to the lysine-producing E. coli strain AJ11442 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain AJ11442-AsfmACDHF-fimZ. The 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-Chôme, 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 Octobe 29, 1987, and received an accession number of FERM BP-1543.

[0111] Both £ coli strains, AJ11442 and AJ11442-AsfmACDHF-fimZ can be cultured in L-medium at 37°C, and 0.3 ml of the obtained culture can be inoculated into 20 ml of the fermentation medium containing the required drugs in a 500-ml flask. The cultivation can be carried out at 37°C for 16 h by using a reciprocal shaker at the agitation speed of 115 rpm. After the cultivation, the amounts of L-lysine and residual glucose in the medium can be measured by a known method (Biotech-analyzer AS210 manufactured by Sakura Seiki Co.). Then, the yield of L-lysine can be calculated relative to consumed glucose for each of the strains.

[0112] The composition of the fermentation medium (g/l) is as follows:

[0113] The pH is adjusted to 7.0 by KOH and the medium is autoclaved at 115°C for 10 min. Glucose and MgS04 7H20 are sterilized separately. CaC03 is dry-heat sterilized at 180°C for 2 hours and added to the medium for a final concentration of 30 g/l.

Example 5 (Reference). Production of L-cvsteine by E. coli JM15(vdeD)-AsfmACDHF-fimZ

[0114] To test the effect of inactivation of the sfmACDHF-fimZ cluster on L-cysteine production, DNA fragments from the chromosome of the above-described £ coli MG1655 AsfmACDHF-fimZ::cat can be transferred to the E. coli L-cysteine-producing strain JM15(ydeD) by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain JM15(ydeD)-AsfmACDHF-fimZ.

[0115] £. coli JM15(ydeD) is a derivative of £. coli JM15 (US Patent No. 6,218,168), which can be transformed with DNA having the ydeD gene encoding 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/).

[0116] Fermentation conditions for evaluation of L-cysteine production were described in detail in Example 6 of US Patent No. 6,218,168.

Example 6 (Reference). Production of L-leucine by £ coli 57-AsfmACDHF-fimZ

[0117] To test the effect of inactivation of the sfmACDHF-fimZ cluster on L-leucine production, DNA fragments from the chromosome of the above-described £. coli strain MG1655 AsfmACDHF-fimZ::cat can be transferred to the £. coli L-leucine-producing strain 57 (VKPM B-7386, US Patent No. 6,124,121) by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain 57-pMWAsfmACDHF-fimZ strain. The strain 57 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on May 19,1997 under accession number VKPM B-7386.

[0118] Both £. coli strains, 57 and 57-AsfmACDHF-fimZ, can be cultured for 18-24 hours at 37°C on L-agar plates. To obtain a seed culture, the strains can be grown on a rotary shaker (250 rpm) at 32°Cfor 18 hours in 20x200-mm test tubes containing 2 ml of L-broth supplemented with 4% sucrose. Then, the fermentation medium can be inoculated with 0.21 ml of seed material (10%). The fermentation can be performed is 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. The amount of L-leucine can be measured by paper chromatography (liquid phase composition: butanol - acetic acid - water = 4:1:1).

[0119] The composition of the fermentation medium (g/l) (pH 7.2) is as follows:

[0120] Glucose and CaC03 are sterilized separately.

Example 7 (Referenced Production of L-histidine by E. coli 80-AsfmACDHF-fimZ

[0121] To test the effect of inactivation of the sfmACDHF-fimZ cluster on L-histidine production, DNA fragments from the chromosome of the above-described E. coli MG1655 AsfmACDHF-fimZ::cat can be transferred to the histidine-producing E. coli strain 80 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 80-AsfmACDHF-fimZ. The strain 80 has been described in Russian patent 2119536 and 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-7270and then converted to a deposit under the Budapest Treaty on July 12, 2004.

[0122] Both E coli strains, 80 and 80-AsfmACDHF-fimZ, can each be cultured in L-broth for 6 h at 29°C. Then, 0.1 ml of obtained culture can be inoculated into 2 ml of fermentation medium in a 20x200-mm test tube and cultivated for 65 hours at 29°C with shaking on a rotary shaker (350 rpm). After cultivation, the amount of histidine which accumulates in the medium can be determined by paper chromatography. The paper can be developed with a mobile phase consisting of n-butanol : acetic acid : water = 4 :1 :1 (v/v). A solution of ninhydrin (0.5%) in acetone can be used as a visualizing reagent.

[0123] The composition of the fermentation medium (g/l) is as follows (pH 6.0):

[0124] Glucose, proline, betaine and CaC03 are sterilized separately. The pH is adjusted to 6.0 before sterilization. Example 8 (Reference). Production of L-alutamate by E. coli VL334thrC+-AsfmACDHF-fimZ

[0125] To test the effect of inactivation of the sfimACDHF-fimZ cluster on L-glutamate production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 AsfmACDHF-fimZ::cat can be transferred to the E. coli L-glutamate-producing strain VL334thrC+ (EP 1172433) by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain VL334thrC+-AsfmACDHF-fimZ. The strain VL334thrC+ has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1 ) on December 6, 2004 under the accession number VKPM B-8961 and then converted to a deposit under the Budapest Treaty on December 8, 2004.

[0126] Both strains, VL334thrC+ and VL334thrC+-AsfinACDHF-fimZ, can be grown for 18-24 hours at 37°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/l), KH2P04 (2g/l), MgS04 (1 g/l), thiamine (0.1 mg/ml), L-isoleucine (70 ^g/ml), and CaCOs (25 g/l). The pH is adjusted to 7.2. Glucose and CaC03 are sterilized separately. Cultivation can be carried out at 30°C for 3 days with shaking. After the cultivation, the amount of L-glutamic acid which is produced can be determined by paper chromatography (liquid phase composition of butanol-acetic acid-water=4:1:1) with subsequent staining by ninhydrin (1% solution in acetone) and further elution of the compounds in 50% ethanol with 0.5% CdCI2.

Example 9 (Referenced. Production of L-phenvlalanine by E. coli AJ12739-AsfmACDHF-fimZ

[0127] To test the effect of inactivation of the ksfmACDHF-fimZ cluster on L-phenylalanine production, DNA fragments from the chromosome of the above-described E. coli MG1655 AsfmACDHF-fimZ::cat can be transferred to the phenylalanine-producing £. co//strain AJ12739 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain AJ12739-AsfmACDHF-fimZ. The strain AJ12739 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on November 6, 2001 under accession no. VKPM B-8197 and then converted to a deposit under the Budapest Treaty on August 23, 2002.

[0128] Both strains, AJ12739-AsfmACDHF-fimZ and AJ12739, can be cultivated at 37°C for 18 hours in a nutrient broth, and 0.3 ml of the obtained culture 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 10x15-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. The Sorbfil plates can be developed with a mobile phase consisting of propan-2-ol : ethylacetate : 25% aqueous ammonia : water = 40 : 40 : 7 :16 (v/v). A solution of ninhydrin (2%) in acetone can be used as a visualizing reagent.

[0129] The composition of the fermentation medium (g/l) is as follows:

[0130] Glucose and magnesium sulfate are sterilized separately. CaC03 is dry-heat sterilized at 180°for2 hours. The pH is adjusted to 7.0.

Example 10 (Referenced Production of L- tryptophan bv E. coli SV164 (pGH51-AsfmACDHF-fimZ

[0131] To test the effect of inactivation of the sfmACDHF-fimZ cluster on L-tryptophan production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 AsfmACDHF-fimZ::cat can be transferred to the tryptophan-producing E. coli strain SV164 (pGH5) by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain SV164(pGH5)-AsfmACDHF-fimZ. 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) was described in detail in US patent No. 6,180,373 or European patent 0662143.

[0132] Both strains, SV164(pGH5)-AsfmACDHF-fimZ and SV164(pGH5), can be cultivated with shaking at 37°C for 18 hours in 3 ml of nutrient broth supplemented with tetracycline (20 mg/l, marker of pGH5 plasmid). The obtained cultures (0.3 ml each) can be inoculated into 3 ml of a fermentation medium containing tetracycline (20 mg/l) in 20 x 200-mm test tubes, and cultivated at 37°C for 48 hours with a rotary shaker at 250 rpm. After cultivation, the amount of tryptophan which accumulates in the medium can be determined by TLC as described in Example 8. The fermentation medium components are listed in Table 2, but should be sterilized in separate groups (A, B, C, D, E, F, and H), as shown, to avoid adverse interactions during sterilization.

Example 11 (Referenced Production of L-proline bv E. coli 702ilvA-AsfmACDHF-fimZ

[0133] To test the effect of inactivation of the sfmACDHF-fimZ cluster on L-proline production, DNA fragments from the chromosome of the above-described £ coli strain MG1655 AsfmACDHF-fimZ::cat can be transferred to the proline-producing £. coli strain 702ilvA by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 702ilvA-AsfmACDHF-fimZ. The strain 702ilvA has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on July 18, 2000 under accession number VKPM B-8012 and then converted to a deposit under the Budapest Treaty on May 18, 2001.

[0134] Both £. coli strains, 702ilvA and 702ilvA-AsfmACDHF-fimZ, can be grown for 18-24 hours at 37°C on L-agar plates. Then, these strains can be cultivated under the same conditions as in Example 8.

Example 12 (Reference). Production of L-arqinine bv £. coli 382-AsfmACDHF-fimZ

[0135] To test the effect of inactivation of the sfmACDHF-fimZ cluster on L-arginine production, DNA fragments from the chromosome of the above-described £. coli strain MG1655 AsfmACDHF-fimZ::cat can be transferred to the arginine-producing £. coli strain 382 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 382-AsfmACDHF-fimZ. The strain 382 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 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.

[0136] Both strains, 382-AsfmACDHF-fimZ and 382, can be 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 3 ml of a fermentation medium in 20 x 200-mm test tubes and cultivated at 32°C for 48 hours on a rotary shaker.

[0137] After the cultivation, the amount of L-arginine which accumulates in the medium can be determined by paper chromatography using the following mobile phase: butanol: acetic acid : water = 4 : 1 : 1 (v/v). 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 CdCI2, and the amount of L-arginine can be estimated spectrophotometrically at 540 nm.

[0138] The composition of the fermentation medium (g/l) is as follows:

[0139] Glucose and magnesium sulfate are sterilized separately. CaCOs is dry-heat sterilized at 180 °C for 2 hours. The pH is adjusted to 7.0.

Example 13. Construction of a strain with the inactivated fimZ gene 1. Deletion of the fimZ gene [0140] A strain with the fimZ gene deleted was constructed by the method initially developed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sei. USA, 2000, 97(12) 6640-6645) called "Red-driven integration". The DNA fragment containing the CmR marker encoded by the cat gene was obtained by PCR, using primers P21 (SEQ ID NO:37) and P22 (SEQ ID NO:38) and plasmid pMW118-attL-Cm-attR as a template (for construction see Example 1). Primer P21 contains both a region complementary to the 36-nt region located at the 5’ end of the fimZ gene and a region complementary to the attL region. Primer P22 contains both a region complementary to the 36-nt region located at the 3’ end of the fimZ gene and a region complementary to the attR region. Conditions for PCR were as follows: dénaturation step: 3 min at 95°C; profile for two first cycles: 1 min at 95°C, 30 sec at 50°C, 40 sec at 72°C; profile for the last 25 cycles: 30 sec at 95°C, 30 sec at 54°C, 40 sec at 72°C; final step: 5 min at 72°C.

[0141] A 1.7 kbp PCR product (Fig. 3) was obtained and purified in agarose gel and was used for electroporation of E. coli MG1655 (ATCC 700926), which contains the plasmid pKD46 having a temperature-sensitive replication. The plasmid pKD46 (Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sei. USA, 2000, 97(12) 6640-6645) includes a 2,154-bp DNA frag ment of phage λ (nucleotide positions 31088 to 33241, GenBank accession No. J02459), and contains genes of the λ Red homologous recombination system (γ, β, exo genes) under the control of the arabinose-inducible ParaB promoter. The plasmid pKD46 is necessary for integration of the PCR product into the chromosome of strain MG1655.

[0142] Electrocompetent cells were prepared as described in the Example 2. Electroporation was performed using 70 μΙ of cells and «100 ng of the PCR product. Cells after electroporation were incubated with 1 ml of SOC medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) at 37°C for 2.5 hours and after that were plated onto L-agar containing chloramphenicol (30 μg/ml) and grown at 37°C to select CmR recombinants. Then, to eliminate the pKD46 plasmid, two passages on L-agar with Cm at 42°C were performed, and the obtained colonies were tested for sensitivity to ampicillin.

2. Verification of the fimZ gene deletion by PCR

[0143] The mutants, which have the fimZ gene deleted and are marked with the Cm resistance gene, were verified by PCR. Locus-specific primers P23 (SEQ ID NO:39) and P24 (SEQ ID NO:40) were used in PCR for verification. Conditions for PCR verification were as follows: dénaturation step: 3 min at 94°C; profile for the 30 cycles: 30 sec at 94°C, 30 sec at 54°C, 1 min at 72°C; final step: 7 min at 72°C. The PCR product obtained in the reaction with the cells of the parental strain fimZ+ MG1655 strain as the template was ~0.7 kb in length. The PCR product obtained in the reaction with the cells of the mutant strain as the template was ~1,8kb in length (Fig.5). The mutant strain was named MG1655 AfimZ::cat.

Example 14. Production of L-threonine by E. coli B-3996-AfimZ

[0144] To test the effect of inactivation of the fimZ gene on threonine production, DNA frag ments from the chromosome of the above-described E. coli MG1655 AfimZ::cat were transferred to the threonine-producing E. coli strain VKPM B-3996 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain B-3996-AfmiZ.

[0145] Both E. coli B-3996 and B-3996-AfimZ, were grown for 18-24 hours at 37°C on L-agar plates. To obtain a seed culture, the strains were grown on a rotary shaker (250 rpm) at 32°C for 18 hours in 20x200-mm test tubes containing 2 ml of L-broth supplemented with 4% glucose. Then, the fermentation medium was inoculated with 0.21 ml (10%) of seed material. The fermentation was performed in 2 ml of minimal medium for fermentation in 20x200-mm test tubes. Cells were grown for 65 hours at 32°C with shaking at 250 rpm.

[0146] After cultivation, the amount of L-threonine which had accumulated in the medium, was determined by paper chromatography using the following mobile phase: butanol: acetic acid : water = 4 :1:1 (v/v). A solution 2% of ninhydrin in acetone was used as a visualizing reagent. A spot containing L-threonine was cut out, L-threonine was eluted in 0.5 % water solution of CdCI2, and the amount of L-threonine was estimated spectrophotometrically at 540 nm. The results of eight independent test tube fermentations are shown in Table 3. As follows from Table 3, B-3996- AfimZ caused accumulation of a higher amount of L-threonine, as compared with B-3996.

[0147] The composition of the fermentation medium (g/l) was as follows:

[0148] Glucose and magnesium sulfate were sterilized separately. CaC03 was sterilized by dry-heat at 180°C for 2 hours. The pH was adjusted to 7.0. The antibiotic was introduced into the medium after sterilization.

Example 15. Production of L-Ivsine bv E. coli AJ11442-AfimZ

[0149] To test the effect of inactivation of the fimZ gene on lysine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 AfimZ: :cat can be transferred to the lysine-producing E. co//'strain AJ11442 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain AJ11442-AfimZ.

[0150] Both E. coli strains AJ11442 and AJ11442-AfimZ can be cultured in L-medium containing streptomycin (20 mg/l) at 37°C, and 0.3 ml of the obtained culture can be inoculated into 20 ml of the fermentation medium containing the required drugs in a 500-ml flask. The cultivation can be carried out at 37°C for 16 h by using a reciprocal shaker at the agitation speed of 115 rpm. After the cultivation, the amounts of L-lysine and residual glucose in the medium can be measured by a known method (Biotech-analyzer AS210 manufactured by Sakura Seiki Co.). Then, the yield of L-lysine can be calculated relative to consumed glucose for each of the strains.

[0151] The composition of the fermentation medium (g/l) is as follows:

[0152] The pH is adjusted to 7.0 by KOH and the medium is autoclaved at 115°C for 10 min. Glucose and MgSQ4 x 7H20 are sterilized separately. CaC03 is dry-heat sterilized at 180°C for 2 hours and added to the medium for a final concentration of 30 g/l.

Example 16. Production of L-cvsteine bv E. coli JM15(vdeD)-AfimZ

[0153] T o test the effect of inactivation of the fimZ gene on L-cysteine production, DNA fragments from the chromosome of the above-described £. coli MG 1655 AfimZ::cat can be transferred to the £. co//L-cysteine-producingstrain JM15(ydeD) by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain JM15(ydeD)-AfimZ.

[0154] E. coli JM15(ydeD) is a derivative of E. coli JM15 (US Patent No. 6,218,168), which can be transformed with DNA having the ydeD gene encoding 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/).

[0155] Fermentation conditions for evaluation of L-cysteine production were described in detail in Example 6 of US Patent No. 6,218,168.

Example 17. Production of L-leucine bv E. coli 57-AfimZ

[0156] To test the effect of inactivation of the fimZ gene on L-leucine production, DNA fragments from the chromosome of the above-described £. coli strain MG1655 A-firmZ::cat can be transferred to the £. coli L-leucine-producing strain 57 (VKPM B-7386, US Patent No. 6,124,121) by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain 57-AfimZ.

[0157] Both £. coli strains, 57 and 57-AfimZ, can be cultured for 18-24 hours at 37°C on L-agar plates. To obtain a seed culture, 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. Then, 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. The amount of L-leucine can be measured by paper chromatography (liquid phase composition: butanol - acetic acid - water = 4:1:1).

[0158] The composition of the fermentation medium (g/l) (pH 7.2) is as follows:

[0159] Glucose and CaC03 are sterilized separately.

Example 18. Production of L-histidine bv E. coli 80-AfimZ

[0160] To test the effect of inactivation of the fimZ gene on L-histidine production, DNA fragments from the chromosome of the above-described £. coli MG 1655 AfimZ::cat can be transferred to the histidine-producing £. coli strain 80 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 80-AfimZ.

[0161] Both £. coli strains, 80 and 80-AfimZ, can each be cultured in L-broth for 6 h at 29°C. Then, 0.1 ml of obtained culture can each be inoculated into 2 ml of fermentation medium in a 20x200-mm test tube and cultivated for 65 hours at 29°C with shaking on a rotary shaker (350 rpm). After cultivation, the amount of histidine which accumulates in the medium can be determined by paper chromatography. The paper can be developed with a mobile phase consisting of n-butanol :aceticacid :water=4 :1:1 (v/v). A solution of ninhydrin (0.5%) in acetone can be used as a visualizing reagent.

[0162] The composition of the fermentation medium (g/l) is as follows (pH 6.0):

[0163] Glucose, proline, betaine and CaC03 are sterilized separately. The pH is adjusted to 6.0 before sterilization. Example 19. Production of L-qlutamate by E. coli VL334thrC+-AfimZ

[0164] To test the effect of inactivation of the fimZ gene on L-glutamate production, DNA fragments from the chromosome of the above-described E. coli strain MG 1655 A-fimZ::cat can be transferred to the E. coli L-glutamate-producing strain VL334thrC+ (EP 1172433) by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain VL334thrC+-AfimZ. Both strains, VL334thrC+ and VL334thrC+-AfimZ, can be grown for 18-24 hours at 37°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/l), KH2P04 (2g/l), MgS04 (1 g/l), thiamine (0.1 mg/ml), L-isoleucine (70 μg/ml), and CaCOs (25 g/l). The pH is adjusted to 7.2. Glucose and CaC03 are sterilized separately. Cultivation can be carried out at 30°C for 3 days with shaking. After the cultivation, the amount of L-glutamic acid which is produced can be determined by paper chromatography (liquid phase composition of butanol-acetic acid-water=4:1:1) with subsequent staining by ninhydrin (1% solution in acetone) and further elution of the compounds in 50% ethanol with 0.5% CdCI2.

Example 20. Production of L- phenylalanine bv E. coli AJ12739-AfimZ

[0165] To test the effect of inactivation of the fimZ gene on L-phenylalanine production, DNA fragments from the chromosome of the above-described E. coli MG1655 Afimz::cat can be transferred to the phenylalanine-producing E. coli strain AJ12739 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain AJ12739-AfimZ.

[0166] Both strains, AJ12739-AfimZ and AJ 12739, can be cultivated at 37°C for 18 hours in a nutrient broth, and 0.3 ml of the obtained culture 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 10x15-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. The Sorbfil plates can be developed with a mobile phase consisting of propan-2-ol : ethylacetate : 25% aqueous ammonia : water = 40 : 40 : 7 : 16 (v/v). A solution of ninhydrin (2%) in acetone can be used as a visualizing reagent.

[0167] The composition of the fermentation medium (g/l) is as follows:

[0168] Glucose and magnesium sulfate are sterilized separately. CaC03 is dry-heat sterilized at 180°for2 hours. The pH is adjusted to 7.0.

Example 21. Production of L- tryptophan by £ coli SV164 (pGH5)- AfimZ

[0169] To test the effect of inactivation of the fimZ gene on L-tryptophan production, DNA fragments from the chromosome of the above-described £ coli strain MG1655 AfimZ::cat can be transferred to the tryptophan-producing £. coli strain SV164 (pGH5) by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain SV164(pGH5)-AfimZ.

[0170] Both strains, SV164(pGH5)-AfimZ and SV164(pGH5), can be cultivated with shaking at 37°C for 18 hours in 3 ml of nutrient broth supplemented with tetracycline (20 mg/l, marker of pGH5 plasmid). The obtained cultures (0.3 ml each) can be inoculated into 3 ml of a fermentation medium containing tetracycline (20 mg/l in 20 x 200-mm test tubes, and cultivated at 37°C for 48 hours with a rotary shaker at 250 rpm. After cultivation, the amount of tryptophan which accumulates in the medium can be determined by TLC as described in Example 8. The fermentation medium components are listed in Table 2, but shoud be sterilized in separate groups (A, B, C, D, E, F, and H), as shown, to avoid adverse interactions during sterilization.

Example 22. Production of L-proline by E. coli 702ilvA- AfimZ

[0171] To test the effect of inactivation of the fimZ gene on L-proline production, DNA frag ments from the chromosome of the above-described £. coli strain MG1655 AfimZ::cat can be transferred to the proline-producing £. coli strain 702ilvA by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain 702ilvA-AfimZ. The strain 702ilvA has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on July 18,2000 under accession number VKPM B-8012 and then converted to a deposit under the Budapest Treaty on May 18, 2001.

[0172] Both £. coli strains 702ilvA and 702i1vA-AfimZ, can be grown for 18-24 hours at 37°C on L-agar plates. Then, these strains can be cultivated under the same conditions as in Example 8.

Example 23. Production of L-arainine by £. coli 382-AfimZ

[0173] To test the effect of inactivation of the ffmZgeneon L-arginine production, DNA fragments from the chromosome of the above-described £. coli strain MG1655 AfimZ::cat can be transferred to the arginine-producing £. coli strain 382 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 382-AfimZ.

[0174] Both strains, 382-AfimZ and 382, can be 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 3 ml of a fermentation medium in 20 x 200-mm test tubes and cultivated at 32°C for 48 hours on a rotary shaker.

[0175] After the cultivation, the amount of L-arginine which accumulates in the medium can be determined by paper chromatography using the following mobile phase: butanol: acetic acid : water = 4 : 1 : 1 (v/v). 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 CdCI2, and the amount of L-arginine can be estimated spectrophotometrically at540nm.

[0176] The composition of the fermentation medium (g/l) is as follows:

[0177] Glucose and magnesium sulfate are sterilized separately. CaC03 is dry-heat sterilized at 180 °C for 2 hours. The pH is adjusted to 7.0.

Example 24. Elimination of the Cm resistance gene /03/06061 from the chromosome of L-amino acid-producing £. coli strains.

[0178] The Cm resistance gene (cat gene) can be eliminated from the chromosome of the L-amino acid-producing strain using the int-xis system. For that purpose, an L-amino acid-producing strain having DNA fragments from the chromosome of the above-described E. coli strain MG1655 AsfmACDHF-fimZ::cat or MG1655 AfimZ::cat transferred by P1 transduction (see Examples 3-23), can be transformed with plasmid pMWts-Int/Xis. Transformant clones can be selected on the LB-medium containing 100 μg/ml of ampicillin. Plates can be incubated overnight at30°C. Transformant clones can be cured from the cat gene by spreading the separate colonies at 37°C (at that temperature repressor Cits is partially inactivated and transcription of the intlxis genes is derepressed) followed by selection of CmsApR variants. Elimination of the cat gene from the chromosome of the strain can be verified by PCR. Locus-specific primers P23 (SEQ ID NO:39) and P24 (SEQ ID NO:40) can be used in PCR for the verification. Conditions for PCR verification can be as described above. The PCR product obtained in reaction with cells having the eliminated cat gene as a template, should be ~0.1 kbp in length. Thus, the L-amino acid-producing strain with the inactivated sfmACDHF-fimZ cluster or fimZ gene and eliminated cat gene can be obtained.

Industrial Applicability [0179] According to the present invention, production of L-amino acid of a bacterium of the Enterobacteriaceae family can be enhanced.

SEQUENCE LISTING

[0180] <110> Ajinomoto Co., Inc..

<120>AMETHOD FOR PRODUCING AN L-AMINO ACID USING ABACTERIUM OFTHE ENTEROBACTERIACEAE FAMILY WITH ATTENUATED EXPRESSION OF THE sfmACDFH-fimZ CLUSTER OR THE fimZ GENE <130> C643-C7009 <150> RU2006112624 <151 > 2006-04-18 <150> US60/829923 <151 > 2006-10-18 <160> 40 <170> Patentln version 3.1 <210> 1 <211> 576

<212> DNA <213> Escherichia coli <220> <221 > CDS <222> (1)..(576) <400> 1 gtg gag teg ata aat gag att gaa gga ata tat atg aaa tta aga ttt 48

Val Glu Ser Ile Asn Glu Ile Glu Gly Ile Tyr Met Lys Leu Arg Phe 15 10 15 att teg tet gcg ctg get gee gea eta ttc gee get aeg ggt agt tat 96

Ile Ser Ser Ala Leu Ala Ala Ala Leu Phe Ala Ala Thr Gly Ser Tyr 20 25 30 get gee gtt gta gat ggc ggt aca att cac ttt gaa ggc gaa ctg gtg 144

Ala Ala Val Val Asp Gly Gly Thr lie His Phe Glu Gly Glu Leu Val 35 40 45 aat get gee tgt tea gtg aat act gac teg gca gac cag gtt gtc aca 192

Asn Ala Ala Cys Ser Val Asn Thr Asp Ser Ala Asp Gin Val Val Thr 50 55 60 etc ggt caa tat cgt ace gat att ttc aat get gtt ggt aat ace tet 240

Leu Gly Gin Tyr Arg Thr Asp lie Phe Asn Ala Val Gly Asn Thr Ser 65 70 75 80 gca tta att cca ttc ace att cag ttg aac gac tgc gat cct gtt gtt 288

Ala Leu Ile Pro Phe Thr Ile Gin Leu Asn Asp Cys Asp Pro Val Val 85 90 95 gee get aat get gee gtt gca ttt tet ggt cag get gat gca ate aat 336

Ala Ala Asn Ala Ala Val Ala Phe Ser Gly Gin Ala Asp Ala lie Asn 100 105 110 gat aat tta ttg gee att gca tee agt ace aat aca aca aca gca aeg 384

Asp Asn Leu Leu Ala lie Ala Ser Ser Thr Asn Thr Thr Thr Ala Thr 115 120 125 ggt gtc ggt att gaa ata ett gat aat aca tee gca att etc aaa cct 432

Gly Val Gly Ile Glu Ile Leu Asp Asn Thr Ser Ala Ile Leu Lys Pro 130 135 140 gat ggg aat age ttc tea ace aac cag aac ttg ate ccc ggg ace aac 480

Asp Gly Asn Ser Phe Ser Thr Asn Gin Asn Leu He Pro Gly Thr Asn 145 150 155 160 gtt ett cat ttt tet gca cgt tat aaa ggc ace ggt aca agt gca tea 528

Val Leu His Phe Ser Ala Arg Tyr Lys Gly Thr Gly Thr Ser Ala Ser 165 170 175 gca ggg caa gca aat get gac gcg act ttt att atg aga tat gaa taa 576

Ala Gly Gin Ala Asn Ala Asp Ala Thr Phe He Met Arg Tyr Glu 180 185 190 <210>2 <211 > 191

<212> PRT <213> Escherichia coli <400>2

Val Glu Ser Ile Asn Glu Ile Glu Gly Ile Tyr Met Lys Leu. Arg Phe 15 10 15

Ile Ser Ser Ala Leu Ala Ala Ala Leu Phe Ala Ala Thr Gly Ser Tyr 20 25 30

Ala Ala Val Val Asp Gly Gly Thr lie His Phe Glu Gly Glu Leu Val 35 40 45

Asn Ala Ala Cys Ser Val Asn Thr Asp Ser Ala Asp Gin Val Val Thr 50 55 60

Leu Gly Gin Tyr Arg Thr Asp lie Phe Asn Ala Val Gly Asn Thr Ser 65 70 75 80

Ala Leu Ile Pro Phe Thr Ile Gin Leu Asn Asp Cys Asp Pro Val Val 85 90 95

Ala Ala Asn Ala Ala Val Ala Phe Ser Gly Gin Ala Asp Ala lie Asn 100 105 110

Asp Asn Leu Leu Ala lie Ala Ser Ser Thr Asn Thr Thr Thr Ala Thr 115 120 125

Gly Val Gly Ile Glu Ile Leu Asp Asn Thr Ser Ala Ile Leu Lys Pro 130 135 140

Asp Gly Asn Ser Phe Ser Thr Asn Gin Asn Leu lie Pro Gly Thr Asn 145 150 155 160

Val Leu His Phe Ser Ala Arg Tyr Lys Gly Thr Gly Thr Ser Ala Ser 165 170 175

Ala Gly Gin Ala Asn Ala Asp Ala Thr Phe lie Met Arg Tyr Glu 180 185 190 <210>3 <211> 693

<212> DNA <213> Escherichia coli <220> <221 > CDS <222> (1)..(693) <400> 3 atg atg act aaa ata aag tta ttg atg etc att ata ttt tat tta ate 48

Met Met Thr Lys Ile Lys Leu Leu Met Leu Ile Ile Phe Tyr Leu lie 15 10 15 att teg gee age gee cat get gee gga ggg ate gca tta ggt gee aeg 96

He Ser Ala Ser Ala His Ala Ala Gly Gly Ile Ala Leu Gly Ala Thr 20 25 30 egt att att tat ccc get gat get aaa cag act geg gta tgg att aga 144

Arg He Ile Tyr Pro Ala Asp Ala Lys Gin Thr Ala Val Trp He Arg 35 40 45 aat age cat ace aat gag ege ttt ctg gtc aat teg tgg att gaa aac 192

Asn Ser His Thr Asn Glu Arg Phe Leu Val Asn Ser Trp Ile Glu Asn 50 55 60 age age ggt gta aaa gaa aag tea tte atc att aca cég cca ctg ttt 240

Ser Ser Gly Val Lys Glu Lys Ser Phe Ile He Thr Pro Pro Leu Phe 65 70 75 80 gtt agt gaa ccc aaa age gaa aat act ttg egt att att tac acc ggt 288

Val Ser Glu Pro Lys Ser Glu Asn Thr Leu Arg He He Tyr Thr Gly 85 90 95 cca cég ctg gca gca gat egt gag tet ctg ttc tgg atg aat gtt aag 336

Pro Pro Leu Ala Ala Asp Arg Glu Ser Leu Phe Trp Met Asn Val Lys 100 105 110 aeg atc cet teg gta gat aaa aat gca ttg aac ggc agg aat gtt ttg 384

Thr He Pro Ser Val Asp Lys Asn Ala Leu Asn Gly‘ Arg Asn Val Leu 115 120 125 caa ctg geg att tta teg ege atg aaa tta ttt etc egt cca att caa 432

Gin Leu Ala He Leu Ser Arg Met Lys Leu Phe Leu Arg Pro Ile Gin 130 135 140 tta caa gaa tta ccc gca gaa geg ccg gac aca etc aag ttt teg ega 480

Leu Gin Glu Leu Pro Ala Glu Ala Pro Asp Thr Leu Lys Phe Ser Arg 145 150 155 160 tcc ggt aae tat ate aat gtt cat aat cca tea cet ttt tat gtc acc 528

Ser Gly Asn Tyr Ile Asn Val His Asn Pro Ser Pro Phe Tyr Val Thr 165 170 175 ctg gtt aac tta caa gtg ggc age caa aag ttg ggg aat get atg get 576

Leu Val Asn Leu Gin Val Gly Ser Gin Lys Leu Gly Asn Ala Met Ala 180 185 190 gca ccc aga gtt aat tea caa att ccc tta ccc tea gga gtg cag gga 624

Ala Pro Arg Val Asn Ser Gin He Pro Leu Pro Ser Gly Val Gin Gly 195 200 205 aag ctg aaa ttt cag acc gtt aat gat tat ggt tea gta act ccg gtc 672

Lys Leu Lys Phe Gin Thr Val Asn Asp Tyr Gly Ser Val Thr Pro Val 210 215 220 aga gaa gtg aac tta aac taa 693

Arg Glu Val Asn Leu Asn 225 230 <210> 4 <211 >230

<212> PRT <213> Escherichia coli <400>4

Met Met Thr Lys He Lys Leu Leu Met Leu He He Phe Tyr Leu He 15 10 15

He Ser Ala Ser Ala His Ala Ala Gly Gly He Ala Leu Gly Ala Thr 20 25 30

Arg He He Tyr Pro Ala Asp Ala Lys Gin Thr Ala Val Trp Ile Arg 35 40 45

Asn Ser His Thr Asn Glu Arg Phe Leu Val Asn Ser Trp Ile Glu Asn 50 55 60

Ser Ser Gly Val Lys Glu Lys Ser Phe Ile Ile Thr Pro Pro Leu Phe 65 70 75 80

Val Ser Glu Pro Lys Ser Glu Asn Thr Leu Arg Ile Ile Tyr Thr Gly 85 90 95

Pro Pro Leu Ala Ala Asp Arg Glu Ser Leu Phe Trp Met Asn Val Lys 100 105 110

Thr Ile Pro Ser Val Asp Lys Asn Ala Leu Asn Gly Arg Asn Val Leu 115 120 125

Gin Leu Ala Ile Leu Ser Arg Met Lys Leu Phe Leu Arg Pro Ile Gin 130 135 140

Leu Gin Glu Leu Pro Ala Glu Ala Pro Asp Thr Leu Lys Phe Ser Arg 145 150 155 160

Ser Gly Asn Tyr Ile Asn Val His Asn Pro Ser Pro Phe Tyr Val Thr 165 170 175

Leu Val Asn Leu Gin Val Gly Ser Gin Lys Leu Gly Asn Ala Met Ala 180 185 190

Ala Pro Arg Val Asn Ser Gin Ile Pro Leu Pro Ser Gly Val Gin Gly 195 200 205

Lys Leu Lys Phe Gin Thr Val Asn Asp Tyr Gly Ser Val Thr Pro Val 210 215 220

Arg Glu Val Asn Leu Asn 225 230 <210>5 <211> 2604

<212> DNA <213> Escherichia coli <220> <221 > CDS <222> (1)..(2604) <400>5 atg aaa ata ccc act act acg gat att ccg cag agg tat acc tgg tgt 48

Met Lys Ile Pro Thr Thr Thr Asp Ile Pro Gin Arg Tyr Thr Trp Cys 15 10 15 ctg gcc gga att tgt tat tea tet ett gcc att tta ccc tcc ttt tta 96

Leu Ala Gly Ile Cys Tyr Ser Ser Leu Ala Ile Leu Pro Ser Phe Leu 20 25 30 agc tat geg gaa agt tat ttc aac ccg gca ttt tta tta gag aat ggc 144

Ser Tyr Ala Glu Ser Tyr Phe Asn Pro Ala Phe Leu Leu Glu Asn Gly 35 40 45 aca tcc gtt get gat tta teg ege ttt gag aga ggt aat cat caa cct 192

Thr Ser Val Ala Asp Leu Ser Arg Phe Glu Arg Gly Asn His Gin Pro 50 55 60 geg ggc gtg tat egg gtg gat ctc tgg egt aat gat gag ttc att ggt 240

Ala Gly Val Tyr Arg Val Asp Leu Trp Arg Asn Asp Glu Phe Ile Gly 65 70 75 80 teg cag gat atc gta ttt gaa teg aca aca gaa aat aca ggt gat aaa 288

Ser Gin Asp Ile Val Phe Glu Ser Thr Thr Glu Asn Thr Gly Asp Lys 85 90 95 tea ggt ggg tta atg ccc tgt ttt aac cag gta ett ett gaa ega att 336

Ser Gly Gly Leu Met Pro Cys Phe Asn Gin Val Leu Leu Glu Arg Ile 100 105 HO ggc ctt aat age agt gca ttt ccc gag tta gcc cag cag caa aac aat 384

Gly Leu Asn Ser Ser Ala Phe Pro Glu Leu Ala Gin Gin Gin Asn Asn 115 120 125 aaa tgc ate aat tta ctg aaa get gta cct gat gcc aca att aac ttt 432

Lys Cys Ile Asn Leu Leu Lys Ala Val Pro Asp Ala Thr lie Asn Phe 130 135 140 gat ttt gca geg atg ege ctg aac ate act att cct cag ata geg ttg 480

Asp Phe Ala Ala Met Arg Leu Asn Ile Thr Ile Pro Gin Ile Ala Leu 145 150 155 160 ttg agt age get cac ggt tac att ccg cct gaa gag tgg gat gaa ggt 528

Leu Ser Ser -Ala His Gly Tyr lie Pro Pro Glu Glu Trp Asp Glu Gly 165 170 175 att cct get tta etc ctg aat tat aat ttc acc ggt aac aga ggt aat 576

Ile Pro Ala Leu Leu Leu Asn Tyr Asn Phe Thr Gly Asn Arg Gly Asn 180 185 190 ggt aac gat age tat ttt ttt agt gag etc age ggg att aat att ggc 624

Gly Asn Asp Ser Tyr Phe Phe Ser Glu Leu Ser Gly Ile Asn Ile Gly 195 200 205 ccg tgg cgt tta ege aac aat ggt tcc tgg aac tat ttt ege gga aat 672

Pro Trp Arg Leu Arg Asn Asn Gly Ser Trp Asn Tyr Phe Arg Gly Asn 210 215 220 gga tat cat tea gaa cag tgg aat aat att ggc acc tgg gta cag ege 720

Gly Tyr His Ser Glu Gin Trp Asn Asn Ile Gly Thr Trp Val Gin Arg 225 230 235 240 gcc att att ccg ctg aaa agt gaa ctg gta atg gga. gac ggc aat aca 768

Ala Ile Ile Pro Leu Lys Ser Glu Leu Val Met Gly Asp Gly Asn Thr 245 250 255 gga agt gat att ttc gat ggc gtt gga ttt cgt ggt gta egg ctt tat 816

Gly Ser Asp Ile Phe Asp Gly Val Gly Phe Arg Gly Val Arg Leu Tyr 260 265 270 tet tet gat. aat atg tat cct gat age cag caa ggg ttt gcc cca aeg 864

Ser Ser Asp Asn Met Tyr Pro Asp Ser Gin Gin Gly Phe Ala Pro Thr 275 280 285 gta cgt ggg att gcc cgt aeg gcg gcc cag cta aeg att cgg caa aat 912

Val Arg Gly Ile Ala Arg Thr Ala Ala Gin Leu Thr Ile Arg Gin Asn 290 295 300 ggt ttt att ate tat caa age tat gtt tcc ccc ggc get ttt gaa att 960

Gly Phe Ile Ile Tyr Gin Ser Tyr Val Ser Pro Gly Ala Phe Glu Ile 305 310 315 320 aca gat ttg cac ccg aca tet tea aat ggc gat ctg gac gtc acc atc 1008

Thr Asp Leu His Pro Thr Ser Ser Asn Gly Asp Leu Asp Val Thr Ile 325 330 335 gac gag ege gat ggc aat cag cag aat tac aca att ccg tat tea aca 1056

Asp Glu Arg Asp Gly Asn Gin Gin Asn Tyr Thr Ile Pro Tyr Ser Thr 340 345 350 gtg cca att tta caa ege gaa ggg cgt ttc aaa ttt gac ctg aeg gcg 1104

Val Pro Ile Leu Gin Arg Glu Gly Arg Phe Lys Phe Asp Leu Thr Ala 355 360 365 ggc gat ttt cgt age ggt aat agt cag caa tea teg cct ttc ttt ttt 1152

Gly Asp Phe Arg Ser Gly Asn Ser Gin Gin Ser Ser Pro Phe Phe Phe 370 375 380 cag ggt aeg gca etc ggc ggt tta cca cag gaa ttt act gcc tac ggc 1200

Gin Gly Thr Ala Leu Gly Gly Leu Pro Gin Glu Phe Thr Ala Tyr Gly 385 390 395 400 ggg acg caa tta tct gcc aat tac acc gcc ttt tta tta ggg ctg ggg 1248

Gly Thr Gin Leu Ser Ala Asn Tyr Thr Ala Phe Leu Leu Gly Leu Gly 405 410 415 cgc aat etc ggg aac tgg ggc gca gtg teg ctg gat gta acg cat geg 1296

Arg Asn Leu Gly Asn Trp Gly Ala Val Ser Leu Asp Val Thr His Ala 420 425 430 cgc agt cag tta gcc gac gcc agt cgt cat gag ggg gat tct att cgc 1344

Arg Ser Gin Leu Ala Asp Ala Ser Arg His Glu Gly Asp Ser Ile Arg 435 440 445 ttc etc tat geg aaa teg atg aac acc ttc ggc acc aat ttt cag tta 1392

Phe Leu Tyr Ala Lys Ser Met Asn Thr Phe Gly Thr Asn Phe Gin Leu 450 455 460 atg ggt tac cgc tat teg aca caa ggt ttt tat acc ett gat gat gtt 1440

Met Gly Tyr Arg Tyr Ser Thr Gin Gly Phe Tyr Thr Leu Asp Asp Val 465 470 475 480 geg tat cgt ega atg gag ggg tac gaa tat gat tac gac ggt gag cat 1488

Ala Tyr Arg Arg Met Glu Gly Tyr Glu Tyr Asp Tyr Asp Gly Glu His 485 490 495 cgc gat gaa ccg ata ate gtg aat tac cac aat tta cgc ttt agc cgt 1536

Arg Asp Glu Pro Ile Ile Val Asn Tyr His Asn Leu Arg Phe Ser Arg 500 505 510 aaa gac cgt ttg cag tta aat gtt tea caa tea ett aat gac ttt ggc 1584

Lys Asp Arg Leu Gin Leu Asn Val Ser Gin Ser Leu Asn Asp Phe Gly 515 520 525 teg ett tat att tct ggt acc cat caa aaa tac tgg aat act teg gat 1632

Ser Leu Tyr Ile Ser Gly Thr His Gin Lys Tyr Trp Asn Thr Ser Asp 530 535 540 tea gat acg tgg tat cag gtg ggg tat acc agc agc tgg gtt ggc atc 1680

Ser Asp Thr Trp Tyr Gin Val Gly Tyr Thr Ser Ser Trp Val Gly Ile 545 550 555 560 agt tat teg etc tea ttt teg tgg aat gaa tct gta ggg atc ccc gat 1728

Ser Tyr Ser Leu Ser Phe Ser Trp Asn Glu Ser Val Gly Ile Pro Asp 565 570 575 aac gaa cgt att gtc gga ett aat gtt tea gtg cct ttc aat gtt ttg 1776

Asn Glu Arg Ile Val Gly Leu Asn Val Ser Val Pro Phe Asn Val Leu 580 585 590 acc aaa cgt cgc tac acc egg gaa aat geg etc gac cgc get tat gcc 1824

Thr Lys Arg Arg Tyr Thr Arg Glu Asn Ala Leu Asp Arg Ala Tyr Ala 595 600 605 tcc ttt aac gcc aac cgt aac agc aac ggg caa aat agc tgg ctg gca 1872

Ser Phe Asn Ala Asn Arg Asn Ser Asn Gly Gin Asn Ser Trp Leu Ala 610 615 620 ggt gta ggt ggg acc tta ctg gaa ggc cac aac ctg agt tat cac gta 1920

Gly Val Gly Gly Thr Leu Leu Glu Gly His Asn Leu Ser Tyr His Val 625 630 635 640 agc cag ggt gat acc teg aat aat ggg tac acg ggc agc gcc acg gca 1968

Ser Gin Gly Asp Thr Ser Asn Asn Gly Tyr Thr Gly Ser Ala Thr Ala 645 650 655 aac tgg cag gcc get tac ggt acg ctg ggg ggc ggg tat aac tac gac 2016

Asn Trp Gin Ala Ala Tyr Gly Thr Leu Gly Gly Gly Tyr Asn Tyr Asp 660 665 670 cgc gat caa cat gac gtt aac tgg cag ctg tct ggc ggt gtg gtc ggg 2064

Arg Asp Gin His Asp Val Asn Trp Gin Leu Ser Gly Gly Val Val Gly 675 680 685 cat gaa aat ggc ata acg ctg agc cag cct tta ggg gat acc aat gtt 2112

His Glu Asn Gly Ile Thr Leu Ser Gin Pro Leu Gly Asp Thr Asn Val 690 695 700 ttg att aaa gcg cct ggc gca ggc ggt gta cgc att gaa aat caa act 2160

Leu Ile Lys Ala Pro Gly Ala Gly Gly Val Arg Ile Glu Asn Gin Thr 705 710 715 720 ggc att tta acc gac tgg cgc ggc tat gcg gtg atg ctg tat gcc acg 2208

Gly Ile Leu Thr Asp Trp Arg Gly Tyr Ala Val Met Leu Tyr Ala Thr 725 730 735 gtt tat egg tat aac cgt atc gcg ctt gat acc aat acg atg ggg aat 2256

Val Tyr Arg Tyr Asn Arg Ile Ala Leu Asp Thr Asn Thr Met Gly Asn 740 745 750 tcc atc gat gtt gaa aaa aat att age agc gtt gtg ccg acg caa ggc 2304

Ser Ile Asp Val Glu Lys Asn Ile Ser Ser Val Val Pro Thr Gin Gly 755 760 765 gcg ttg gtt cgt gcc aat ttt gat acc cgc ata ggc gtg cgg gcg etc 2352

Ala Leu Val Arg Ala Asn Phe Asp Thr Arg Ile Gly Val Arg Ala Leu 770 775 780 att acc gtt acc cag ggc gga aaa ccg gtg ccg ttt gga tea ctg gta 2400

Ile Thr Val Thr Gin Gly Gly Lys Pro Val Pro Phe Gly Ser Leu Val 785 790 795 800 cgg gaa aac agt acc gga ata acc agt atg gtg ggt gat gac ggg caa 2448

Arg Glu Asn Ser Thr Gly Ile Thr Ser Met Val Gly Asp Asp Gly Gin 805 810 815 gtt tat tta agt ggt gcg cca ttg tet ggt gaa tta ctg gtt cag tgg 2496

Val Tyr Leu Ser Gly Ala Pro Leu Ser Gly Glu Leu Leu Val Gin Trp 820 825 830 gga gac ggc gcg aac tea cgc tgc att gcg cac tat gta ttg ccg aag 2544

Gly Asp Gly Ala Asn Ser Arg Cys Ile Ala His Tyr Val Leu Pro Lys 835 840 845 caa agc tta cag caa gcc gtc act gtt att teg gca gtt tgc aca cat 2592

Gin Ser Leu Gin Gin Ala Val Thr Val Ile Ser Ala Val Cys Thr His 850 855 860 cct ggc tea taa 2604

Pro Gly Ser 865 <210> 6 <211> 867

<212> PRT <213> Escherichia coli <400>6

Met Lys Ile Pro Thr Thr Thr Asp Ile Pro Gin Arg Tyr Thr Trp Cys 15 10 15

Leu Ala Gly Ile Cys Tyr Ser Ser Leu Ala Ile Leu Pro Ser Phe Leu 20 25 30

Ser Tyr Ala Glu Ser Tyr Phe Asn Pro Ala Phe Leu Leu Glu Asn Gly 35 40 45

Thr Ser Val Ala Asp Leu Ser Arg Phe Glu Arg Gly Asn His Gin Pro 50 55 60

Ala Gly Val Tyr Arg Val Asp Leu Trp Arg Asn Asp Glu Phe Ile Gly 65 70 75 80

Ser Gin Asp Ile Val Phe Glu Ser Thr Thr Glu Asn Thr Gly Asp Lys 85 90 95

Ser Gly Gly Leu Met Pro Cys Phe Asn Gin Val Leu Leu Glu Arg Ile 100 105 HO

Gly Leu Asn Ser Ser Ala Phe Pro Glu Leu Ala Gin Gin Gin Asn Asn 115 120 125

Lys Cys Ile Asn Leu Leu Lys Ala Val Pro Asp Ala Thr lie Asn Phe 130 135 140

Asp Phe Ala Ala Met Arg Leu Asn Ile Thr Ile Pro Gin Ile Ala Leu 145 150 155 160

Leu Ser Ser Ala His Gly Tyr lie Pro Pro Glu Glu Trp Asp Glu Gly 165 170 175

Ile Pro Ala Leu Leu Leu Asn Tyr Asn Phe Thr Gly Asn Arg Gly Asn 180 185 190

Gly Asn Asp Ser Tyr Phe Phe Ser Glu Leu Ser Gly Ile Asn Ile Gly 195 200 205

Pro Trp Arg Leu Arg Asn Asn Gly Ser Trp Asn Tyr Phe Arg Gly Asn 210 215 220

Gly Tyr His Ser Glu Gin Trp Asn Asn Ile Gly Thr Trp Val Gin Arg 225 230 235 240

Ala Ile Ile Pro Leu Lys Ser Glu Leu Val Met Gly Asp Gly Asn Thr 245 250 255

Gly Ser Asp Ile Phe Asp Gly Val Gly Phe. Arg Gly Val Arg Leu Tyr 260 265 270

Ser Ser Asp Asn Met Tyr Pro Asp Ser Gin Gin Gly Phe Ala Pro Thr 275 280 285

Val Arg Gly Ile Ala Arg Thr Ala Ala Gin Leu Thr Ile Arg Gin Asn 290 295 300

Gly Phe Ile Ile Tyr Gin Ser Tyr Val Ser Pro Gly Ala Phe Glu Ile 305 310 315 320

Thr Asp Leu His Pro Thr Ser Ser Asn Gly Asp Leu Asp Val Thr Ile 325 330 335

Asp Glu Arg Asp Gly Asn Gin Gin Asn Tyr Thr Ile Pro Tyr Ser Thr 340 345 350

Val Pro Ile Leu Gin Arg Glu Gly Arg Phe Lys Phe Asp Leu Thr Ala 355 360 365

Gly Asp Phe Arg Ser Gly Asn Ser Gin Gin Ser Ser Pro Phe Phe Phe 370 375 380

Gin Gly Thr Ala Leu Gly Gly Leu Pro Gin Glu Phe Thr Ala Tyr Gly 385 390 395 400

Gly Thr Gin Leu Ser Ala Asn Tyr Thr Ala Phe Leu Leu Gly Leu Gly 405 410 415

Arg Asn Leu Gly Asn Trp Gly Ala Val Ser Leu Asp Val Thr His Ala 420 425 430

Arg Ser Gin Leu Ala Asp Ala Ser Arg His Glu Gly Asp Ser Ile Arg 435 440 445

Phe Leu Tyr Ala Lys Ser Met Asn Thr Phe Gly Thr Asn Phe Gin Leu 450 455 460

Met Gly Tyr Arg Tyr Ser Thr Gin Gly Phe Tyr Thr Leu Asp Asp Val 465 470 475 480

Ala Tyr Arg Arg Met Glu Gly Tyr Glu Tyr Asp Tyr Asp Gly Glu His 485 490 495

Arg Asp Glu Pro Ile Ile Val Asn Tyr His Asn Leu Arg Phe Ser Arg 500 505 510

Lys Asp Arg Leu Gin Leu Asn Val Ser Gin Ser Leu Asn Asp Phe Gly 515 520 525

Ser Leu Tyr Ile Ser Gly Thr His Gin Lys Tyr Trp Asn Thr Ser Asp 530 535 540

Ser Asp Thr Trp Tyr Gin Val Gly Tyr Thr Ser Ser Trp Val Gly Ile 545 550 555 . 560

Ser Tyr Ser Leu Ser Phe Ser Trp Asn Glu Ser Val Gly Ile Pro Asp 565 570 575

Asn Glu Arg Ile Val Gly Leu Asn Val Ser Val Pro Phe Asn Val Leu 580 585 590

Thr Lys Arg Arg Tyr Thr Arg Glu Asn Ala Leu Asp Arg Ala Tyr Ala 595 600 605

Ser Phe Asn Ala Asn Arg Asn Ser Asn Gly Gin Asn Ser Trp Leu Ala 610 615 620

Gly Val Gly Gly Thr Leu Leu Glu Gly His Asn Leu Ser Tyr His Val 625 630 635 640

Ser Gin Gly Asp Thr Ser Asn Asn Gly Tyr Thr Gly Ser Ala Thr Alá 645 650 655

Asn Trp Gin Ala Ala Tyr Gly Thr Leu Gly Gly Gly Tyr Asn Tyr Asp 660 665 670

Arg Asp Gin His Asp Val Asn Trp Gin Leu Ser Gly Gly Val Val Gly 675 680 685

His Glu Asn Gly Ile Thr Leu Ser Gin Pro Leu Gly Asp Thr Asn Val 690 695 700

Leu Ile Lys Ala Pro Gly Ala Gly Gly Val Arg Ile Glu Asn Gin Thr 705 710 715 720

Gly Ile Leu Thr Asp Trp Arg Gly Tyr Ala Val Met Leu Tyr Ala Thr 725 730 735

Val Tyr Arg Tyr Asn Arg Ile Ala Leu Asp Thr Asn Thr Met Gly Asn 740 745 750

Ser Ile Asp Val Glu Lys Asn Ile Ser Ser Val Val Pro Thr Gin Gly 755 760 765

Ala Leu Val Arg Ala Asn Phe Asp Thr Arg Ile Gly Val Arg Ala Leu 770 775 780

Ile Thr Val Thr Gin Gly Gly Lys Pro Val Pro Phe Gly Ser Leu Val 785 790 795 800

Arg Glu Asn Ser Thr Gly Ile Thr Ser Met Val Gly Asp Asp Gly Gin 805 810 815

Val Tyr Leu Ser Gly Ala Pro Leu Ser Gly Glu Leu Leu Val Gin Trp 820 825 830

Gly Asp Gly Ala Asn Ser Arg Cys Ile Ala His Tyr Val Leu Pro Lys 835 840 845

Gin Ser Leu Gin Gin Ala Val Thr Val Ile Ser Ala Val Cys Thr His 850 855 860

Pro Gly Ser 865 <210>7 <211> 978

<212> DNA <213> Escherichia coli <220> <221 > CDS <222> (1)..(978) <400> 7 atg gca tgt ttg tgt ctg gca aac ata tcc tgg get act gtt tgt gea 48

Met Ala Cys Leu Cys Leu Ala Asn Ile Ser Trp Ala Thr Val Cys Ala 15 10 15 aat agt act ggc gta gca gaa gat gaa cac tat gat etc tea aat atc 96

Asn Ser Thr Gly Val Ala Glu Asp Glu His Tyr Asp Leu Ser Asn Ile 20 25 30 ttt aat agc acc aat aac cag cca ggg cag att gtt gtt tta ccg gaa 144

Phe Asn Ser Thr Asn Asn Gin Pro Gly Gin Ile Val Val Leu Pro Glu 35 40 45 aaa tcc ggc tgg gta ggt gtc tea gea att tgt cca ccc ggt aeg ctg 192

Lys Ser Gly Trp Val Gly Val Ser Ala Ile Cys Pro Pro Gly Thr Leu 50 55 60 gtg aat tat aca tac egt agt tat gtc acc aac ttt att gtt cag gaa 240

Val Asn Tyr Thr Tyr Arg Ser Tyr Val Thr Asn Phe Ile Val Gin Glu 65 70 75 80 act atc gat aat tat aaa tat atg caa tta cat gat tat cta tta ggt 288

Thr Ile Asp Asn Tyr Lys Tyr Met Gin Leu His Asp Tyr Leu Leu Gly 85 90 95 geg atg agt ctg gtt gat agt gtg atg gat att cag ttc ccc ccg caa 336

Ala Met Ser Leu Val Asp Ser Val Met Asp Ile Gin Phe Pro Pro Gin 100 105 110 aat tat att egg atg gga aca gat cct aac gtt teg caa aac ett cca 384

Asn Tyr Ile Arg Met Gly Thr Asp Pro Asn Val Ser Gin Asn Leu Pro 115 120 125 ttc ggg gtg atg gat tet egt tta ata ttt egt tta aag gtt att egt 432

Phe Gly Val Met Asp Ser Arg Leu Ile Phe Arg Leu Lys Val Ile Arg 130 135 140 ccc ttt att aac atg gtg gag atc ccc aga cag gtg atg ttt acc gtg 480

Pro Phe Ile Asn Met Val Glu Ile Pro Arg Gin Val Met Phe Thr Val 145 150 155 160 tat gtg aca tea aeg cct tac gat ccg ttg gtt aca cct gtt tat acc 528

Tyr Val Thr Ser Thr Pro Tyr Asp Pro Leu Val Thr Pro Val Tyr Thr 165 170 175 att agt ttt ggt ggc egg gtt gaa gta ccg caa aac tgc gaa tta aat 576

Ile Ser Phe Gly Gly Arg Val Glu Val Pro Gin Asn Cys Glu Leu Asn 180 185 190 gcc ggg cag att gtt gaa ttt gat ttt ggt gat atc ggc gca teg tta 624

Ala Gly Gin Ile Val Glu Phe Asp Phe Gly Asp Ile Gly Ala Ser Leu 195 200 205 ttt agt geg gca ggg ccg ggt aat ega cct get ggt gtc atg ccg caa 672

Phe Ser Ala Ala Gly Pro Gly Asn Arg Pro Ala Gly Val Met Pro Gin 210 215 220 acc aag age att geg gtc aaa tgt aeg aat gtt get geg cag get tat 720

Thr Lys Ser Ile Ala Val Lys Cys Thr Asn Val Ala Ala Gin Ala Tyr 225 230 235 240 tta aca atg egt ctg gaa gcc agt gcc gtt tet ggt cag geg atg gtg 768

Leu Thr Met Arg Leu Glu Ala Ser Ala Val Ser Gly Gin Ala Met Val 245 250 255 teg gac aat cag gat tta ggt ttt att gtc gcc gat cag aac gat aeg 816

Ser Asp Asn Gin Asp Leu Gly Phe Ile Val Ala Asp Gin Asn Asp Thr 260 265 270 ccg atc aeg cct aac gat ctc aat agc gtt att cct ttc egt ctg gat 864

Pro Ile Thr Pro Asn Asp Leu Asn Ser Val Ile Pro Phe Arg Leu Asp 275 280 285 gca get geg gca gcc aat gtc aca ett ege gcc tgg cct atc agt att 912

Ala Ala Ala Ala Ala Asn Val Thr Leu Arg Ala Trp Pro Ile Ser Ile 290 295 300 acc ggt caa aaa ccg acc gaa ggg ccg ttt agc geg ctg ggg tat tta 960

Thr Gly Gin Lys Pro Thr Glu Gly Pro Phe Ser Ala Leu Gly Tyr Leu 305 310 315 320 ege gtc gat tat caa tga 978

Arg Val Asp Tyr Gin 325 <210> 8 <211> 325

<212> PRT <213> Escherichia coli <400>8

Met Ala Cys Leu Cys Leu Ala Asn Ile Ser Trp Ala Thr Val Cys Ala 15 10 15

Asn Ser Thr Gly Val Ala Glu Asp Glu His Tyr Asp Leu Ser Asn Ile 20 25 30

Phe Asn Ser Thr Asn Asn Gin Pro Gly Gin Ile Val Val Leu Pro Glu 35 40 45

Lys Ser Gly Trp Val Gly Val Ser Ala Ile Cys Pro Pro Gly Thr Leu 50 55 ' 60

Val Asn Tyr Thr Tyr Arg Ser Tyr Val Thr Asn Phe Ile Val Gin Glu 65 70 75 80

Thr Ile Asp Asn Tyr Lys Tyr Met Gin Leu His Asp Tyr Leu Leu Gly 85 90 95

Ala Met Ser Leu Val Asp Ser Val Met Asp Ile Gin Phe Pro Pro Gin 100 105 110

Asn Tyr Ile Arg Met Gly Thr Asp Pro Asn Val Ser Gin Asn Leu Pro 115 120 125

Phe Gly Val Met Asp Ser Arg Leu Ile Phe Arg Leu Lys Val Ile Arg 130 135 140

Pro Phe Ile Asn Met Val Glu Ile Pro Arg Gin Val Met Phe Thr Val 145 150 155 160

Tyr Val Thr Ser Thr Pro Tyr Asp Pro Leu Val Thr Pro Val Tyr Thr 165 170 175

Ile Ser Phe Gly Gly Arg Val Glu Val Pro Gin Asn Cys Glu Leu Asn 180 185 190

Ala Gly Gin Ile Val Glu Phe Asp Phe Gly Asp Ile Gly Ala Ser Leu 195 200 205

Phe Ser Ala Ala Gly Pro Gly Asn Arg Pro Ala Gly Val Met Pro Gin 210 215 220

Thr Lys Ser Ile Ala Val Lys Cys Thr Asn Val Ala Ala Gin Ala Tyr 225 230 235 240

Leu Thr Met Arg Leu Glu Ala Ser Ala Val Ser Gly Gin Ala Met Val 245 250 255

Ser Asp Asn Gin Asp Leu Gly Phe Ile Val Ala Asp Gin Asn Asp Thr 260 265 270

Pro Ile Thr Pro Asn Asp Leu Asn Ser Val Ile Pro Phe Arg Leu Asp 275 280 285

Ala Ala Ala Ala Ala Asn Val Thr Leu Arg Ala Trp Pro Ile Ser Ile 290 295 300

Thr Gly Gin Lys Pro Thr Glu Gly Pro Phe Ser Ala Leu Gly Tyr Leu 305 310 315 320

Arg Val Asp Tyr Gin 325 <210> 9 <211> 516

<212> DNA <213> Escherichia coli <220> <221 > CDS <222> (1)..(516) <400> 9 atg aga aga gta etc ttt age tgt ttc tgc ggg eta ctg tgg agt tee 48

Met Arg Arg Val Leu Phe Ser Cys Phe Cys Gly Leu Leu Trp Ser Ser 15 10 15 agt gga tgg gca gtt gac cet tta gga aeg att aat atc aat ttg cac 96

Ser Gly Trp Ala Val Asp Pro Leu Gly Thr Ile Asn Ile Asn Leu His 20 25 30 ggt aac gtt gtt gat ttc tcc tgt acc gta aac aca geg gat att gat 144

Gly Asn Val Val Asp Phe Ser Cys Thr Val Asn Thr Ala Asp Ile Asp 35 40 45 aag aeg gta gat tta ggc aga tgg cet aeg aca caa cta ctg aac get 192

Lys Thr Val Asp Leu Gly Arg Trp Pro Thr Thr Gin Leu Leu Asn Ala 50 55 60 ggc gat acc aeg gca etc gtc cct ttt agc ctg egg ctg gag gga tgt 240

Gly Asp Thr Thr Ala Leu Val Pro Phe Ser Leu Arg Leu Glu Gly Cys 65 70 75 80 cct ccg ggt tea gtt geg att tta ttt aeg gga aeg ccg gca tcc gat 288

Pro Pro Gly Ser Val Ala Ile Leu Phe Thr Gly Thr Pro Ala Ser Asp 85 90 95 acc aac ctg ctg get ctg gat gat ccc gca atg gca caa acc gtc gcc 336

Thr Asn Leu Leu Ala Leu Asp Asp Pro Ala Met Ala Gin Thr Val Ala 100 105 110 atc gaa tta egt aat agc gat ege tcc egg ctc gca ctg ggg gag geg 384

Ile Glu Leu Arg Asn Ser Asp Arg Ser Arg Leu Ala Leu Gly Glu Ala 115 120 125 age ccg act gag gaa gta gat gca aat ggc aat gtc aca cta aac ttt 432

Ser Pro Thr Glu Glu Val Asp Ala Asn Gly Asn Val Thr Leu Asn Phe 130 135 140 ttt gcc aat tat ega geg tta gcc agc ggt gtt egg cca ggt gtg geg 480

Phe Ala Asn Tyr Arg Ala Leu Ala Ser Gly Val Arg Pro Gly Val Ala 145 150 155 160 aaa geg gat geg ata ttt atg atc aat tat aat taa 516

Lys Ala Asp Ala Ile Phe Met Ile Asn Tyr Asn 165 170 <210 10 <211> 171

<212> PRT <213> Escherichia coli <400> 10

Met Arg Arg Val Leu Phe Ser Cys Phe Cys Gly Leu Leu Trp Ser Ser 15 10 15

Ser Gly Trp Ala Val Asp Pro Leu Gly Thr Ile Asn Ile Asn Leu His 20 25 30

Gly Asn Val Val Asp Phe Ser Cys Thr Val Asn Thr Ala Asp Ile Asp 35 40 45

Lys Thr Val Asp Leu Gly Arg Trp Pro Thr Thr Gin Leu Leu Asn Ala 50 55 60

Gly Asp Thr Thr Ala Leu Val Pro Phe Ser Leu Arg Leu Glu Gly Cys 65 70 75 80

Pro Pro Gly Ser Val Ala Ile Leu Phe Thr Gly Thr Pro Ala Ser Asp 85 90 95

Thr Asn Leu Leu Ala Leu Asp Asp Pro Ala Met Ala Gin Thr Val Ala 100 105 110

Ile Glu Leu Arg Asn Ser Asp Arg Ser Arg Leu Ala Leu Gly Glu Ala 115 120 125

Ser Pro Thr Glu Glu Val Asp Ala Asn Gly Asn Val Thr Leu Asn Phe 130 135 140

Phe Ala Asn Tyr Arg Ala Leu Ala Ser Gly Val Arg Pro Gly Val Ala 145 150 155 160

Lys Ala Asp Ala Ile Phe Met Ile Asn Tyr Asn 165 170 <210> 11 <211> 633

<212> DNA <213> Escherichia coli <220> <221 > CDS <222> (1)..(633) <400> 11 atg aaa cca acg teg gtg ate att atg gat act cat cct ate ate aga 48

Met Lys Pro Thr Ser Val Ile Ile Met Asp Thr His Pro Ile Ile Arg 15 10 15 atg tet att gaa gtt ctg ttg caa aaa aac agt gaa ttg cag att gtc 96

Met Ser Ile Glu Val Leu Leu Gin Lys Asn Ser Glu Leu Gin Ile Val 20 25 30 ctg aaa acg gat gat tat ege ata acc atc gat tat etc ega acc cgt 144

Leu Lys Thr Asp Asp Tyr Arg Ile Thr Ile Asp Tyr Leu Arg Thr Arg 35 40 45 cct gtt gat tta atc att atg gat ata gac ttg ccc gga aca gac ggt 192

Pro Val Asp Leu Ile Ile Met Asp Ile Asp Leu Pro Gly Thr Asp Gly 50 55 60 ttt acc ttc ctg aaa agg atc aaa caa atc cag age aca gtg aaa gtg 240

Phe Thr Phe Leu Lys Arg Ile Lys Gin Ile Gin Ser Thr Val Lys Val 65 70 75 80 tta ttt tta tea teg aaa tea gaa tgc ttt tat get ggc aga geg ata 288

Leu Phe Leu Ser Ser Lys Ser Glu Cys Phe Tyr Ala Gly Arg Ala Ile 85 90 95 caa get ggt get aac ggt ttt gtc agt aaa tgc aat gat cag aat gat 336

Gin Ala Gly Ala Asn Gly Phe Val Ser Lys Cys Asn Asp Gin Asn Asp 100 105 110 att ttt cat gcc gtt cag atg atc etc tcc gga tac acg ttt ttt ccc 384

Ile Phe His Ala Val Gin Met Ile Leu Ser Gly Tyr Thr Phe Phe Pro 115 120 125 agc gaa acg ett aac tat ata aaa agc aat aaa tgt agt acg aat agt 432

Ser Glu Thr Leu Asn Tyr Ile Lys Ser Asn Lys Cys Ser Thr Asn Ser 130 135 140 tea acg gtc act gtg cta tet aat cgt gaa gtg acc ata tta cgt tat 480

Ser Thr Val Thr Val Leu Ser Asn Arg Glu Val Thr Ile Leu Arg Tyr 145 150 155 160 ctg gtt age gga tta tet aat aaa gaa att gcc gat aag tta tta ctt 528

Leu Val Ser Gly Leu Ser Asn Lys Glu Ile Ala Asp Lys Leu Leu Leu 165 170 175 age aat aaa aca gtt agt geg cat aaa tet aat att tat ggc aag cta 576

Ser Asn Lys Thr Val Ser Ala His Lys Ser Asn Ile Tyr Gly Lys Leu 180 185 190 ggt ttg cat tea att gta gag ctt atc gac tac gcc aaa tta tac gaa 624

Gly Leu His Ser Ile Val Glu Leu Ile Asp Tyr Ala Lys Leu Tyr Glu 195 200 205 tta ata taa 633

Leu Ile 210 <210> 12 <211> 210

<212> PRT <213> Escherichia coli <400> 12

Met Lys Pro Thr Ser Val Ile Ile Met Asp Thr His Pro Ile Ile Arg 15 10 15

Met Ser Ile Glu Val Leu Leu Gin Lys Asn Ser Glu Leu Gin Ile Val 20 25 30

Leu Lys Thr Asp Asp Tyr Arg Ile Thr Ile Asp Tyr Leu Arg Thr Arg 35 40 45

Pro Val Asp Leu Ile Ile Met Asp Ile Asp Leu Pro Gly Thr Asp Gly 50 55 60

Phe Thr Phe Leu Lys Arg Ile Lys Gin Ile Gin Ser Thr Val Lys Val 65 70 75 80

Leu Phe Leu Ser Ser Lys Ser Glu Cys Phe Tyr Ala Gly Arg Ala Ile 85 90 95

Gin Ala Gly Ala Asn Gly Phe Val Ser Lys Cys Asn Asp Gin Asn Asp 100 105 110

Ile Phe His Ala Val Gin Met Ile Leu Ser Gly Tyr Thr Phe Phe Pro 115 120 125

Ser Glu Thr Leu Asn Tyr Ile Lys Ser Asn Lys Cys Ser Thr Asn Ser 130 135 140

Ser Thr Val Thr Val Leu Ser Asn Arg Glu Val Thr Ile Leu Arg Tyr 145 150 155 160

Leu Val Ser Gly Leu Ser Asn Lys Glu Ile Ala Asp Lys Leu Leu Leu 165 170 175

Ser Asn Lys Thr Val Ser Ala His Lys Ser Asn Ile Tyr Gly Lys Leu 180 185 190

Gly Leu His Ser Ile Val Glu Leu Ile Asp Tyr Ala Lys Leu Tyr Glu 195 200 205

Leu Ile 210 <210> 13 <211 > 120 <212> DNA <213> Artificial <220 <223> DNA fragment containing attL <400 13 agatcttgaa gcctgctttt ttatactaag ttggcattat aaaaaagcat tgcttatcaa 60 tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttga tttcgaattc 120 <210> 14 <211 >40 <212> DNA <213> Artificial <220> <223> primer P1 <400> 14 ctagtaagat cttgaagcct gcttttttat actaagttgg 40 <210> 15 <211 >41 <212> DNA <213> Artificial <220 <223> primer P2 <400> 15 atgatcgaat tcgaaatcaa ataatgattt tattttgact g 41 <210> 16 <211 > 184 <212> DNA <213> Artificial <220> <223> DNA fragment containing attR <400> 16 ctgcagtctg ttacaggtca ctaataccat ctaagtagtt gattcatagt gactgcatat 60 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga 120 tatttatatc attttacgtt tctcgttcag cttttttata ctaacttgag cgtctagaaa 180 gctt 184 <210> 17 <211>41 <212> DNA <213> Artificial <220> <223> primer P3 <400> 17 atgccactgc agtctgttac aggtcactaa taccatctaa g 41 <210> 18 <211> 46 <212> DNA <213> Artificial <220> <223> primer P4 <400> 18 accgttaagc tttctagacg ctcaagttag tataaaaaag ctgaac 46 <210> 19 <211> 38 <212> DNA <213> Artificial <220 <223> primer P5 <400> 19 ttcttagacg tcaggtggca cttttcgggg aaatgtgc 38 <210> 20 <211> 37 <212> DNA <213> Artificial <220> <223> primer P6 <400> 20 taacagagat ctcgcgcaga aaaaaaggat ctcaaga 37 <210> 21 <211> 46 <212> DNA <213> Artificial <220> <223> primer P7 <400> 21 aacagagatc taagcttaga tcctttgcct ggcggcagta gcgcgg 46 <210> 22 <211> 35 <212> DNA <213> Artificial <220> <223> primer P8 <400> 22 ataaactgca gcaaaaagag tttgtagaaa cgcaa 35

<210> 23 <211 > 1388 <212> DNA <213> Artificial <220> <223> DNA fragment containing Tc gene and ter_thrL <400> 23 gaattctcat gtttgacagc ttatcatcga taagctttaa tgcggtagtt tatcacagtt 60 aaattgctaa cgcagtcagg caccgtgtat gaaatctaac aatgcgctca tcgtcatcct 120 cggcaccgtc accctggatg ctgtaggcat aggcttggtt atgccggtac tgccgggcct 180 cttgcgggat atcgtccatt ccgacagcat cgccagtcac tatggcgtgc tgctagcgct 240 atatgcgttg atgcaatttc tatgcgcacc cgttctcgga gcactgtccg accgctttgg 300 ccgccgccca gtcctgctcg cttcgctact tggagccact atcgactacg cgatcatggc 360 gaccacaccc gtcctgtgga tcctctacgc cggacgcatc gtggccggca tcaccggcgc 420 cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg 480 ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg 540 gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg 600 cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgacc 660 gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat 720 cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc 780 gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc 840 gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac 900 caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccgacg cgctgggcta 960 cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc cccattatga ttcttctcgc 1020 ttccggcggc atcgggatgc ccgcgttgca ggccatgctg tccaggcagg tagatgacga 1080 ccatcaggga cagcttcaag gatcgctcgc ggctcttacc agcctaactt cgatcactgg 1140 accgctgatc gtcacggcga tttatgccgc ctcggcgagc acatggaacg ggttggcatg 1200 gattgtaggc gccgccctat accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag 1260 ccgggccacc tcgacctgaa tggaagccgg cggcacctcg ctaacggatt caccactcca 1320 actagaaagc ttaacacaga aaaaagcccg cacctgacag tgcgggcttt ttttttcgac 1380 cactgcag 1388 <210> 24 <211> 36 <212> DNA <213> Artificial <220> <223> primer P9 <400> 24 agtaattcta gaaagcttaa cacagaaaaa agcccg 36 <210> 25 <211> 43 <212> DNA <213> Artificial <220> <223> primer P10 <400> 25 ctagtaggat ccctgcagtg gtcgaaaaaa aaagcccgca ctg 43

<210> 26 <211 > 1162 <212> DNA <213> Artificial <220 <223> DNA fragment containing Pa2 promoter <400 26 agatctccgg ataagtagac agcctgataa gtcgcacgaa aaacaggtat tgacaacatg 60 aagtaacatg cagtaagata caaatcgcta ggtaacacta gcagcgtcaa ccgggcgotc 120 tagctagagc caagctagct tggccggatc cgagattttc aggagctaag gaagctaaaa 180 tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac 240 attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata 300 ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc 360 acattcttgc ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg 420 agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa 480 cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt 540 cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga 600 atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg 660 ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg 720 acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtctgtgat ggcttccatg 780 tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaat 840 ttttttaagg cagttattgg tgcccttaaa cgcctggtgc tacgcctgaa taagtgataa 900 taagcggatg aatggcagaa attcgtcgaa gcttaacaca gaaaaaagcc cgcacctgac 960 agtgcgggct ttttttttcg accactgcag tctgttacag gtcactaata ccatctaagt 1020 agttgattca tagtgactgc atatgttgtg ttttacagta ttatgtagtc tgttttttat 1080 gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt tcagcttttt 1140 tatactaact tgagcgtcta ga 1162 <210> 27 <211> 37 <212> DNA <213> Artificial <220> <223> primer P11 <400> 27 atcgaggtac cagatctccg gataagtaga cagcctg 37 <210> 28 <211> 32 <212> DNA <213> Artificial <220> <223> primer P12 <400> 28 gaaggtctag agcgcccggt tgacgctgct ag 32 <210> 29 <211 >27 <212> DNA <213> Artificial <220> <223> primer P13 <400 29 ctaatatcga tgaagattct tgctcaa 27 <210> 30 <211> 34 <212> DNA <213> Artificial <220> <223> primer P14 <400> 30 gcgttgaatt ccatacaacc tccttagtac atgc 34 <210> 31 <211> 34 <212> DNA <213> Artificial <220> <223> primer P15 <400> 31 gtactagaat tcgtgtaatt gcggagactt tgcg 34 <210 32 <211> 41 <212> DNA <213> Artificial <220> <223> primer P16 <400> 32 aatagcctgc agttatttga tttcaatttt gtcccactcc c 41 <210> 33 <211> 57 <212> DNA <213> Artificial <220> <223> primer P17 <400> 33 aacggaaaat tgtccgctcc tatgagactg gtaacttagt aagccagtat acactcc 57 <210> 34 <211> 57 <212> DNA <213> Artificial <220> <223> primer P18 <400> 34 cagtgtctta aataaagtaa tcggttatat acggatttaa gggcaccaat aactgcc 57 <210> 35 <211> 21 <212> DNA <213> Artificial <220 <223> primer P19 <400 35 cgtctgaatc aagaaaaccc g 21 <210> 36 <211> 21 <212> DNA <213> Artificial <220> <223> primer P20 <400> 36 cgcggaagta ttcatctaac g 21 <210> 37 <211> 63 <212> DNA <213> Artificial <220> <223> primer P21 <400> 37 ttatattaat tcgtataatt tggcgtagtc gataagtgaa gcctgctttt ttatactaag 60 ttg 63 <210> 38 <211> 63 <212> DNA <213> Artificial <220> <223> primer P22 <400> 38 catgtatcaa agtacaattt cccgacctaa cggaaacgct caagttagta taaaaaagct 60 gaa 63 <210> 39 <211> 22 <212> DNA <213> Artificial <220> <223> primer P23 <400 39 cgtataattt ggcgtagtcg at 22 <210> 40 <211> 24 <212> DNA <213> Artificial <220> <223> primer P24 <400> 40 catgtatcaa agtacaattt cccg 24

Claims 1. A method for producing an L-amino acid comprising: - cultivating an L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein said bacterium has been modified to attenuate expression of a fimZ gene by inactivation of the gene, in a medium to produce and excrete said L-amino acid into the medium, and - collecting said L-amino acid from the medium. 2. The method according to claim 1, wherein said bacterium belongs to the genus Escherichia. 3. The method according to claim 1, wherein said bacterium belongs to genus Pantoea. 4. The method according to any one of claims 1 to 3, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid. 5. The method according to claim 4, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan. 6. The method according to claim 4, wherein said 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.

Patentansprüche 1. Verfahren zur Herstellung einer L-Aminosäure, umfassend: - Kultivieren eines L-Aminosäure-produzierenden Bakteriums der Familie Enterobacteriaceae, wobei das Bakterium modifiziert wurde, um die Expression des fimZ-Gens durch Inaktivierung des Gens abzuschwächen, in einem Medium, um die L-Aminosäure herzustellen und in das Medium auszuscheiden, und - Sammeln der L-Aminosäure aus dem Medium. 2. Verfahren gemäss Anspruch 1, wobei das Bakterium zur Gattung Escherichia gehört. 3. Verfahren gemäss Anspruch 1, wobei das Bakterium zur Gattung Pantoea gehört. 4. Verfahren gemäss irgendeinem der Ansprüche 1 bis 3, wobei die L-Aminosäure aus der Gruppe bestehend aus aromatischer L-Aminosäure und nicht-aromatischer L-Aminosäure ausgewählt ist. 5. Verfahren gemäss Anspruch 4, wobei die aromatische L-Aminosäure aus der Gruppe bestehend aus L-Phenylalanin, L-Tyrosin und L-Tryptophan ausgewählt ist. 6. Verfahren gemäss Anspruch 4, wobei die nicht-aromatische L-Aminosäure aus der Gruppe bestehend aus L-Thre-onin, L-Lysin, L-Cystein, L-Methionin, L-Leucin, L-Isoleucin, L-Valin, L-Histidin, Glycin, L-Serin, L-Alanin, L-Aspa-ragin, L-Asparaginsäure, L-Glutamin, L-Glutaminsäure, L-Prolin und L-Arginin ausgewählt ist.

Revendications 1. Procédé de production d’un acide L-aminé comprenant : - la culture d’une bactérie de la famille des Enterobacteriaceae produisant un acide L-aminé, dans laquelle ladite bactérie a été modifiée pour atténuer l’expression d’un gène fimZ par l’inactivation du gène, dans un milieu pour produire et excréter ledit acide L-aminé dans le milieu, et - le recueil dudit acide L-aminé à partir du milieu. 2. Procédé selon la revendication 1, dans lequel ladite bactérie appartient au genre Escherichia. 3. Procédé selon la revendication 1, dans lequel ladite bactérie appartient au genre Pantoea. 4. Procédé selon l’une quelconque des revendications 1 à 3, dans lequel ledit acide L-aminé est choisi dans le groupe constitué par un acide L-aminé aromatique et un acide L-aminé non aromatique. 5. Procédé selon la revendication 4, dans lequel ledit acide L-aminé aromatique est choisi dans le groupe constitué par la L-phénylalanine, la L-tyrosine et le L-tryptophane. 6. Procédé selon la revendication 4, dans lequel ledit acide L-aminé non aromatique est choisi dans le groupe constitué parla L-thréonine, la L-lysine, la L-cystéine, la L-méthionine, la L-leucine, la L-isoleucine, la L-valine, la L-histidine, la glycine, la L-sérine, la L-alanine, la L-asparagine, l’acide L-aspartique, la L-glutamine, l’acide L-glutamique, la L-proline et la L-arginine.

Claims (5)

Claims 1. For the production of Epràs Lmmhtossrz. which contains: »kContinue: ··· L-stnic acid producing, Émemhaeteriaceas essíéhhs cultivation in a culture medium :, sx production of L-amoacetic acid - and: removal of broth in the medium, where the bacterium is modified so that IlmZ geo express? gene inactivating csbkkeptetk # ··: L-Amioic Acid Ripper Medium 2. The method according to point 1, shot, belongs to the PktérPo m EscPrichia. 3. The {. The method of claim 1, wherein said Pkem belongs to the Eastoea genus. 4. The 1 -3. The process according to any one of claims 1 to 3, wherein the L-emmossv is a leg-shaped l-amoic acid and a leg-like l-amoic acid group. 5. The vaporous, api-asthmatic α-amino acid of claim 4 according to claim 4, wherein L-tyrosine is selected from the group consisting of L-triptane and esofxop. The epras according to claim 4. api is the gender: gummy acid from the following group ·. L-threonine, L-cysteine, L-cysteine, X1-metsonisS :,l, - isocin, L-cholesterol, L-valine, Ldstidine, gPin, L-serum, I.-aPP, L-asparagus L ~ aspartic acid, t-patamine, I-gpamiosne, L-prohn and L ~ arginine.