WO2008096837A1

WO2008096837A1 - A method for producing an l-amino acid using a bacterium of the enterobacteriaceae family with attenuated expression of the tolc gene

Info

Publication number: WO2008096837A1
Application number: PCT/JP2008/052092
Authority: WO
Inventors: Valery Vasilievich Samsonov; Alla Markovna Kaplan; Valeriy Zavenovich Akhverdian; Elvira Borisovna Voroshilova; Mikhail Markovich Gusyatiner
Original assignee: Ajinomoto Co., Inc.
Priority date: 2007-02-07
Filing date: 2008-02-04
Publication date: 2008-08-14
Also published as: RU2007104648A; RU2366703C2

Abstract

The present invention provides a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, particularly a bacterium belonging to genus Escherichia or Pantoea, which has been modified to attenuate expression of the tolC gene.

Description

DESCRIPTION

A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE ENTEROBACTERIACEAE FAMILY WITH ATTENUATED EXPRESSION OF THE tolC GENE

Technical Field

The present invention relates to the microbiological industry, and specifically to a method for producing an L-amino acid using a bacterium of the Enter ^•obacteriaceae family which has been modified to attenuate expression of the tolC gene.

Background Art

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.

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).

Another way to enhance L-amino acid production yields is to attenuate expression of a gene or several genes involved in degradation of the target L-amino acid, genes diverting the precursors of the target L-amino acid from the L-amino acid biosynthetic pathway, genes involved in the redistribution of carbon, nitrogen, and phosphate fluxes, and genes coding for toxins etc.

TolC is an outer membrane porin involved in the efflux of several hydrophobic and amphipathic molecules. TolC is a beta barrel with 18 membrane spanning beta strands and functions as a trimer. It is a common outer membrane component of several multi-drug efflux systems.

The tolC gene product was determined to localize to the outer membrane (Morona R.,et al., J.Bacteriol.,153(2):693-699 (1983)). TolC was purified from the Escherichia coli outer membrane as a trimer and its structure was determined by two dimensional projection at a resolution of 12 angstroms. ToIC was found to be an outer membrane protein with each monomer comprising a membrane domain, predicted to be beta-barrel, and a C-terminal periplasmic domain. Targeting of ToIC to the Sec-translocase for transport across the inner membrane is SecB-dependent (Baars L.,et al., J.Biol.Chem., 281(15): 10024- 10034 (2006)).

Mutations in tolC have been shown to result in a reduction in the synthesis of OmpF porin, and an increase in the level of OmpC porin synthesis (Misra R.et al., J.Bacteriol.,169(10):4722-4730 (1987)). Reconstitution studies suggest that ToIC is an outer membrane channel for peptides. ToIC has been found to be required for the functioning of the AcrAB multidrug efflux system (Fralick J.,J.Bacteriol., 178 (19):5803- 5805 (1996)) and its homologs AcrEF (Kobayashi K. et α/.,J.Bacteriol, 183(8): 2646-2653 (2001)) and YhiUV (Nishino K.,et al., J.Bacteriol., 184(8):2319-2323 (2002)), the EmrAB drug efflux system (Borges-Walmsley M.I.,ef α/.,J.Biol.Chem.,278 (15):12903-12912 (2003)), the EmrAB homolog, EmrKY (Tanabe R.,et α/.,J.Gen.Appt.Microbiol.,43:257- 263 (1997)), the MdtABC drug efflux system (Nagakubo S.,et al, J.Bacteriol.,184(15):4161-4167 (2002)) and the MacAB macrolide extrusion system (Kobayashi N., et al, J.Bacteriol., 183(19):5639-5644 (2001)).

The tolC mutants become tolerant to colicin El, show an altered bacteriophage sensitivity pattern, lack the OmpF protein, and become hypersensitive to detergents, dyes, and certain antibiotics.

But currently, there have been no reports of attenuating expression of the tolC gene for the purpose of producing L-amino acids.

Disclosure of the Invention

Aspects 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.

The above Aspects were achieved by finding that attenuating expression of the tolC 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- phenylalanine, L-tyrosine, and L-tryptophan. The present invention provides a bacterium of the Enterobacteriaceae family having an increased ability to produce amino acids, such asL-threonine, L-lysine, L- cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L- alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine, L-phenylalanine, L-tyrosine, and L-tryptophan.

It is an aspect of the present invention to provide an L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified to attenuate expression of the tolC gene.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the expression of the tolC gene is attenuated by inactivating the tolC gene.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium belongs to the genus Escherichia.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium belongs to the genus Pantoea.

It is a further aspect of the present invention to provide 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.

It is a further aspect of the present invention to provide 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.

It is a further aspect of the present invention to provide 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.

It is a further aspect of the present invention to provide a method for producing an L-amino acid comprising:

- cultivating the bacterium as described above in a medium, and

- collecting said L-amino acid from the medium.

It is a further aspect 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. It is a further aspect 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.

It is a further aspect of the present invention to provide the method 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.

The present invention is described in detail below.

Brief Description of Drawings

Figure 1 shows the relative positions of primers Pl and P2 on plasmid pMWl 18- attL-Cm-attR which is used as a template for PCR amplification of the cat gene.

Figure 2 shows the construction of the chromosomal DNA fragment containing the inactivated tolC gene.

Detailed Description of the Preferred Embodiments 1. Bacterium of the present invention

The bacterium of the present invention is an L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified to attenuate expression of the tolC gene.

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

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 microorganism is able to cause accumulation in a medium of an amount not less than 0.5 g/L, more preferably not less than 1.0 g/L, of the target L-amino acid. 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. The term "aromatic L-amino acid" includes L-phenylalanine, L-tyrosine, and L- tryptophan. The term "non-aromatic L-amino acid" includes L-threonine, L-lysine, L- cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L- alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L- arginine. L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L- phenylalanine, L-tryptophan, L-proline, and L-arginine are particularly preferred.

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://wΛvw.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id-91347) can be used. A bacterium belonging to the genus Escherichia or Pantoea is preferred.

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. colϊ).

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.

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)).

The phrase "bacterium has been modified to attenuate expression of the tolC gene" means that the bacterium has been modified in such a way that a modified bacterium contains a reduced amount of the ToIC protein as compared with an unmodified bacterium, or is unable to synthesize the ToIC protein. The phrase "inactivation of the tolC 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, 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 a promoter, enhancer, attenuator, ribosome-binding site, etc..

The level of gene expression can be determined by measuring the amount of mRNA transcribed from the gene using various known methods including Northern blotting, quantitative RT-PCR, and the like. Amount of the protein coded by the gene can be measured by known methods including SDS-PAGE followed by immunoblotting assay (Western blotting analysis) and the like.

The tolC gene (synonyms: ECK3026, weeA, b3035, colEl-i, mtcB, mukA, refl, toe) encodes the ToIC protein (synonyms: ToIC outer membrane channel, B3Q35 , WeeA , Toe , Reft , MukA , MtcB). The tolC gene (nucleotides in positions from 3,176,137 to 3,177,618; GenBank accession no. NC_000913.2; gi: 49175990) is located between the ycal gene and the ipxK gene on the chromosome of E. coli K-12. The nucleotide sequence of the tolC gene and the amino acid sequence of ToIC encoded by the tolC gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

Since there may be some differences in DNA sequences between the genera or strains of the Enterobacteriaceae family, the tolC gene to be inactivated on the chromosome is not limited to the gene shown in SEQ ID No:l, but may include genes homologous to SEQ ID No:l encoding a variant protein of the ToIC protein. The phrase "variant protein" as used in the present invention means a protein which has changes in the sequence, whether they are deletions, insertions, additions, or substitutions of amino acids, but still maintains the activity of the product as the ToIC protein. The number of changes in the variant protein depends on the position or the type of amino acid residues in the three dimensional structure of the protein. It may be 1 to 30, preferably 1 to 15, and more preferably 1 to 5 in SEQ ID NO: 2. These changes in the variants can occur in regions of the protein which are not critical for the 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. These changes in the variant protein can occur in regions of the protein which are not critical for the function of the protein. Therefore, the protein variant encoded by the tolC gene 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 sequence shown in SEQ ID NO. 2, as long as the activity of the ToIC protein prior to inactivation is maintained.

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

Moreover, the tolC gene may be a variant which hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: I₅ or a probe which can be prepared from the nucleotide sequence, provided that it encodes a functional ToIC protein prior to inactivation. "Stringent conditions" include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, preferably not less than 70%, more preferably not less than 80%, still more preferably not less than 90%, and most preferably not less than 95%, is formed and a 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 1 X SSC, 0.1% SDS, preferably 0.1 X SSC, 0.1% SDS at 60⁰C. Duration of washing depends on the type of membrane used for blotting and, as a rule, may 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 is usually 100 bp to 1 kbp.

Homology between two amino acid sequences can be determined using the well- known methods, for example, the computer program BLAST 2.0.

Expression of the tolC gene can be attenuated by introducing a mutation into the gene on the chromosome so that the intracellular amount of the TolC protein encoded by the gene is decreased as compared to an unmodified strain. Such a mutation can be introduction of insertion of a drug-resistance gene, or deletion of a part of the gene or the entire gene (Qiu, Z. and Goodman, M.F., J. Biol. Chem., 272, 8611-8617 (1997); Kwon, D. H. et al, J. Antimicrob. Chemother., 46, 793-796 (2000)). Expression of the tolC gene can also be attenuated by modifying an expression regulating sequence such as the promoter, the Shine-Dalgarno (SD) sequence, etc. (WO95/34672, Carrier, T.A. and Keasling, J.D., Biotechnol Prog 15, 58-64 (1999)).

For example, the following methods may be employed to introduce a mutation by gene recombination. A mutant gene is prepared, and the bacterium to be modified is transformed with a DNA fragment containing the mutant gene. Then, the native gene on the chromosome is replaced with the mutant gene by homologous recombination, and the resulting strain is selected. Such gene replacement by homologous recombination can be conducted by employing a linear DNA, which is known as "Red-driven integration" (Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)), or by employing a plasmid containing a temperature-sensitive replication control region (U.S. Patent 6,303,383 or JP 05-007491 A). Furthermore, the incorporation of a site-specific mutation by gene substitution using homologous recombination such as set forth above can also be conducted with a plasmid lacking the ability to replicate in the host. When a marker gene such as antibiotic resistant gene is used to prepare the mutant gene or to detect recombination between the mutant gene and the native gene on the chromosome, the marker gene can be eliminated from the chromosome by, for example, the method described in Examples section.

Expression of the gene can also be attenuated by insertion of a transposon or an IS factor into the coding region of the gene (U.S. Patent No. 5,175,107), or by conventional methods, such as mutagenesis using UV irradiation or nitrosoguanidine (N-methyl-N¹- nitro-N-nitrosoguanidine) treatment.

Inactivation of the gene can be performed by conventional methods, such as mutagenesis treatment using 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. et al., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 5978-83 and Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 6640-45) also called "Red-driven integration".

Production of the ToIC protein in assemblage with Membrane Fusion Proteins (MFPs) provides facilitating drugs transport across Escherichia coli outer membranes. Strains with deletions of tolC gene become hypersensitive to hydrophobic agents as a result of inactivation of the arc AB multiple drug-resistance efflux system (Fralick J.,J.Bacteriol., 178 (19):5803-5805 (1996)).Therefore, the reduced or absent activity of the ToIC protein in the bacterium can be determined when compared to the parent unmodified bacterium

The presence or absence of the tolC 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 gene using various known methods including Northern blotting, quantitative RT-PCR, and the like. Amount of the protein coded by the gene can be measured by known methods including SDS-PAGE followed by immunoblotting assay (Western blotting analysis) and the like. For example, identification of the tolC gene product was done by making autoradiographs of a linear 11% polyacrylamide gel run of the labeled minicells samples (Morona R.,et al, J.Bacteriol.,153(2):693-699 (1983)).

Methods for preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer, and the like may be ordinary methods well-known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).

L-amino acid-producing bacteria

As a bacterium of the present invention which is modified to attenuate expression of the tolC gene, bacteria which are able to produce either an aromatic or a non-aromatic L-amino acids may be used.

The bacterium of the present invention can be obtained by attenuating expression of the tolC gene in a bacterium 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 tolC gene.

L-threonine-producing bacteria

Examples of parent strains for deriving the L-threonine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli TDH-6/pVIC40 (VKPM B-3996) (U.S. Patent No. 5, 175, 107, U.S. Patent No. 5,705,371), E. coli 472T23/pYN7 (ATCC 98081) (U.S. Patent No.5,631,157), E. coli NRRL-21593 (U.S. Patent No. 5,939,307), E. coli FERM BP-3756 (U.S. Patent No. 5,474,918), E. coli FERM BP-3519 and FERM BP-3520 (U.S. Patent No. 5,376,538), E. coli MG442 (Gusyatiner et al, Genetika (in Russian), 14, 947-956 (1978)), E. coli VL643 and VL2055 (EP 1149911 A), and the like.

The strain TDH-6 is deficient in the thrC gene, as well as being sucrose- assimilative, and the UvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene, which imparts resistance to high concentrations of threonine or homoserine. The strain B-3996 contains the plasmid pVIC40 which was obtained by inserting a thrA*BC operon which includes a mutant thrA gene into a RSFlOlO-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which is substantially desensitized to feedback inhibition by threonine. The strain B-3996 was deposited on November 19, 1987 in the Ail-Union Scientific Center of Antibiotics (Russia, 117105 Moscow, Nagatinskaya Street 3-A) under the accession number RIA 1867. The strain was also deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on April 7, 1987 under the accession number VKPM B-3996.

E. coli VKPM B-5318 (EP 0593792B) may also be used as a parent strain for deriving L-threonine-producing bacteria of the present invention. The strain B-5318 is prototrophic with regard to isoleucine, and a temperature-sensitive lambda-phage Cl repressor and PR promoter replaces the regulatory region of the threonine operon in plasmid pVIC40. The strain VKPM B-5318 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) on May 3, 1990 under accession number of VKPM B-5318.

Preferably, the bacterium of the present invention is additionally modified to enhance expression of one or more of the following genes: the mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine; the thrB gene which codes for homoserine kinase; the thrC gene which codes for threonine synthase; the rhtA gene which codes for a putative transmembrane protein; the asd gene which codes for aspartate-β-semialdehyde dehydrogenase; and the aspC gene which codes for aspartate aminotransferase (aspartate transaminase); The thrA gene which encodes aspartokinase homoserine dehydrogenase I of Escherichia coli has been elucidated (nucleotide positions 337 to 2799, GenBank accession No. NC_000913.2, gi: 49175990). The thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K-12. The thrB gene which encodes homoserine kinase of Escherichia coli has been elucidated (nucleotide positions 2801 to 3733, GenBank accession No. NC_000913.2, gi: 49175990). The thrB gene is located between the thrA and thrC genes on the chromosome of E. coli K-12. The thrC gene which encodes threonine synthase of Escherichia coli has been elucidated (nucleotide positions 3734 to 5020, GenBank accession No. NC_000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaX open reading frame on the chromosome of E. coli K- 12. All three genes function as a single threonine operon. To enhance expression of the threonine operon, the attenuator region which affects the transcription is desirably removed from the operon (WO2005/049808, WO2003/097839).

A mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine, as well as, the thrB and thrC genes can be obtained as one operon from well-known plasmid pVIC40 which is present in the threonine producing E. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S. Patent No. 5,705,371.

The rhtA gene exists at 18 min on the E. coli chromosome close to the glnHPQ operon, which encodes components of the glutamine transport system. The rhtA gene is identical to ORPl (ybiF gene, nucleotide positions 764 to 1651, GenBank accession number AAA218541, gi:440181) and located between the pexB and ompX genes. The unit expressing a protein encoded by the ORPl 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 17^th 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).

The asd gene of E. coli has already been elucidated (nucleotide positions 3572511 to 3571408, GenBank accession No. NC_000913.1, gi:16131307), and can be obtained by PCR (polymerase chain reaction; refer to White, TJ. 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. Also, the aspC gene of E. coli has already been elucidated (nucleotide positions 983742 to 984932, GenBank accession No. NC_000913.1, gi:16128895), and can be obtained by PCR. The aspC genes of other microorganisms can be obtained in a similar manner.

L-lysine-producing bacteria

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, α-chlorocaprolactam and so forth. Mutants having resistance to these lysine analogues can be obtained by subjecting bacteria belonging to the genus Escherichia to a conventional artificial mutagenesis treatment. Specific examples of bacterial strains useful for producing L-lysine include Escherichia coli AJl 1442 (FERM BP-1543, NRRL B-12185; see U.S. Patent No. 4,346,170) and Escherichia coli VL611. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is desensitized.

The strain WC 196 may be used as an L-lysine producing bacterium of Escherichia coli. This bacterial strain was bred by conferring AEC resistance to the strain W3110, which was derived from Escherichia coli K- 12. The resulting strain was designated Escherichia coli AJl 3069 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).

Examples of parent strains for deriving L-lysine-producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L- lysine biosynthetic enzyme are enhanced. Examples of such genes include, but are not limited to, genes encoding dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (tysA), diaminopimelate dehydrogenase (ddh) (U.S. Patent No. 6,040,160), phosphoenolpyrvate carboxylase (ppc), aspartate semialdehyde dehydrogenase (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), theybjE gene (WO2005/073390), or combinations thereof.

Examples of parent strains for deriving L-lysine-producing bacteria of the present invention also include strains having decreased or eliminated activity of an enzyme that catalyzes a reaction for generating a compound other than L-lysine by branching off from the biosynthetic pathway of L-lysine. Examples of the enzymes that catalyze a reaction for generating a compound other than L-lysine by branching off from the biosynthetic pathway of L-lysine include homoserine dehydrogenase, lysine decarboxylase (U.S. Patent No. 5,827,698), and the malic enzyme (WO2005/010175).

L-cysteine-producing bacteria

Examples of parent strains for deriving L-cysteine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JMl 5 which is transformed with different cysE alleles coding for feedback- resistant serine acetyltransferases (U.S. Patent No. 6,218,168, Russian patent application 2003121601); E. coli W3110 having over-expressed genes which encode proteins suitable for secreting substances toxic for cells (U.S. Patent No. 5,972,663); E. coli strains having lowered cysteine desulfohydrase activity (JPl 1155571 A2); E. coli W3110 with increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene (WO0127307A1), and the like.

L-leucine-producing bacteria

Examples of parent strains for deriving L-leucine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strains resistant to leucine (for example, the strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121)) or leucine analogs including 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. The bacterium 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 lenA gene coding for isopropylmalate synthase which is not subject to feedback inhibition by L- leucine (US Patent 6,403,342). In addition, the bacterium of the present invention may be improved by enhancing the expression of one or more genes coding for proteins which excrete L-amino acid from the bacterial cell. Examples of such genes include the b2682 and b2683 genes (ygaZH genes) (EP 1239041 A2).

- L-histidine-producing bacteria

Examples of parent strains for deriving L-histidine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 24 (VKPM B-5945, RU2003677); E. coli strain 80 (VKPM B-7270, RU2119536); £. cø/z NRRL B-12116 - B12121 (U.S. Patent No. 4,388,405); E. coli H- 9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Patent No. 6,344,347); E. coli H-9341 (FERM BP-6674) (EP1085087); £. coli AI80/pFM201 (U₅S. Patent No. 6,258,554) and the like.

Examples of parent strains for deriving L-histidine-producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-histidine biosynthetic enzyme are enhanced. Examples of such genes include genes encoding ATP phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase (MsT), phosphoribosyl-ATP pyrophosphohydrolase (MsIE), phosphoribosylformimino-5- aminoimidazole carboxamide ribotide isomerase (MsA), amidotransferase (MsH), histidinol phosphate aminotransferase (MsC), histidinol phosphatase (MsB), histidinol dehydrogenase (MsD), and so forth.

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

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 (EPl 01671 OA), E. coli 80 strain imparted with sulfaguanidine, DL- l,2,4-triazole-3 -alanine, and streptomycin-resistance (VKPM B-7270, Russian Patent No. 2119536), and so forth.

L-glutamic acid-producing bacteria

Examples of parent strains for deriving L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli VL334thrC⁺ (EP 1172433). E. coli VL334 (VKPM B-1641) is an L- isoleucine and L-threonine auxotrophic strain having mutations in thrC and UvA genes (U.S. Patent No. 4,278,765). A wild-type allele of the thrC gene was transferred by the method of general transduction using a bacteriophage Pl which was grown on the wild- type E. coli 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.

Examples of parent strains for deriving the L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains which are deficient in α- ketoglutarate dehydrogenase activity, or strains in which 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 ipyc), pyruvate dehydrogenase (aceEF, ipdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA, pgml), phosphoglycerate kinase (pgk), glyceraldehyde-3-phophate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pβA, ppxB), and glucose phosphate isomerase (pgi).

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.

Examples of strains which have been modified so that expression of the citrate synthetase gene and/or the phosphoenolpyruvate carboxylase gene are reduced, and/or are deficient in a-ketoglutarate dehydrogenase activity include those disclosed in EP1078989A, EP955368A, and EP952221 A. Examples of parent strains for deriving the L-glutamic acid-producing bacteria of the present invention also include strains having decreased or eliminated activity of an enzyme that catalyzes synthesis of a compound other than L-glutamic acid by branching off from an L-glutamic acid biosynthesis pathway. Examples of such genes include genes encoding isocitrate lyase (aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (ptά), acetate kinase (ack), acetohydroxy acid synthase (UvG), acetolactate synthase (UvI), formate acetyltransferase (pβ), lactate dehydrogenase (ϊdh), and glutamate decarboxylase (gadAE). Bacteria belonging to the genus Escherichia deficient in α-ketoglutarate 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. co/z W3110sucA::Km^R

E. coli AJ12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJl 2949 (FERM BP-4881)

E. coli W3110sucA::Km^R is a strain obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter referred to as "sue A gene") of E. coli W3110. This strain is completely deficient in the α-ketoglutarate dehydrogenase.

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 the α-ketoglutarate dehydrogenase activity and include, for example, E. coli AJ13199 (FERM BP-5807) (U.S. Patent No. 5,908,768), FFRM P- 12379, which additionally has a low L-glutamic acid decomposing ability (U.S. Patent No. 5,393,671); AJ13138 (FERM BP-5565) (U.S. Patent No. 6,110,714), and the like.

Examples of L-glutamic acid-producing bacteria include mutant strains belonging to the genus Pantoea which are deficient in the α-ketoglutarate dehydrogenase activity or have a decreased α-ketoglύtarate dehydrogenase activity, and can be obtained as described above. Such strains include Pantoea ananatis AJ13356. (U.S. Patent No. 6,331,419). Pantoea ananatis AJl 3356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on February 19, 1998 under an accession number of FERM P- 16645. It was then converted to an international deposit under the provisions of Budapest Treaty on January 11, 1999 and received an accession number of FERM BP-6615. Pantoea ananatis AJ13356 is deficient in the α-ketoglutarate dehydrogenase activity as a result of disruption of the αKGDH-El subunit gene (sucA). The above strain was identified as Enterobacter agglomerans when it was isolated and deposited as the Enterobacter agglomerans AJ13356. However, it was recently re- classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth. Although AJl 3356 was deposited at the aforementioned depository as Enterobacter agglomerans, for the purposes of this specification, they are described as Pantoea ananatis.

L-phenylalanine-producinfi bacteria

Examples of parent strains for deriving L-phenylalanine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli AJ12739 (tyrA::TnlO, tyrR) (VKPM B-8197); E. coli HW1089 (ATCC 55371) harboring the mutant pheA34 gene (U.S. Patent No. 5,354,672); E. coli MWEC101-b (KR8903681); E. co//NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (U.S. Patent No. 4,407,952). Also, as a parent strain, E. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP- 12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used (EP 488424 Bl). Furthermore, L-phenylalanine producing bacteria belonging to the genus Escherichia with an enhanced activity of the protein encoded by the yedA gene or XhsyddG gene may also be used (U.S. patent applications 2003/0148473 Al and 2003/0157667 Al).

L-trvptophan-producing bacteria

Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) which is deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Patent No. 5,756,345); E. coli SVl 64 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase not subject to feedback inhibition by serine and a trpE allele encoding anthranilate synthase not subject to feedback inhibition by tryptophan (U.S. Patent No. 6,180,373); E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient in the enzyme tryptophanase (U.S. Patent No. 4,371,614); E. coli AGX17/pGX50,pACKG4-pps in which a phosphoenolpyruvate-producing ability is enhanced (WO9708333, U.S. Patent No. 6,319,696), and the like may be used. L- tryptophan-producing bacteria belonging to the genus Escherichia with an enhanced activity of the identified protein encoded by and the yedA gene or the yddG gene may also be used (U.S. patent applications 2003/0148473 Al and 2003/0157667 Al).

Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention also include strains in which one or more activities of the enzymes selected from anthranilate synthase, phosphoglycerate dehydrogenase, and tryptophan synthase are enhanced. The anthranilate synthase and phosphoglycerate dehydrogenase are both subject to feedback inhibition by L-tryptophan and L-serine, so that a mutation desensitizing the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such a mutation include a E. coli SVl 64 which harbors desensitized anthranilate synthase and a transformant strain obtained by introducing into the E. coli SVl 64 the plasmid pGH5 (WO 94/08031), which contains a mutant serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase.

Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention also include strains into which the tryptophan operon which contains a gene encoding desensitized anthranilate synthase has been introduced (JP 57-71397 A, JP 62-244382 A, U.S. Patent No. 4,371,614). Moreover, L-tryptophan-producing ability may be imparted by enhancing expression of a gene which encodes tryptophan synthase, among tryptophan operons (JrpBA). The tryptophan synthase consists of α and β 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 (WO2005/103275).

L-proline-producinfi bacteria

Examples of parent strains for deriving L-proline-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli 702ilvA (VKPM B-8012) which is deficient in the UvA gene and is able to produce L-proline (EP 1172433). The bacterium of the present invention may be improved by enhancing the expression of one or more genes involved in L-proline biosynthesis. Examples of such genes for L-proline producing bacteria which are preferred include the proB gene coding for glutamate kinase of which feedback inhibition by L-proline is desensitized (DE Patent 3127361). In addition, the bacterium of the present invention may be improved by enhancing the expression of one or more genes coding for proteins excreting L-amino acid from bacterial cell. Such genes are exemplified by b2682 and b2683 genes (ygaZH genes) (EP1239041 A2).

Examples of bacteria belonging to the genus Escherichia, which have an activity to produce L-proline include the following E. coli strains: NRRL B-12403 and NRRL B- 12404 (GB Patent 2075056), VKPM B-8012 (Russian patent application 2000124295), plasmid mutants described in DE Patent 3127361, plasmid mutants described by Bloom F.R. et al (The 15^th Miami winter symposium, 1983, p. 34), and the like.

L-arRinine-producing bacteria

Examples of parent strains for deriving L-arginine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 237 (VKPM B-7925) (U.S. Patent Application 2002/058315 Al) and its derivative strains harboring mutant N-acetylglutamate synthase (Russian Patent Application No. 2001112869), E. coli strain 382 (VKPM B-7926) (EPl 170358A1), an arginine-producing strain into which argA gene encoding N-acetylglutamate synthetase is introduced therein (EPl 170361 Al), and the like.

Examples of parent strains for deriving L-arginine producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L- arginine biosynthetic enzyme are enhanced. Examples of such genes include genes encoding N-acetylglutamyl phosphate reductase (argC), ornithine acetyl transferase (argj), N-acetylglutamate kinase (argB), acetylornithine transaminase (argD), ornithine carbamoyl transferase (argF), argininosuccinic acid synthetase (argG), argininosuccinic acid lyase (argH), and carbamoyl phosphate synthetase {car AB).

L-valine-producing bacteria

Example of parent strains for deriving L-valine-producing bacteria of the present invention include, but are not limited to, strains which have been modified to overexpress the UvGMEDA operon (U.S. Patent No. 5,998,178). It is desirable to remove the region of the UvGMEDA operon which is required for attenuation so that expression of the operon is not attenuated by L- valine that is produced. Furthermore, the UvA gene in the operon is desirably disrupted so that threonine deaminase activity is decreased.

Examples of parent strains for deriving L-valine-producing bacteria of the present invention include also include mutants having a mutation of amino-acyl t-RNA synthetase (U.S. Patent No. 5,658,766). For example, E. coli VLl 970, which has a mutation in the UeS gene encoding isoleucine tRNA synthetase, can be used. E. coli VLl 970 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny Proezd, 1) on June 24, 1988 under accession number VKPM B-4411.

Furthermore, mutants requiring lipoic acid for growth and/or lacking H⁺-ATPaSe can also be used as parent strains (WO96/06926).

L-isoleucine-producing bacteria

Examples of parent strains for deriving L-isoleucine producing bacteria of the present invention include, but are not limited to, mutants having resistance to 6- dimethylaminopurine (JP 5-304969 A), mutants having resistance to an isoleucine analogue such as thiaisoleucine and isoleucine hydroxamate, and mutants additionally having resistance to DL-ethionine and/or arginine hydroxamate (JP 5-130882 A). In addition, recombinant strains transformed with genes encoding proteins involved in L- isoleucine biosynthesis, such as threonine deaminase and acetohydroxate 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

The method of the present invention is a method for producing an L-amino acid by cultivating the bacterium of the present invention in a culture medium to produce and excrete the L-amino acid into the medium, and collecting the L-amino acid from the medium.

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. The medium used for culture may be either synthetic or natural, so long as it includes a carbon source, a nitrogen source, minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth. The carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the mode of assimilation of the chosen microorganism, alcohol, including ethanol and glycerol, may be used. As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism can be used. 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.

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 ⁰C. 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.

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.

Examples

The present invention will be more concretely explained below with reference to the following non-limiting Examples.

Example 1. Construction of a strain with an inactivated tolC gene

1. Deletion of the tolC gene

A strain in which the tolC gene had been deleted was constructed by the method initially developed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) called "Red-driven integration". The DNA fragment containing the Cm^R marker encoded by the cat gene was obtained by PCR, using primers Pl (SEQ ID NO: 3) and P2 (SEQ ID NO: 4) and plasmid pMWl 18-attL-Cm-attR as a template (WO 05/010175). Primer Pl contains both a region complementary to the region located at the 5' end of the tolC gene and a region complementary to the attR region. Primer P2 contains both a region complementary to the region located at the 3' end of the tolC gene and a region complementary to the attL region. Conditions for PCR were as follows: denaturation step for 3 min at 95 °C; profile for two first cycles: 1 min at 95 °C, 30 sec at 50 ⁰C, 40 sec at 72 ⁰C; profile for the last 25 cycles: 30 sec at 95 °C, 30 sec at 54 ⁰C, 40 sec at 72 °C; final step: 5 min at 72 °C.

A 1699-bp PCR product (Fig. 1) was obtained and purified in agarose gel and was used for electroporation of the E. coli strain MGl 655 (ATCC 700926), which contains the plasmid pKD46 having a temperature-sensitive replication control region. The plasmid pKD46 (Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 2000, 97:12:6640- 45) includes a 2,154 nucleotide DNA fragment of phage 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 P₃T₃B 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, United States of America).

Electrocompetent cells were prepared as follows: E. coli MG1655/pKD46 was grown overnight at 30 °C in LB medium containing ampicillin (100 mg/1), and the culture was diluted 100 times with 5 ml of SOB medium (Sambrook et al, "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, 1989) containing ampicillin and L-arabinose (1 mM). The cells were grown with aeration at 30 °C to an OD₆oo of «0.6 and then were made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H₂O. Electroporation was performed using 70 μl 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 ⁰C for 2.5 hours and then were plated onto L-agar containing chloramphenicol (30 μg/ml) and grown at 37 °C to select Cm^R recombinants. Then, to eliminate the pKD46 plasmid, two passages on L-agar with Cm at 42°C were performed and the resulting colonies were tested for sensitivity to ampicillin.

2. Verification of the tolC gene deletion by PCR The mutants having the tolC gene deleted and marked with the Cm resistance gene were verified by PCR. Locus-specific primers P3 (SEQ ID NO: 5) and P4 (SEQ ID NO: 6) were used in PCR for the verification. Conditions for PCR verification were as follows: denaturation step for 3 min at 94 °C; profile for 30 cycles: 30 sec at 94 °C, 30 sec at 54 °C, 1 min at 72 °C; final step: 7 min at 72 ⁰C. The PCR product obtained in the reaction with the cells of parental tolC ⁺ strain MGl 655 as a template, was ~2.0 kbp in length. The PCR product obtained in the reaction with the cells of mutant strain as the template was -1.7 kbp in length (Fig. 2). The mutant strain was named MGl 655 ΔtolC::cat.

Example 2. Production of L-threonine by E. coli strain B-3996-ΔtolC

To test the effect of inactivation of the tolC gene on threonine production, DNA fragments from the chromosome of the above-described E. coli MG1655 ΔtolC::cat was transferred to the threonine-producing E. coli strain VKPM B-3996 by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain B-3996ΔtolC.

Both E. coli strains, B-3996 and B-3996-ΔtolC, 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 0.2 ml (10%) of seed material. The fermentation was performed in 2 ml of minimal medium for fermentation in 20x200-mm test tubes. Cells were grown for 65 hours at 32 ⁰C with shaking at 250 rpm.

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 CdCl₂, and the amount of L-threonine was estimated spectrophotometrically at 540 nm. The results of five independent test tube fermentations are shown in Table 1. As follows from Table 1, B-3996-ΔtolC produced a higher amount of L- threonine, as compared with B-3996.

The composition of the fermentation medium (g/1) is as follows:

Glucose 80.0 (NH₄)₂SO₄ 22.0

NaCl 0.8

KH₂PO₄ 2.0

MgSO₄-7H₂O 0.8

FeSO₄-7H₂O 0.02

MnSO₄-5H₂O 0.02

Thiamine HCl 0.0002

Yeast extract 1.0

CaCO₃ 30.0

Glucose and magnesium sulfate are sterilized separately. CaCO₃ is sterilized by dry- heat at 180 ⁰C for 2 hours. The pH is adjusted to 7.0. The antibiotic is introduced into the medium after sterilization.

Table 1

Example 3. Production of L-lysine by E. coli AJl 1442-ΔtolC To test the effect of inactivation of the tolC gene on lysine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔtolC::cat can be transferred to the Iy sine-producing E. coli strain AJl 1442 by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain AJl 1442-ΔtolC. The strain AJl 1442 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Tsukuba Central 6, 1-1, Higashi 1- Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on May 1, 1981 and received an accession number of FERM P-5084. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on October 29, 1987, and received an accession number of FERM BP- 1543. Both E. coli strains, AJl 1442 and AJl 1442-ΔtolC, 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.

The composition of the fermentation medium (g/1) is as follows:

Glucose 40

(NH₄)₂SO₄ 24

K₂HPO₄ 1.0

MgSO₄-7H₂O 1.0

FeSO₄-7H₂O 0.01

MnSO₄-5H₂O 0.01

Yeast extract 2.0

The pH is adjusted to 7.0 by KOH and the medium is autoclaved at 115 ⁰C for 10 min. Glucose and MgSO₄-7H₂O are sterilized separately. CaCO₃ is dry-heat sterilized at 180 ⁰C for 2 hours and added to the medium for a final concentration of 30 g/1.

Example 4. Production of L-cysteine by E. coli strain JM15(ydeD)- ΔtolC

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

E. coli strain JM15(ydeD) is a derivative of E. coli strain JM 15 (US Patent No. 6,218,168) which can be transformed with DNA having the y deD gene, which codes for a membrane protein, and is not involved in a biosynthetic pathway of any L-amino acid (U.S. Patent No. 5,972,663). The strain JMl 5 (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/).

Fermentation conditions for evaluation of L-cysteine production were described in detail in Example 6 of US Patent No. 6,218,168. Example 5. Production of L-leucine by E. coli 57-ΔtolC

To test the effect of inactivation of the tolC gene on L-leucine production, DNA fragments from the chromosome of the above-described E. coli strain MGl 655 ΔtolC::cat can be transferred to the E. coli L-leucine-producing strain 57 (VKPM B-7386, US Patent No. 6,124,121) by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain 57-pMW-ΔtolC. 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.

Both E. coli strains, 57 and 57-ΔtolC, can be cultured for 18-24 hours at 37 ⁰C 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).

The composition of the fermentation medium (g/1) (pH 7.2) is as follows:

Glucose 60.0

(NH₄)₂SO₄ 25.0

K₂HPO₄ 2.0

MgSO₄-7H₂O 1.0

Thiamine 0.01

CaCO₃ 25.0

Glucose and CaCO₃ are sterilized separately.

Example 6. Production of L-histidine by E. coli strain 80-ΔtolC To test the effect of inactivation of the tolC gene on L-histidine production, DNA fragments from the chromosome of the above-described E. coli MGl 655 ΔtolC::cat can be transferred to the histidine-producing E. coli strain 80 by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 80-ΔtolC. 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- 7270 and then converted to a deposit under the Budapest Treaty on July 12, 2004.

Both E. coli strains, 80 and 80-ΔtolC, 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.

The composition of the fermentation medium (g/1) is as follows (pH 6.0):

Glucose 100.0

Mameno (soybean hydrolysate) 0.2 of as total nitrogen

L-proline 1.0

(NH₄)₂SO₄ 25.0

KH₂PO₄ 2.0

MgSO₄-7H₂0 1.0

FeSO₄-7H₂0 0.01

MnSO₄ 0.01

Thiamine 0.001

Betaine 2.0

CaCO₃ 60.0

Glucose, proline, betaine and CaCO₃ are sterilized separately. The pH is adjusted to 6.0 before sterilization.

Example 7. Production of L-glutamate by E. coli strain VL334thrC⁺-ΔtolC To test the effect of inactivation of the tolC gene on L-glutamate production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔtolC::cat can be transferred to the E. coli L-glutamate-producing strain VL334thrC⁺ (EP 1172433) by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain VL334thrC⁺-ΔtolC. 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.

Both strains, VL334thrC⁺ and VL334thrC⁺-ΔtolC, can be grown for 18-24 hours at 37 ⁰C on L-agar plates. Then, one loop of the cells can be transferred into test tubes containing 2ml of fermentation medium. The fermentation medium contains glucose (60g/l), ammonium sulfate (25 g/1), KH₂PO₄ (2g/l), MgSO₄ (I g/1), thiamine (0.1 mg/ml), L-isoleucine (70 μg/ml), and CaCO₃ (25 g/1). The pH is adjusted to 7.2. Glucose and CaCO₃ 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:l:l) with subsequent staining by ninhydrin (1% solution in acetone) and further elution of the compounds in 50% ethanol with 0-5% CdCl₂.

Example 8. Production of L-phenylalanine by E. coli strain AJ12739-ΔtolC To test the effect of inactivation of the tolC gene on L-phenylalanine production, DNA fragments from the chromosome of the above-described E. coli MG1655 ΔtolC::cat can be transferred to the phenylalanine-producing E. coli strain AJl 2739 by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain AJ12739-ΔtolC. The strain AJl 2739 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.

Both strains, AJ12739-ΔtolC 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.

The composition of the fermentation medium (g/1) is as follows:

Glucose 40.0

(NH₄)₂SO₄ 16.0

K₂HPO₄ 0.1

MgSO₄-7H₂O 1.0

FeSO₄-7H₂O 0.01

MnSO₄-5H₂O 0.01

Thiamine HCl 0.0002

Yeast extract 2.0

Tyrosine 0.125

CaCO₃ 20.0

Glucose and magnesium sulfate are sterilized separately. CaCO₃ is dry-heat sterilized at 180 ⁰C for 2 hours. The pH is adjusted to 7.0.

Example 9. Production of L- tryptophan bv E. coli strain SVl 64 fpGH5)-ΔtolC To test the effect of inactivation of the tolC gene on L-tryptophan production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔtolC::cat can be transferred to the tryptophan-producing E, coli strain SVl 64 (pGH5) by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain SV164(pGH5)-ΔtolC. The strain SV164 has the trpE allele encoding anthranilate synthase not subject to feedback inhibition by tryptophan. The plasmid pGH5 harbors a mutant serA gene encoding phosphoglycerate dehydrogenase not subject to feedback inhibition by serine. The strain SVl 64 (pGH5) was described in detail in US patent No. 6,180,373 or European patent 0662143.

Both strains, SV164(ρGH5)-ΔtolC and SV164(pGH5), can be cultivated with shaking at 32 °C for 18 hours in 3 ml of nutrient broth supplemented with tetracycline (10 mg/1, marker of pGH5 plasmid). The obtained cultures (0.3 ml each) can be inoculated into 3 ml of a fermentation medium containing tetracycline (10 mg/1) in 20 x 200-mm test tubes, and cultivated at 32 °C for 72 hours with a rotary shaker at 250 rpm. After cultivation, the amount of tryptophan which accumulates in the medium can be determined by TLC as described in Example 9.

The fermentation medium components are listed in Table 2, but should be sterilized in separate groups (A, B, C₅ D, E, F₃ G and H), as shown, to avoid adverse interactions during sterilization.

Table 2

The pH of solution A is adjusted to 7.1 with NH₄OH. Each group is sterilized separately, chilled, and then mixed together.

Example 10. Production of L-proline by E. coli strain 702ilvA-ΔtolC To test the effect of inactivation of the tolC gene on L-proline production, DNA fragments from the chromosome of the above-described E. coli strain MGl 655 ΔtolC::cat can be transferred to the proline-producing E. coli strain 702ilvA by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 702ilvA-ΔtolC. 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.

Both E. coli strains, 702ilvA and 702ilvA-ΔtolC, 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 11. Production of L-arginine by E. coli strain 382-ΔtolC

To test the effect of inactivation of the tolC gene on L-arginine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔtolC::cat can be transferred to the arginine-producing E. coli strain 382 by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 382-ΔtolC. 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.

Both strains, 382-ΔtolC 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 2 ml of a fermentation medium in 20 x 200-mm test tubes and cultivated at 32 °C for 48 hours on a rotary shaker.

After the cultivation, the amount of L-arginine which had 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 CdCl₂, and the amount of L-arginine can be estimated spectrophotometrically at 540 run.

The composition of the fermentation medium (g/1) is as follows:

Glucose 48.0

(NH4)₂SO₄ 35.0

KH₂PO₄ 2.0

MgSO₄-7H₂O 1.0

Thiamine HCl 0.0002

Yeast extract 1.0 L-isoleucine 0.1

CaCO₃ 5.0

Glucose and magnesium sulfate are sterilized separately. CaCO₃ is dry-heat sterilized at 180°C for 2 hours. The pH is adjusted to 7.0.

Example 12. Elimination of Cm resistance gene (cat gene) from the chromosome of L-amino acid-producing E. coli strains.

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 ΔtolC::cat transferred by Pl transduction 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 at 30°C. Transformant clones can be cured from the cat gene by spreading the separate colonies at 37⁰C (at that temperature repressor Cits is partially inactivated and transcription of the int/xis genes is derepressed) followed by selection of Cm^sAp^R variants. Elimination of the cat gene from the chromosome of the strain can be verified by PCR. Locus-specific primers P3 (SEQ ID NO: 5) and P4 (SEQ ID NO: 6) 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 much shorter (~0.3-0.5 kbp in length) then strain with cat gene.

While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. All the cited references herein are incorporated as a part of this application by reference.

Industrial Applicability

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

Claims

1. An L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein said bacterium has been modified to attenuate expression of the tolC gene.

2. The bacterium according to claim 1, wherein said expression of the tolC gene is attenuated by inactivating the tolC gene.

3. The bacterium according to claim 1, wherein said bacterium belongs to the genus

Escherichia.

4. The bacterium according to claim 1, wherein said bacterium belongs to the genus

Pantoea.

5. The bacterium according to any of claims 1 to 4, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

6. The bacterium according to claim 5, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.

7. The bacterium according to claim 5, 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.

8. A method for producing an L-amino acid comprising:

- cultivating the bacterium according to any of claims 1 to 7 in a medium, and

- collecting said L-amino acid from the medium.

9. The method according to claim 8, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

10. The method according to claim 9, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.

11. The method according to claim 9, 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.