WO2008044714A1

WO2008044714A1 - Process for the preparation of l-threonine employing a bacterium of the enterobacteriaceae family with enhanced mdte and mdtf expression

Info

Publication number: WO2008044714A1
Application number: PCT/JP2007/069782
Authority: WO
Inventors: Yoshinori Tajima; Shintaro Iwatani; Yoshihiro Usuda; Kazuhiko Matsui
Original assignee: Ajinomoto Co., Inc.
Priority date: 2006-10-04
Filing date: 2007-10-02
Publication date: 2008-04-17

Abstract

The present invention provides a method for producing an L-amino acid by cultivating a bacterium having an L-amino acid-producing ability in a medium, and collecting the L-amino acid from the medium, wherein said bacterium belongs to the Enterobacteriaceae family, and is modified so that expression of the mdtE and mdtF genes is enhanced.

Description

DESCRIPTION

PROCESS FOR THE PREPARATION OF L-THREONINE EMPLOYING A BACTERIUM OF THE ENTEROBACTERIACEAE FAMILY WITH ENHANCED MDTE AND MDTF EXPRESSION

Technical Field [0001]

The present invention relates to a method for producing an L-amino acid using a bacterium, and more particularly, to a method for producing an L-amino acid including L-lysine, L-threonine, and L-tryptophan. L-lysine, L-threonine, and L-tryptophan are industrially useful as additives in animal feeds, components of health foods, amino acid infusions, and the like.

Background Art [0002]

Known methods for producing a target substance such as an L-amino acid by fermentation using a bacterium include using a wild-type bacterium (wild-type strain), using a nutrient-auxotrophic strain derived from a wild-type strain, using a metabolic regulation mutant strain such as a drug-resistant mutant strain derived from a wild-type strain, and using a strain having both nutrient-auxotrophy and metabolic regulation mutant. [0003]

In recent years, production of a target substance by fermentation can also be performed using a recombinant strain which is modified by recombinant DNA techniques. For example, the L-amino acid-producing ability of a bacterium can be enhanced by increasing the expression of genes encoding L-amino acid biosynthetic enzymes (U.S. Patent No. 5,168,056 and U.S. Patent No. 5,776,736) or by enhancing the influx of a carbon source into the L-amino acid biosynthetic pathway (U.S. Patent No. 5,906,925). [0004]

Multidrug resistance transporters have been discovered in many bacteria, such as Escherichia coli, and are known to protect against the invasion of environmentally harmful substances of various structures. Many multidrug resistance transporters have been discovered in Escherichia coli, and the transporters are known to be inducibly expressed and involved in the excretion of various drugs (Microbil. MoI. Biol. Rev. 2002. 66(4): 671-701. Grkovic, S., Brown, M. H., and Skurray, R. A. Regulation of bacterial drug export systems.). MdtEF, a member of the resistance nodulation cell division family (RND), includes the MdtE and MdtF proteins, encoded by the mdtE and mdtF genes, respectively. The mdtE and mdtF genes are a part of the mdtEF operon (also called the yhiUV operon), which is known to impart resistance to drugs such as doxorubicin, rhodamine 6G, and benzalkonium to the host bacterium (J. Bacteriol. 2001. 183(20): 5803-5812. Nishino, K., and Yamaguchi, A. Analysis of a complete library of putative drug transporter genes in Escherichia coli.). However, there are no reports of the production of an L-amino acid using a bacterium which is modified so that the expression of the mdtEF operon is enhanced.

Disclosure of the Invention [0005]

An object of the present invention is to provide a bacterium that belongs to the Enterobacteriaceae family and is capable of effectively producing an L-amino acid, and to provide a method of effectively producing an L-amino acid using the bacterium. [0006]

The inventors of the present invention have made intensive studies to solve the above-mentioned object. As a result, they have found that the ability of a bacterium to produce L-amino acids is improved by modifying the bacterium so that expression of the mdtE and mdtF genes encoding the multidrug resistance transporter is enhanced, thus completed the present invention. [0007]

It is an object of the present invention to provide a method for producing an L-amino acid comprising cultivating a bacterium having an L-amino acid-producing ability in a medium, and collecting the L-amino acid from the medium, wherein said bacterium belongs to the Enterobacteriaceae family, and is modified so that expression of the mdtE and mdtF genes is enhanced.

It is a further object of the present invention to provide the method as described above, wherein the mdtE gene is a DNA selected from the group consisting of ):

(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1; and

(b) a DNA that is able to hybridize with the nucleotide sequence complementary to SEQ ID NO: 1, or with a probe which can be prepared from the nucleotide sequence of SEQ ID NO: 1, under stringent conditions, and encodes a protein with multidrug resistance transporter activity in the presence of the MdtF protein. It is a further object of the present invention to provide the method as described above, wherein the mdtF gene is a DNA selected from the group consisting of :

(c) a DNA comprising the nucleotide sequence of SEQ ID NO: 3; and

(d) a DNA that is able to hybridize with the nucleotide sequence complementary to SEQ ID NO: 3, or with a probe which can be prepared from the nucleotide sequence of SEQ ID NO: 3, under stringent conditions, and encodes a protein with multidrug resistance transporter activity in the presence of the MdtE protein.

It is a further object of the present invention to provide the method as described above, wherein the mdtE gene encodes a protein selected from the group consisting of:

(A) a protein comprising the amino acid sequence of SEQ ID NO: 2; and

(B) a protein comprising an amino acid sequence which includes substitutions, deletions, insertions, additions, or inversions of one or several amino acids in SEQ ID NO: 2 and has multidrug resistance transporter activity in the presence of the MdtF protein.

It is a further object of the present invention to provide the method as described above, wherein the mdtF gene encodes a protein selected from the group consisting of:

(C) a protein comprising the amino acid sequence of SEQ ID NO: 4; and

(D) a protein comprising an amino acid sequence which includes substitutions, deletions, insertions, additions, or inversions of one or several amino acids in SEQ ID NO: 4 and has multidrug resistance transporter activity in the presence of the MdtE protein.

It is a further object of the present invention to provide the method as described above, wherein expression of the mdtE and mdtF genes is enhanced by increasing the copy numbers of the genes or by modifying expression regulatory sequences of the genes.

It is a further object of the present invention to provide the method as described above, wherein the bacterium belongs to a genus selected from the group consisting of Escherichia, Enterobacter, Pantoea, Klebsiella, and Serratia.

It is a further object of the present invention to provide the method as described above, wherein the L-amino acid is selected from the group consisting of L-lysine, L-threonine, and L-tryptophan.

Description of the Preferred Embodiments [0008]

Hereinafter, the present invention will be described in detail. [0009]

The bacterium of the present invention is a member of the Enterobacteriaceae family, has an L-amino acid-producing ability, and is modified so that expression of the mdtE and mdtF genes is enhanced. Herein, the term "L-amino acid-producing ability" refers to the ability to produce and accumulate an L-amino acid at a sufficient level to be collected from a medium or bacterial cells when the bacterium is cultured in the medium. The bacterium of the present invention may have the ability to produce a plurality of L-amino acids. The ability of the bacterium to produce L-amino acids may be a native ability, or may be obtained by modifying any one of the bacteria mentioned below with a mutagenesis treatment or a recombinant DNA technique. [0010]

Meanwhile, the phrase "expression of a gene is enhanced" refers to the enhancement of a transcription and/or translation level of a gene. [0011]

The kind of the L-amino acid is not particularly limited, but examples thereof include: basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, and L-citrulline; aliphatic amino acids such as L-isoleucine, L-alanine, L-valine, L-leucine, and glycine; hydroxy monoaminocarboxylic acids such as L-threonine and L-serine; cyclic amino acid such as L-proline; aromatic amino acids such as L-phenylalanine, L-tyrosine, and L-tryptophan; sulfur-containing amino acids such as L-cysteine, L-cystine, and L-methionine; and acidic amino acids such as L-glutamic acid, L-aspartic acid, L-glutamine, and L-asparagine. In particular, L-lysine, L-threonine, and L-tryptophan are preferred. [0012] <1-1> Imparting L-amino acid-producing ability

Hereinafter, methods of imparting an L-amino acid-producing ability will be described, as well as examples of bacteria to which an L-amino acid-producing ability have been imparted. However, the bacterium is not limited thereto, as long as it has an L-amino acid-producing ability. [0013]

Bacteria to be used in the present invention include, but are not limited to, bacteria belonging to the Enterobacteriaceae family such as those belonging to the genus Escherichia, Enter obacter, Pantoea, Klebsiella, Serratia, Erwinia, Salmonella, or Morganellcrwhich are able to produce L-amino acids. Specifically, bacteria belonging to the Enterobacteriaceae family according to the classification shown in NCBI (National Center for Biotechnology Information) database

(www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) can be used. Bacteria belonging to the genus Escherichia, Enterobacter, or Pantoea can be preferably used as the parent strain which is modified. [0014]

Escherichia bacteria which can be used as the parent strain to derive the bacterium of the present invention include, but are not limited to, Escherichia bacteria reported in Neidhardt et al. (Backmann, B.J. 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, p. 2460-2488. Table 1. In F. D. Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology/Second Edition, American Society for Microbiology Press, Washington, D. C), such as Escherichia coli. Specific examples of Escherichia coli include Escherichia coli W3110 strain (ATCC No. 27325), and MGl 655 strain (ATCC No. 47076), which are derived from the wild-type (prototype) Escherichia coli Kl 2 strain. [0015]

These strains are available from the American Type Culture Collection (ATCC) (Address: P.O. Box 1549, Manassas, VA 20108, 1, United States of America). That is, each strain is given a unique registration number which is listed in the catalogue of the ATCC (www.atcc.org/). Strains can be ordered using this registration number. [0016]

Examples of Enterobacter bacteria include Enter obacter agglomerans and Enterobacter aerogenes, and an example of Pantoea bacteria is Pantoea ananatis. Recently, Enterobacter agglomerans was reclassified in some cases as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, or the like, based on an analysis of the nucleotide sequence of 16S rRNA. Therefore, bacteria of the present invention may belong to either the genus Enterobacter or the genus Pantoea, as long as they are classified in the Enterobacteriaceae family. When Pantoea ananatis is bred using genetic engineering techniques, Pantoea ananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207), and derivatives thereof may be used. These strains were identified and deposited as Enterobacter agglomerans when they were isolated, but as described above, these strains have been reclassified as Pantoea ananatis based on an analysis of the nucleotide sequence of 16S rRNA. [0017]

Examples of methods of imparting or enhancing the ability to produce L-amino acids to bacteria belonging to the Enterobacteriaceae family are described below. [0018]

In order to impart the L-amino acid-producing ability, methods may be used which are conventional in the breeding of Escherichia bacteria or the like, such as by acquiring nutrient-auxotrophic mutant strains, analogue resistant strains, or metabolic regulation mutant strains, or by creating recombinant strains having enhanced expression of L-amino acid biosynthetic enzymes (Amino Acid Fermentation, Japan Scientific Societies Press, first edition publication: May 30, 1986, p.77 to 100). In the present invention, properties such as nutrient-auxotrophy, analogue-resistance, and metabolic regulation mutation may be imparted alone or in combination. Furthermore, expression of one or more L-amino acid biosynthetic enzymes may be enhanced. Furthermore, imparting of such properties as nutrient-auxotrophy, analogue-resistance, and metabolic regulation mutation may be combined with enhancing the expression of the L-amino acid biosynthetic enzymes. [0019]

Nutrient-auxotrophic mutant strains, L-amino acid-analogue resistant strains, and metabolic regulation mutant strains that have an L-amino acid-producing ability can be obtained as follows. A parent strain or a wild-type strain is subjected to a typical mutation treatment, such as irradiation with X-rays or ultraviolet rays, or by treating with a mutagen, including N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ethylmethanesulfonate (EMS), followed by selection of the strains that exhibit nutrient-auxotrophy, analogue-resistance, or a metabolic regulation mutation, and have an L-amino acid-producing ability. [0020]

Hereinafter, L-lysine-producing bacteria and methods of constructing L-lysine-producing bacteria are exemplified.

Examples of parent strains which can be used to derive the L-lysine-producing bacteria of the present invention include bacteria resistant to an L-lysine analogue and bacteria having a metabolic regulation mutation. Examples of an L-lysine analogue include oxalysine, lysinehydroxamate, S-(2-aminoethyl)-L-cysteine (AEC), γ-methyllysine, and α-chlorocaprolactam. L-lysine analogue resistant strains can be obtained by treating a bacterium of the Enterobacteriaceae family with a conventional mutagenesis. Specific examples of an L-lysine analogue resistant strain and metabolic regulation mutant strain having an L-lysine-producing ability include Escherichia coli AJl 1442 strain (FERM BP-1543, NRRL B-12185; JP 56-18596 A and U.S. Patent No. 4346170) and Escherichia coli VL611 strain (JP 2000-189180 A). WC 196 strain (WO 96/17930) may be used as an L-lysine producing strain of Escherichia coli. WC 196 strain has been obtained by imparting AEC (S-(2-aminoethyl)-cysteine)-resistance to W3110 strain which was derived from Escherichia coli K- 12 strain. The WC 196 strain was named Escherichia coli AJl 3069 strain and deposited at the National Institute of Bioscience and Human- Technology, Agency of Industrial Science and Technology (currently, International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan) on December 6, 1994 and given an accession number of FERM P- 14690, and the deposit was then converted to an international deposit under the provisions of Budapest Treaty on September 29, 1995 and given an accession number of FERM BP-5252. [0021]

L-lysine producing bacteria can be constructed by enhancing the activity of an L-lysine biosynthetic enzyme. The activity of an L-lysine biosynthetic enzyme can be enhanced by increasing the copy number of the gene encoding the L-lysine biosynthetic enzyme or by modifying an expression regulatory sequence of the gene encoding the enzyme. Increasing the copy number or modifying the expression regulatory sequence can be performed in the same way as for the mdtE and mdtF genes as described below. [0022]

Examples of genes encoding L-lysine biosynthetic enzymes include, but are not limited to, genes encoding an enzyme in the diaminopimelate pathway such as the dihydrodipicolinate synthase gene {dap A: hereinafter, the words in parentheses represent the gene names), aspartokinase gene (lysC), dihydrodipicolinate reductase gene (dapB), diaminopimelate decarboxylase gene (lysA), diaminopimelate dehydrogenase gene (ddh) (WO96/40934), phosphoenolpyruvate carboxylase gene (ppc) (JP 60-87788 A), aspartate aminotransferase gene (aspC) (JP 06-102028 B), diaminopimelate epimerase gene (dapF) (JP 2003-135066), and aspartate semialdehyde dehydrogenase gene (asd) (WO 00/61723). Also included are genes encoding enzymes in the aminoadipic acid pathway such as homoaconitate hydratase (JP 2000-157276 A). [0023]

It is known that wild-type DDPS derived from Escherichia coli is regulated by feedback inhibition by L-lysine, while wild-type aspartokinase derived from Escherichia coli is regulated by suppression and feedback inhibition by L-lysine. Therefore, mutated forms oϊdapA and lysC genes encoding these enzymes are preferably used so that the enzymes encoded by the genes are not subject to feedback inhibition. [0024]

An example of a DNA encoding mutant DDPS desensitized to feedback inhibition by L-lysine includes a DNA encoding DDPS which has the amino acid sequence in which the histidine at position 118 is replaced by tyrosine. Meanwhile, an example of a DNA encoding mutant aspartokinase III (AKIII) desensitized to feedback inhibition by L-lysine includes a DNA encoding an AKIII having an amino acid sequence in which the threonine at position 352, the glycine at position 323, and the methionine at position 318 are replaced by isoleucine, asparagine and isoleucine, respectively (U.S. Patent No. 5661012 and U.S. Patent No. 6040160). Such mutant DNAs can be obtained by a site-specific mutation using PCR or the like. [0025]

Wide host-range plasmids RSFD80, pCABl, and pCABD2 are known to contain a mutant dapA gene encoding a mutant DDPS and a mutant lysC gene encoding a mutant AKIII (U.S. Patent No. 6040160). Escherichia coli JM 109 strain transformed with RSFD80 was named AJ12396 (U.S. Patent No. 6040160), and the strain was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology) on October 28, 1993 and given an accession number of FERM P- 13936, and the deposit was then converted to an international deposit under the provisions of Budapest Treaty on November 1, 1994 and given an accession number of FERM BP-4859. RSFD80 can be obtained from the AJ12396 strain by a conventional method. [0026]

Furthermore, in the bacterium of the present invention, the activity of an enzyme that catalyzes a reaction which branches off from the L-lysine biosynthetic pathway and produces a compound other than L-lysine may be decreased or may be made deficient. Examples of such an enzyme include homoserine dehydrogenase, lysine decarboxylase (cadA, idcC), and malic enzyme, and strains in which activities of such enzymes are decreased or deficient are described in WO 95/23864, WO 96/17930, WO 2005/010175, and the like. [0027]

Activities of these enzymes can be decreased or eliminated by introducing a mutation to the genes encoding the enzymes on the chromosome using a known mutation treatment, to thereby decrease or eliminate the activities of the enzymes in a cell. For example, decreasing or eliminating the activities of the enzymes can be attained by disrupting the genes encoding the enzymes on the chromosome by gene recombination or by modifying an expression regulatory sequence such as a promoter or Shine-Dalgarno (SD) sequence. In addition, this can also be attained by introducing an amino acid substitution (missense mutation) to the region encoding the enzymes on the chromosome, introducing a stop codon (nonsense mutation), introducing a frameshift mutation that adds or deletes one or two nucleotides, or deleting part of the gene (Journal of biological Chemistry 272: 8611-8617 (1997)). Meanwhile, the activities of the enzymes can also be decreased or eliminated by constructing a mutant gene which has a deletion in the coding region, and then replacing the normal gene on the chromosome with the mutant gene by homologous recombination, or introducing the mutant gene using a transposon or an IS factor. [0028]

For example, the following gene recombination methods can be used to introduce a mutation that decreases or eliminates the activities of the above-mentioned enzymes. A mutant gene is prepared by modifying a partial sequence of a target gene so that it does not encode an enzyme that can function normally. Then, a bacterium belonging to the Enterobacteriaceae family is transformed with a DNA containing the mutant gene to cause recombination of a gene on the bacterial chromosome with the mutant gene, thereby substituting the target gene on the chromosome with the mutant gene. Examples of this type of gene substitution using homologous recombination include the method using a linear DNA called "Red-driven integration" (Datsenko, K.A, and Wanner, B. L. Proc. Natl. Acad. Sci. USA. 97: 6640-6645 (2000), a combination of Red-driven integration and a cleavage system derived from λ phage (Cho, E.H., Gumport, R.I., Gardner, J.F.J. Bacteriol. 184: 5200-5203 (2002)) (WO 2005/010175), a method using a plasmid containing a temperature-sensitive replication origin (Datsenko, K.A, and Wanner, B. L. Proc. Natl. Acad. Sci. USA. 97: 6640-6645 (2000); U.S. Patent No. 6303383; JP 05-007491 A), and the like. Meanwhile, a site-specific mutation by gene substitution using homologous recombination can also be performed by using a plasmid which is not able to replicate in a host cell. [0029]

The above-described methods for enhancing the expression of the L-lysine biosynthetic enzymes' genes and for decreasing the activities of enzymes can also be applied to genes encoding other L-amino acid synthetic enzymes so that the ability to produce another L-amino acid is imparted to the bacterium of the Enterobacteriaceae family. Hereinafter, a bacterium to which the ability to produce an L-amino acid other than L-lysine is imparted will be exemplified. [0030]

Examples of parent strains which can be used to derive the

L-tryptophan-producing bacteria of the present invention 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 a mutation which results in resistance to the feedback inhibition may be introduced into these enzymes. Specifically, a bacterium belonging to the Enterobacteriaceae family and harboring the feedback resistant enzymes can be obtained by mutating the anthranilate synthase and phosphoglycerate dehydrogenase so as to be resistant to the feedback inhibition and introducing the mutant enzymes into the bacterium. Specific examples of strains having such a mutation include a strain obtained by introducing the plasmid pGH5 (WO 94/08031) which contains a serA gene which has been mutated so that it encodes feedback-desensitized phosphoglycerate dehydrogenase into E. coli SV 164 strain. SVl 64 strain was obtained by introducing a mutant gene encoding feedback-desensitized anthranilate synthase into E. coli KB862 (DSM7196) strain which is deficient in trpE (WO94/08031). [0031]

Examples of parent strains which can be used to derive the

L-tryptophan-producing bacteria of the present invention also include strains transformed with the tryptophan operon which contains a gene encoding desensitized anthranilate synthase (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 (trpBA). The tryptophan synthase consists of α and β subunits which are encoded by trp A and trpB, respectively. [0032] A strain which is deficient in trpR (a repressor of the tryptophan operon) and a strain having a mutation in trpR are also preferable as a tryptophan-producing strain (U.S. Patent No. 4,371,614 and WO2005/056776). [0033]

Strains in which malate synthase-isocitrate lyase-isocitrate dehydrogenasekinase/phosphatase operon (ace operon) is constitutively expressed or expression of the operon is enhanced are also preferable as a L-tryptophan-producing strain. Specifically, it is preferable that the promoter of the ace operon is not suppressed by the repressor iclR, or the suppression by iclR is inhibited or eliminated. Such strains can be obtained by disrupting the iclR gene or by modifying the expression regulatory sequence of the ace operon. A strain in which the expression of the ace operon is enhanced can be obtained by connecting a DNA comprising the ace operon to a strong promoter, and introducing it into cells by a plasmid or homologous recombination or by transferring it so that multiple copies of the DNAs are integrated into the chromosomal DNA. The ace operon includes aceB, aceA, and aceK. [0034]

Examples of parent strains which can be used to derive the

L-tryptophan-producing bacteria of the present invention also include E. coli AGX 17 (pGX44) strain (NRRL B- 12263), which is auxotrophic for L-phenylalanine and L-tyrosine, and AGX6(pGX50)aroP strain (NRRL B- 12264) which harbors plasmid pGX50 comprising tryptophan operon (U.S. Patent No. 4,371,614). These strains are available from Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research (Peoria, Illinois 61604, USA).

Examples of parent strains which can be used to derive the

L-tryptophan-producing bacteria of the present invention also include a strain which has enhanced activity of 3-phosphoserine phosphatase (serB) (US4,371,614), a strain which has enhanced activity of phosphoenolpyruvate carboxykinase (pckA) (WO2004/090125), and a strain which constitutively expresses the glyoxylate pathway (WO2005/103275). [0035]

L-tryptophan, L-phenylalanine, and L-tyrosine are aromatic amino acids which have a common synthetic pathway. Examples of aromatic amino acid synthetic enzymes include 3-deoxyarabino-heptulosonic acid 7-phosphate synthase (aroG), 3-dehydrokinate synthase (aroB), shikimic acid dehydratase, shikimic acid kinase (aroL), 5-enol-pyruvylshikimic acid 3-phosphate synthase (aroA), and chorismic acid synthase (aroC) (EP763127A). Thus, the ability to produce these aromatic amino acids can be enhanced by increasing the copy number of a gene encoding one or more of these enzymes with a plasmid or on a chromosome. Furthermore, these genes are regulated by tyrosine repressor (tyrR) and therefore the ability to produce these aromatic amino acids may be enhanced by disrupting the tyrR gene (EP763127A). In order to enhance the ability to produce one of these aromatic amino acids, the biosynthetic pathway for the other aromatic amino acids may be attenuated. For example, the biosynthetic pathways for L-phenylalanine and L-tyrosine may be attenuated for the purpose of producing L-tryptophan (US4,371,614). [0036]

3-deoxyarabino-heptulosonic acid 7-phosphate synthase (aroF and αroG) is sensitive to feeback inhibition by aromatic amino acids, so the enzyme may be modified so as to be resistant to the feedback inhibition. For example, aromatic amino acids can be efficiently produced by introducing into a host a mutant aroF gene which encodes a mutant enzyme in which the aspartic acid residue at position 147 and the serine residue at position 181 are replaced with another amino acid residue and a mutant aroG gene encoding a mutant enzyme in which one of aspartic acid residue at position 146, methionine residue at position 147, proline residue at position 150, alanine residue at position 202 is replaced with another amino acid residue, or both the methionine residue at position 157 and the alanine residue at position 219 are replaced with another amino acid residue (EP0488424). [0037]

Examples of parent strains which can be used to derive the

L-phenylalanine-producing bacteria of the present invention include, but are not limited to, AJ12739 (tyrA::Tnl0, tyrR) (VKPM B-8197) strain which is deficient in tyrA and tyrR and a strain in which a phenylalanine exporting gene such as yddG gene and yedA gene is amplified (WO03/044192, and US2003/0148473A1, respectively). [0038]

Examples of parent strains which can be used to derive the L-threonine-producing bacteria of the present invention include, but are not limited to, bacteria belonging to the Enterobαcteriαceαe family in which activities of L-threonine biosynthetic enzymes are enhanced. Examples of genes encoding L-threonine synthetic enzymes include aspartokinase III gene (lysC), aspartate semialdehyde dehydrogenase (αsd), and aspartokinase I gene (thrA), homoserine kinase gene (thrB), and threonine synthase gene (thrC) which are encoded by the threonine operon. Two or more of the genes may be introduced. The genes encoding the L-threonine synthetic enzymes may be introduced into a bacterium belonging to the Enterobacteriaceae family in which threonine decomposition is decreased. An example of an E. coli strain in which threonine decomposition is decreased includes TDH6 strain which is deficient in threonine dehydrogenase activity (JP2001-346578A). [0039]

The activities of the L-threonine biosynthetic enzymes are inhibited by endoproduct L-threonine, so L-threonine biosynthetic enzymes are preferably modified so as to be desensitized to feedback inhibition by L-threonine for constructing L-threonine producing strains. The above-described thrA gene, thrB gene and thrC gene constitute a threonine operon with a promoter which has an attenuator structure. Since the expression of threonine operon is inhibited by isoleucine and threonine in the culture medium and also inhibited by attemuation, the threonine operon is preferably modified by removing leader sequence or attenuator in the attenuation region (Lynn, S. P., Burton, W. S., Donohue, T. J., Gould, R. M., Gumport, R. L, and Gardner, J. F. J. MoI. Biol. 194:59-69 (1987); WO02/26993; WO2005/049808). [0040]

The native promoter of the threonine operon may be replaced by a non-native promoter (WO98/04715), or the threonine operon may be connected to the repressor and promoter of λ-phage so that expression of the threonine synthetic genes is controlled by the repressor and promoter of λ-phage (EP0593792).

Furthermore, mutant Escherichia bacteria that are desensitized to feedback inhibition by L-threonine can be obtained by screening for strains resistant to α-amino β-hydroxy isovaleric acid (AHV). [0041]

It is preferable to increase the copy number of the thereonine operon that is modified so as to be desensitized to feedback inhibition by L-threonine in a host bacterium or increase the expression of the modified operon by connecting it to a potent promoter. The copy number can be increased by using, in addition to a plasmid, an transposon or Mu-phage so that the operon is transferred onto a chromosome of a host bacterium. [0042]

The gene encoding aspartokinase {lysC) is preferably modified to be desensitized to feedback inhibition by L-lysine. Such a modified lysC gene can be obtained by the method described in U.S. Patent No. 5,932,453.

[0043]

L-threonine producing bacterium can also be obtained by enhancing the expression of genes involved in glycolytic pathway, TCA cycle, or respiratory chain, or genes that regulate the expression of these genes, or genes involved in sugar uptake. Examples of these genes that are effective for L-threonine production include the transhydrogenase gene (pntAB)(EP7337l2B), phosphoenolpyruvate carboxylase gene (pepC)(WO95/06114), phosphoenolpyruvate synthase gene (p/λs)(EP877090B), pyruvate carboxylase gene derived from coryneform bacterium or Bacillus bacterium (WO99/18228, EP 1092776 A). [0044]

L-threonine producing bacterium can also be obtained by enhancing the expression of a gene that imparts L-threonine resistance and/or a gene that imparts L-homoserine resistance, or by imparting L-threonine resistance and/or L-homoserine resistance to the host bacterium. Examples of the genes that impart L-threonine resistance include the rhtA gene (Res. Microbiol. 154:123-135 (2003)), rhtB gene (EP0994190A), rhtC gene (EPlOl 3765A), yfiK gene, and yeaS gene (EP1016710A). Methods for imparting L-threonine resistance to a host bacterium are described in EP0994190A or WO90/04636. [0045]

E. coli VKPM B-3996 (U.S. Patent No. 5175107) may also be used as a parent strain to derive L-threonine-producing bacteria of the present invention. The strain B-3996 was deposited on April 7, 1987 in the the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika, (Russia, 117545 Moscow 1, Dorozhny proezd. 1) under the accession number VKPM B-3996. The strain B-3996 contains the plasmid pVIC40 (WO90/04636) which was obtained by inserting threonine biosynthetic genes (threonine operon: thrABC) into a wide host range plasmid vector pAYC32 containing the streptomycin resistance marker (Chistorerdov, A. Y., and Tsygankov, Y. D. Plasmid, 16, 161-167 (1986)). In pVIC40, the threonine operon contains a mutant thrA gene which encodes aspartokinase homoserine dehydrogenase I which is substantially desensitized to feedback inhibition by threonine. [0046]

E. coli VKPM B-5318 (EP 0593792B) also may be used as a parent strain to derive L-threonine-producing bacteria of the present invention. The VKPM B-5318 strain was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow 1, Dorozhny proezd. 1) on May 3, 1990 under accession number of VKPM B-5318. The VKPM B-5318 strain is prototrophic with regard to L-isoleucine, and harbors a plasmid DNA which contains the threonine operon encoding the threonine biosynthesis enzyme located downstream from the Cl temperature-sensitive represser, PR-promoter, and N-terminal of Cro protein derived from λ phage so that the expression of the threonine operon is regulated by the promoter and the repressor derived from λ phage. [0047]

Examples of parent strains which can be used to derive the L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains in which expression of one or more genes encoding an L-glutamic acid biosynthetic enzyme are enhanced. Examples of the enzymes involved in L-glutamic acid biosynthesis include glutamate dehydrogenase (GDH), glutamine synthetase, glutamate synthase, isocitrate dehydrogenase, aconitate hydratase, citrate synthase (CS), phosphoenolpyruvate carboxylase (PEPC), pyruvate carboxylase, pyruvate dehydrogenase, pyruvate kinase, phosphoenolpyruvate synthase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3-phophate dehydrogenase, triose phosphate isomerase, fructose bisphosphate aldolase, phosphofructokinase, and glucose phosphate isomerase. Among them, one or more of GDH, CS and PEPC is preferable, and all three of the enzymes are preferable. [0048]

Examples of strains belonging to the Enterobacteriaceae family which are 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 U.S. Patent No. 6197559, U.S. Patent No. 6331419, and EP0999282A. [0049]

Bacterium belonging to the Enterobacteriaceae family which is modified so that 6-phosphogluconate dehydratase activity and/or 2-keto-3-deoxy-6-phosphogluconate aldorase activity is/are enhanced (EP1352966A) may also be used. [0050]

Examples of parent strains which can be used to derive the L-glutamic acid-producing bacteria of the present invention also include strains which have decreased or no activity of an enzyme that catalyzes synthesis of a compound other than L-glutamic acid, and branches off from the L-glutamic acid biosynthetic pathway. Examples of such enzymes include 2-oxoglutarate dehydrogenase, isocitrate lyase, phosphotransacetylase, acetate kinase, acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, and 1 -pyrophosphate dehydrogenase. Among them, it is preferable to decrease or eliminate the 2-oxoglutarate dehydrogenase activity. [0051]

Methods for decreasing or eliminating 2-oxoglutarate dehydrogenase activity in a bacterium belonging to the Enterobacteriaceae family are described in U.S. Patent No. 5573945, U.S. Patent No. 6197559, and U.S. Patent No. 6331419. Examples of bacteria belonging to the Enterobacteriaceae family which are deficient in 2-oxoglutarate dehydrogenase activity or have a reduced 2-oxoglutarate dehydrogenase activity include the following:

Pantoea ananatis AJ 13601 (FERM BP-7207) Klebsiella planticola AJ 13410 (FERM BP-6617) Pantoea ananatis AJ13355 (FERM BP-6614) E. coli AJ12949 (FERM BP-4881) [0052]

Specific examples of strains which are able to produce L-histidine include E. coli FERM-P 5038 and 5048, which have been transformed with a vector carrying DNA encoding an L-histidine-biosynthetic enzyme (JP 56-005099 A), E. coli strains transformed with rht, which is a gene active in amino acid export (EPl 016710A), E. coli 80 strain imparted with sulfaguanidine, DL-l,2,4-triazole-3-alanine, and streptomycin-resistance (VKPM B-7270, Russian Patent No. 2119536). [0053]

Examples of parent strains which can be used to derive 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 these L-histidine-biosynthetic enzymes include ATP phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase (hisl), phosphoribosyl-ATP pyrophosphohydrolase (hisIE), phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (hisA), amidotransferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD), and so forth. [0054]

Examples of parent strains which can be used to derive L-cysteine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as an E. coli strain with decreased activity of cystathione beta lyase (JP2003-169668) and an E. coli strain harboring serine acetyltransferases that is resistant to feedback inhibition by L-cysteine (JPl 1-155571 A), and the like. [0055]

Examples of parent strains which can be used to derive L-proline-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli 702 (VKPM B-8011) which is resistant to 3,4-dehydroxyproline and azathidine-2-carboxylate, and E. coli 702ilvA (VKPM B-8012) which is derived from the 702 strain and deficient in the UvA gene (JP2002-300874). [0056]

Examples of parent strains which can be used to derive L-arginine-producing bacteria of the present invention include, but are not limited to, mutant strains of Escherichia coli, which are resistant to α-methylmethionine, p-fluorophenylalanine, D-arginine, arginine hydroxamate, S-(2-aminoethyl)-cysteine, α-methylserine, β-2-thienylalanine, or sulfaguanidine (JP56- 106598 A). E. coli strain 237 strain (VKPM B-7925) (Russian Patent Application No. 2000117677), which has a mutation that imparts resistance to feedback inhibition by L-arginine and has high N-acetylglutamate synthase activity, is also preferably used as an L-arginine producing strain. E. coli 237 strain was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) on April 10, 2000 under accession number VKPM B-7925 and then converted to an international deposit under the provisions of Budapest Treaty on May 18, 2001. E. coli 382 strain (JP2002-017342A), which is derived from the 237 strain and has enhanced acetate assimilating ability, may also be used to produce arginine. E. coli 382 strain was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) on April 10, 2000 under accession number VKPM B-7926. [0057]

Examples of parent strains which can be used to derive L-arginine-producing bacteria of the present invention also include strains in which expression is enhanced of one or more genes encoding an L-arginine biosynthetic enzyme. Examples of L-arginine biosynthetic enzymes include N-acetylglutamate synthase (argA), N-acetylglutamyl phosphate reductase (argC), ornithine acetyl transferase (argJ), N-acetylglutamate kinase (argB), acetylornithine transaminase (argD), acetylornithine deacetylase (argE), ornithine carbamoyl transferase (argF), argininosuccinic acid synthetase (argG), argininosuccinic acid lyase (argH), and carbamoyl phosphate synthetase {car AB). Among these, a mutant gene encoding N-acetylglutamate synthase (argA) in which the amino acid sequence at positions 15 to 19 is replaced resulting in inhibition of feedback inhibition by L-arginine is preferably used (EPl 170361 A). [0058]

Examples of parent strains which can be used to derive L-isoleucine-producing bacteria of the present invention include, but are not limited to, an Escherichia bacterium in which the branched chain amino acid transaminase encoded by the HvE gene is inactivated and the aromatic acid transaminase encoded by the tyrB gene is enhanced (JP2004-024259A), Escherichia coli strains which are resistant to 4-azaleucine or 5,5,5-trifluoroleucine, including the Escherichia coli H-9068 strain (ATCC21530), Escherichia coli H-9070 strain (FERM BP-4704), Escherichia coli H-9072 strain (FERM BP-4706)(U.S. Patent No. 5,744,331), an Escherichia coli strain which harbors isopropylmalate synthase desensitized to feedback inhibition by L-leucine (EPl 067191 B), and the Escherichia coli AJl 1478 strain which is resistant to β-2-thienylalanine and β-hydroxyleucine (U.S. Patent No. 5,763,231). [0059]

Examples of parent strains which can be used to derive L-isoleucine-producing bacteria of the present invention include, but are not limited to, mutants which are resistant to 6-dimethylaminopurine (JP 5-304969 A), mutants which are resistant to an isoleucine analogue such as isoleucine hydroxamate, thiaisoleucine, 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 (JP 2-458 A, JP2-42988 A, and JP8-47397 A). [0060]

An example of a parent strain which can be used to derive L-valine-producing bacteria of the present invention includes the Escherichia coli VL1970 strain (U.S. Patent No. 5,658,766). L-valine producing strains with a lipoic acid-auxortophic mutation and/or a proton ATPase-deficient mutation as disclosed in WO96/06926, and a strain which has been transformed with a DNA fragment including the HvGMEDA operon and which expresses at least the UvG, HvM, HvE, and HvD genes are also preferably used. It is desirable to remove the region of the HvGMEDA operon which is required for attenuation so that expression of the operon is not attenuated by the L-valine that is produced (U.S. Patent No. 5,998,178). Furthermore, the ilvA gene in the operon is desirably disrupted so that threonine deaminase activity is decreased. E. coli VL 1970, which has a mutation in the ileS gene encoding isoleucine tRNA synthetase, can also be used. E. coli VL 1970 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 113545 Moscow, 1 Dorozhny Proezd.) on June 24, 1988 under accession number VKPM B-4411. [0061]

Expression of genes other than L-amino acid biosynthetic genes may also be enhanced in the L-amino acid producing bacterium of the present invention, and examples of such genes include those encoding enzymes involved in sugar uptake, sugar metabolism (glycolytic pathway), and energy metabolism. [0062]

Genes involved in sugar metabolism include genes encoding enzymes in the glycolytic pathway or enzymes involved in sugar uptake. Examples thereof include the glucose-6-phosphate isomerase gene (pgi; WO 01/02542), phosphoenolpyruvate synthase gene (pps; EP 877090 A), phosphoglucomutase gene (pgm; WO 03/04598), fructose bisphosphate aldolase gene (fba; WO 03/04664), pyruvate kinase gene (pykF; WO 03/008609), transaldolase gene (talB; WO 03/008611), fumarase gene (fum; WO 01/02545), phosphoenolpyruvate synthase gene {pps; EP 877090 A), non-PTS sucrose uptake gene (esc; EP 149911 A), and sucrose-assimilating gene (scrAB operon; WO 90/04636). [0063]

Examples of genes encoding enzymes involved in energy metabolism include transhydrogenase gene (pntAB; U.S. Patent No. 5,830,716) and cytochromoe bo type oxidase gene (cyoB; EP 1070376). [0064]

The bacterium of the present invention can be obtained by modifying a bacterium having the L-amino acid-producing ability as described above so that expression of the mdtE and mdtF genes, which encode a multidrug resistance transporter, is enhanced. The L-amino acid-producing ability may be imparted or enhanced after the bacterium is modified so that expression of the mdtE and mdtF genes is enhanced. Either expression of the endogenous mdtE and mdtF genes may be enhanced by modification of an expression regulatory region such as a promoter, or expression of the mdtE and mdtF genes may be enhanced by introducing these genes on a plasmid . These methods may be combined. [0065]

The MdtE protein encoded by the mdtE gene and the MdtF protein encoded by the mdtF gene exhibit a multidrug resistance transporter activity in cooperation with each other.

In the present invention, the multidrug resistance transporter means a transport carrier protein having an activity to secrete at least one drug. The kind of the drug is not particularly limited, but examples thereof include doxorubicin, rhodamine 6G, and benzalkonium. The multidrug resistance transporter activity may be confirmed by expressing the mdtE and mdtF genes in a host bacterium and verifying that the resistance of the bacterium to the above-mentioned drugs is increased compared with that of an unmodified strain such as the wild-type strain. For example, a resistance to doxorubicin, rhodamine 6G, or benzalkonium may be evaluated by the method described in J. Bacterid. 183. 5803 (2001). [0066]

The increased expression of the mdtE and mdtF genes may be confirmed by comparison of the mRNA levels of the genes in the wild-type or unmodified strain. Examples of methods for measuring expression include Northern hybridization and Reverse-Transcriptase PCR (RT-PCR) (Sambrook, J., and Russell, D.W., Molecular Cloning A Laboratory Manual/Third Edition. New York: Cold Spring Harbor Laboratory Press (2001)). The expression may be at any level as long as it is increased compared with a wild-type or unmodified strain, and for example, the level is desirably increased 1.5-fold or more, more preferably 2-fold or more, and particularly preferably 3-fold or more compared with a wild-type or unmodified strain. An increase in expression may be confirmed by an increase in the level of the target proteins compared with a wild-type or unmodified strain, and the level may be detected by, for example, Western blotting using an antibody (Sambrook, J., and Russell, D. W., Molecular Cloning A Laboratory Manual/Third Edition. New York: Cold Spring Harbor Laboratory Press (2001)). [0067]

The mdtE and mdtF genes of the present invention include the mdtE and mdtF genes of, or native to, an Escherichia bacterium, and homologues thereof. An example of the mdtE gene native to Escherichia coli includes the gene (SEQ ID NO: 1) that encodes the protein of the amino acid sequence of SEQ ID NO: 2 (GenBank Accession No. AAC76538 [GI: 1789929]). An example of the mdtF gene native to Escherichia coli includes the gene (SEQ ID NO: 3) that encodes the protein of the amino acid sequence of SEQ ID NO: 4 (GenBank Accession No. AAC76539 [GI: 1789930]). [0068]

The phrase "homologues of mdtE and mdtF genes" indicates genes that are derived from, or native to, another microorganism, have high structural homology to the mdtE and mdtF genes native to Escherichia coli, and when introduced into a host, improve the L-amino acid-producing ability and impart a multidrug resistance transporter activity to the host bacterium. Examples of homologues of the mdtE and mdtF genes include the mdtE and mdtF genes native to a Shigella bacterium or the like, registered in Genbank. In addition, the mdtE and mdtF genes may be obtained by cloning, based on homology to the nucleotide sequence of SEQ ID NO: 1 or 3, from an Escherichia bacterium such as Escherichia coli, a Salmonella bacterium such as Salmonella Typhimurium, a Pseudomonas bacterium, or the like. The homologues may have different gene names as long as they have high homology to mdtE gene of SEQ ID NO: 1 or mdtF gene of SEQ ID NO: 3. For example, the homologue of mdtE gene includes a gene obtained by cloning using synthetic oligonucleotides of SEQ ID NOS: 6 and 8. The homologue of the mdtF gene also includes a gene obtained by cloning using synthetic oligonucleotides of SEQ ID NOS: 7 and 9.

The mdtE and mdtF genes may be derived from different microorganisms. [0069]

Furthermore, homologues of the mdtE and mdtF genes can be obtained by searching for genes having high homology from known databases based on the above-mentioned sequence information. The homology of amino acid sequences and nucleotide sequences may be determined by using, for example, an algorithm BLAST (Proc. Natl. Acad. Sci. USA, 90, 5873 (1993)) or FASTA (Methods EnzymoL, 183, 63 (1990)) created by Karlin and Altschul. Based on the algorithm BLAST, programs called BLASTN and BLASTX have been developed (//www.ncbi.nlm.nih.gov). [0070]

The mdtE gene to be used in the present invention is not limited to a wild-type gene and may be a mutant or an artificially modified gene that encodes a protein having the amino acid sequence of SEQ ID NO: 2 and which may include substitutions, deletions, insertions, additions of one or several amino acids at one or a plurality of positions, as long as the function of the MdtE protein encoded by the gene, that is, the function as a multidrug resistance transporter in cooperation with the MdtF protein, is maintained. The mdtF gene to be used in the present invention is not limited to a wild-type gene and may be a mutant or artificially modified gene that encodes a protein having an amino acid sequence of SEQ ID NO: 4 and which may include substitution, deletion, insertion, addition of one or several amino acids at one or a plurality of positions, as long as the function of the MdtF protein encoded by the gene, that is, the function as a multidrug resistance transporter in cooperation with the MdtE protein, is maintained.

In the present invention, the term "one or several" in referring to amino acids specifically means 1 to 20, preferably 1 to 10, and more preferably 1 to 5, although this determination depends on the position in the protein's tertiary structure and/or the types of amino acid residues in the protein. The above-mentioned substitution is preferably a conservative substitution, which may include substitutions between aromatic amino acids such as substitution among Phe, Tip and Tyr, substitution between hydrophobic amino acids such as substitution among Leu, He and VaI, substitution between polar amino acids such as substitution between GIn and Asn, substitution between basic amino acids such as substitution among Lys, Arg and His, substitution between acidic amino acids such as substitution between Asp and GIu, substitution between hydroxyl group-containing amino acids such as substitution between Ser and Thr. Examples of conservative substitutions include substitution of Ser or Thr for Ala; substitution of GIn, His or Lys for Arg; substitution of GIu, GIn, Lys, His or Asp for Asn; substitution of Asn, GIu or GIn for Asp; substitution of Ser or Ala for Cys; substitution of Asn, GIu, Lys, His, Asp or Arg for GIn; substitution of GIy, Asn, GIn, Lys or Asp for GIu; substitution of Pro for GIy; substitution of Asn, Lys, GIn, Arg or Tyr for His; substitution of Leu, Met, VaI or Phe for He; substitution of He, Met, VaI or Phe for Leu; substitution of Asn, GIu, GIn, His or Arg for Lys; substitution of He, Leu, VaI or Phe for Met; substitution of Trp, Tyr, Met, He or Leu for Phe; substitution of Thr or Ala for Ser; substitution of Ser or Ala for Thr; substitution of Phe or Tyr for Trp; substitution of His, Phe or Trp for Tyr; and substitution of Met, He or Leu for VaI. The above-mentioned amino acid substitution, deletion, insertion, addition or inversion may be a result of a naturally-occurring mutation (mutant or variant) due to an individual difference, or a difference of species of a bacterium harboring the mdtE gene or mdtF gene. Such a homologue gene can be obtained by modifying the nucleotide sequence of SEQ ID NO: 1 or 3 with site-specific mutagenesis so that the modified gene encodes a protein that has a substitution, deletion, insertion or addition of the amino acid residue at a specific position. [0071] The mdtE gene includes genes that have homology of not less than 80%, preferably not less than 90%, more preferably not less than 95%, particularly preferably not less than 97% to the entire amino acid sequences of SEQ ID NO: 2, and encode a protein which exhibits a multidrug resistance transporter activity in cooperation with the MdtF protein.

The mdtF gene includes genes that have homology of not less than 80%, preferably not less than 90%, more preferably not less than 95%, particularly preferably not less than 97% to the entire amino acid sequences of SEQ ID NO: 4, and encode a protein which exhibits a multidrug resistance transporter activity in cooperation with the MdtE protein.

The mdtE gene mdtF genes may be modified so that the genes comprise codons which are easily translated into amino acids in a host cell. Furthermore, each of the mdtE and mdtF genes may encode a protein which has a deletion or addition of an amino terminal portion or carboxy terminal portion of the MdtE protein and MdtF protein as long as the multidrug resistance transporter activity is maintained. The length of the amino acids to be deleted from the amino terminus or carboxy terminus or to be added at the amino terminus or carboxy terminus of the the MdtE protein and MdtF protein is not more than 50, preferably not more than 20, more preferably not more than 10, particularly preferably not more than 5. Specifically, the the MdtE protein and MdtF protein may have an amino acid sequence of SEQ ID NO: 2 and 4 in which 5 to 50 amino acids are deleted from the amino terminus or carboxy terminus, or an amino acid sequence of SEQ ID NO: 2 or 4 in which 5 to 50 amino acids are added to the amino terminus or carboxy terminus. [0072]

Homologues of the mdtE and mdtF genes can also be obtained by conventional mutagenesis technique. Examples of the mutagenesis technique include a method of treating the mdtE and mdtF genes with hydroxylamine in vitro and a method of treating a bacterium such as Escherichia bacterium which harbors the mdtE and mdtF genes with ultraviolet rays, or with a mutagen, including N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ethylmethanesulfonate (EMS). Whether the obtained gene encodes a protein having the multidrug resistance transporter activity can be confirmed by expressing the gene into a suitable host and evaluating that the host has been imparted with the multidrug resistance transporter activity. [0073] Meanwhile, the mdtE gene may hybridize with a complementary strand of the nucleotide sequence of SEQ ID NO: 1, or with a probe that can be prepared from the sequence under stringent conditions, and encodes a protein which exhibits a multidrug resistance transporter activity in the presence of the MdtF protein. The mdtF gene may hybridize with a complementary strand of the nucleotide sequence of SEQ ID NO: 3, or with a probe that can be prepared from the sequence under stringent conditions and encodes a protein which exhibits a multidrug resistance transporter activity in cooperation with the MdtE protein.

In the present invention, the term "stringent conditions" refers to conditions where a so-called specific hybrid is formed and a non-specific hybrid is not formed. It is difficult to clearly define the conditions by numerical value, but examples thereof include conditions where DNAs having high homology, for example, at least 80%, preferably 90%, more preferably 95%, and further more preferably 97% homology hybridize with each other and DNAs having homology less than the value do not hybridize with each other; and specifically include washing conditions typical of Southern hybridization, e.g., washing at 60°C, I xSSC, 0.1% SDS, preferably 60°C, 0.I xSSC, 0.1% SDS, more preferably 68°C, 0.1 *SSC, 0.1% SDS, once or preferably twice or three times. [0074]

As a probe, a partial sequence of the nucleotide sequence complementary to SEQ ID NO: 1 or 3 can also be used. Such a probe can be prepared by PCR using oligonucleotides produced based on the nucleotide sequence of SEQ ID NO: 1 or 3 as primers, and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 1 or 3 as a template. When a DNA fragment of a length of about 300 bp is used as the probe, the conditions of washing for the hybridization consist of, for example, 50°C, 2^χSSC, and 0.1% SDS. [0075]

Expression of the above-mentioned mdtE and mdtF genes can be increased by, for example, increasing the copy number of the genes in a cell using a gene recombination technique. For example, a DNA fragment containing the mdtE and mdtF genes is ligated to a vector that functions in the host bacterium, preferably a multi-copy vector, to thereby prepare a recombinant DNA, and the recombinant DNA is used to transform the host bacterium.

The mdtE and mdtF genes may be introduced into a host bacterium with separate vectors each containing mdtE gene or mdtF gene, or with a single vector containing both of the genes. When these genes are introduced with a single vector, these genes are preferably introduced as a part of the mdtEF operon. The mdtEF operon can be amplified using primers of SEQ ID NOs: 6 and 7 from the chromosomal DNA of E. coli. [0076]

The mdtE and mdtF genes of E. coli can be obtained by PCR (polymerase chain reaction; White, T.J. et al., Trends Genet. 5, 185 (1989)) using primers based on the nucleotide sequence of SEQ ID NOS: 1 or 3, for example, primers of SEQ ID NOS: 6 and 8 {mdtE gene), or 7 and 9 {mdtF gene) and the chromosomal DNA of Escherichia coli as a template. The mdtE and mdtF genes from other bacteria can also be obtained by PCR from the chromosomal DNA or a genomic DNA library of the chosen bacterium using, as primers, oligonucleotides prepared based on the known sequences of the mdtE gene or mdtF gene of the bacterium, or of the mdtE gene or mdtF gene of another kind of bacterium, or the known sequence of other multidrug resistance transporters; or hybridization using an oligonucleotide prepared based on the sequence as a probe. A chromosomal DNA can be prepared from a bacterium that serves as a DNA donor by the method of Saito and Miura (see H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Experiment Manual for Biotechnology, edited by The Society for Biotechnology, Japan, p97-98, Baifukan Co., Ltd., 1992) or the like. [0077]

Then, a recombinant DNA is prepared by ligating the mdtE and mdtF genes which have been amplified by PCR to a vector DNA which is capable of functioning in the host bacterium. Examples of the vector capable of functioning in the host bacterium include vectors autonomously replicable in the host bacterium. Examples of a vector which is autonomously replicable in Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pACYC184, (pHSG and pACYC are available from Takara Bio Inc.), RSFlOlO, pBR322, pMW219, pMWl 19 (pMW is available form Nippon Gene Co., Ltd.), and pSTV29 (Takara Bio Inc.). [0078]

To introduce a recombinant DNA prepared as described above into a microorganism, any known transformation method reported so far can be employed. For example, treating recipient cells with calcium chloride so as to increase the permeability of DNA, which has been reported for Escherichia coli (Mandel, M. and Higa, A., J. MoI. Biol., 53, 159 (1970)), and using competent cells prepared from growing cells to introduce a DNA, which has been reported for Bacillus subtilis (Duncan, C. H., Wilson, G. A. and Young, F.E., Gene, 1, 153 (1977)) can be employed. In addition to these methods, introducing a recombinant DNA into protoplast- or spheroplast-like recipient cells, which have been reported to be applicable to Bacillus subtilis, actinomycetes, and yeasts (Chang, S. and Choen, S.N., Molec. Gen. Genet., 168, 111 (1979); Bibb, M.J., Ward, J.M. and Hopwood, O.A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J.B. and Fink, G.R., Proc. Natl. Sci., USA, 75, 1929 (1978)), can be employed. [0079]

The copy number of the mdtE and mdtF genes can also be increased by introducing multiple copies of the genes into the chromosomal DNA of a bacterium. In this case, multiple copies of the mdtE gene and the mdtF gene may be introduced into the chromosomal DNA in a separate procedure by using fragments or vectors containing each of the genes or in a single procedure by using a fragment or a vector containing both of the genes. Introduction of multiple copies of the genes into the chromosomal DNA of a bacterium can be attained by homologous recombination using a target sequence present on the chromosomal DNA in multiple copies. Such a sequence present on a chromosomal DNA in multiple copies may be a repetitive DNA or an inverted repeat present on the edge of a transposing element. The mdtE and mdtF genes may be integrated tandemly in a region adjacent to the chromosomal mdtE and mdtF genes, or integrated into a region redundantly which is not necessary for the function of the host bacterium. Such kind of gene integration can be performed with a temperature-sensitive plasmid or integration vector. [0080]

Alternatively, as disclosed in JP 2-109985 A, multiple copies of the mdtE and mdtF genes can be introduced into the chromosomal DNA by inserting the genes into a transposon, and transferring it so that multiple copies of the gene are integrated into the chromosomal DNA. Integration of these genes into the chromosome can be confirmed by Southern hybridization using a portion of the genes as a probe. [0081]

Furthermore, expression of the mdtE and mdtF genes may also be enhanced by, as described in WO 00/18935, substituting an expression regulatory sequence such as a promoter of the genes on a chromosomal DNA or of the genes on a plasmid with a stronger promoter, modifying the sequences of "-35 region" and "-10 region" so that the sequences become a consensus sequence, amplifying a regulator that increases expression of the genes, or deleting or attenuating a regulator that decreases expression of the genes. Examples of known strong promoters include the lac promoter, tip promoter, trc promoter, tac promoter, araBA promoter, lambda phage PR promoter, PL promoter, tet promoter, T7 promoter, and Φ10 promoter. A promoter or SD sequence of the mdtE and mdtF genes can be modified so as to become a more potent promoter and a more potent SD sequence. Examples of a method of evaluating the strength of a promoter and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1995, 1, 105-128) or the like. In addition, it is known that a spacer sequence between the ribosome binding site (RBS) and translation initiation codon, especially, several nucleotides just upstream of the initiation codon, has a great influence on translation efficiency. Therefore, this sequence may be modified. Expression regulatory sequences of the mdtE and mdtF genes may be identified using a vector for promoter identification or genetic analysis software such as GENETYX. By substituting or modifying an expression regulatory sequence such as a promoter as described above, expression of the mdtE and mdtF genes is enhanced. Substitution of the expression regulatory sequence can also be performed by using a temperature sensitive plasmid or by Red-driven integration (WO2005/010175).

The mdtE and mdtF genes of E. coli form an mdtEF operon, and expression of the mdtE and mdtF genes is regulated by the same promoter. Therefore, when the mdtE and mdtF genes of E. coli are used, expression of both of the genes can be enhanced by modifying the promoter upstream of the mdtE gene. [0082] <2> Method of producing L-amino acid

The method of producing an L-amino acid of the present invention includes cultivating the bacterium of the present invention as described above in a medium to produce and accumulate an L-amino acid in the medium or bacterial cells, and collecting the L-amino acid from the medium or the bacterial cells. [0083]

A conventional medium to be used for fermentative production of an L-amino acid using a bacterium can be used. That is, a general medium containing a carbon source, nitrogen source, inorganic ion, and if necessary, other organic components can be used. In the present invention, examples of the carbon source include sugars such as glucose, sucrose, lactose, galactose, fructose and a starch hydrolysate; alcohols such as glycerol and sorbitol; and organic acids such as fumaric acid, citric acid and succinic acid. Examples of the nitrogen source include inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate; an organic nitrogen such as a soybean hydrolysate; ammonia gas; and aqueous ammonia. As organic trace nutrients, auxotrophic substances such as vitamin B 1 and L-homoserine, yeast extract, and the like are preferably contained in the medium in an appropriate amount. Besides such substances, if necessary, potassium phosphate, magnesium sulfate, iron ion, manganese ion, or the like may be added in small amounts. The medium to be used in the present invention may be a natural medium or a synthetic medium as long as it contains a carbon source, nitrogen source, inorganic ion, and if necessary, other organic trace nutrients. [0084]

L-amino acids which improve the growth or productivity may be added. For example, L-threonine, L-homoserine, or L-isoleucine is preferably added in L-lysine fermentation, and L-isoleucine, L-lysine, L-glutamic acid, or L-homoserine is preferably added in L-threonine fermentation, and L-phenylalanine, or L-tyrosine is preferably added in L-tryptophan fermentation. These amino acids are usually added at a concentration of 0.01-lOg/L. [0085]

The culture is preferably performed under aerobic conditions for 1 to 7 days at a temperature of 24°C to 37°C and a pH of 5 to 9. The pH can be adjusted with an inorganic or organic acidic or alkaline substance, ammonia gas or the like. The L-amino acid can be collected from the fermentation liquid by a conventional method such as ion-exchange resin, precipitation, and other known methods. When the L-amino acid accumulates in the bacterial cells, the L-amino acid can be collected, for example, by disrupting the bacterial cells by ultrasonication or the like to release L-amino acid into the supernatant fraction, and then the bacterial cells are removed by centrifugation, followed by subjecting the resulting supernatant to an ion-exchange resin or the like. [0086]

Hereinafter, the present invention will be described in more detail by referring to examples. [0087] Example 1

Construction of a plasmid for enhancing the mdtEF operon> <1-1> Construction of a plasmid for gene amplification

The entire nucleotide sequences of the genomes of Escherichia coli (Escherichia coli K- 12 strain) (Genbank Accession No. U00096) have been reported (Science, 277, 1453- 1474 ( 1997)). The plasmid pM WPthr was used for gene amplification. The plasmid was obtained by inserting a promoter region (SEQ ID NO: 5) of a threonine operon (thrABC) which is present on the genome of Escherichia coli in between the HmdIII site and Xbal site of the pMWl 18 vector (manufactured by Nippon Gene Co., Ltd.). This plasmid can be used to amplify a target gene by cloning the gene downstream of the promoter of the threonine operon. [0088] <l-2> Construction of a plasmid for enhancing the mdtEF operon

Based on the nucleotide sequence of the mdtEF operon which is present on the genome of Escherichia coli (3657255..3661550 of GenBank Accession No. U00096), PCR was performed using a synthetic oligonucleotide of SEQ ID NO: 6 with a Smal site as a 5'-primer, and a synthetic oligonucleotide of SEQ ID NO: 7 with a Sac/ site as a 3'-primer, and using the genomic DNA of Escherichia coli W3110 strain as a template. The PCR product was treated with restriction enzymes Smal and Sad, to thereby yield a gene fragment containing the mdtEF operon. The fragment was purified and ligated to the vector pMWPthr digested with Smal and Sad to construct the plasmid pMWmdtEF for amplifying the mdtEF operon. [0089] Example 2

Escherichia coli B-5318 strain (EP 0,593,792 A) was used as an L-threonine-producing strain of Escherichia coli. [0090]

The B-5318 strain was transformed with the plasmid pMWmdtEF prepared in Example 1 to amplify the mdtEF operon, and an ampicillin-resistant strain was selected. Introduction of the plasmid was confirmed, and the strain introduced with pMWmdtEF was named B-5318/mdtEF strain. A strain introduced with pMWl 18 was named B-5318/pMWl 18 strain and used as a control. [0091]

The strains prepared as decribed above were cultured at 37°C in an LB medium containing 50 mg/L ampicillin until the OD600 reached about 0.6, and then a 40% glycerol solution was added in the same amount as that of the culture medium, followed by mixing. Then, the solution was dispensed in appropriate amounts and stored in glycerol at -80°C (glycerol stock). [0092]

The glycerol stocks of the strains were thawed, and 100 μL of each of the glycerol stocks was uniformly applied on an L-plate containing 50 mg/L ampicillin, and the strains were cultured at 37°C for 24 hours. About one eighth of the bacterial cells on the plate were inoculated into 20 mL of the fermentation medium (L-threonine production medium shown below) with 50 mg/L ampicillin contained in a 500 mL-Sakaguchi flask, and the cells were cultured at 40°C for 18 hours using a reciprocal shaker. After the culture, the level of L-threonine which had accumulated in the medium was determined using the amino acid analyzer L-8500 (manufactured by Hitachi, Ltd.). The composition of the medium used in the culture is shown below. [0093]

[L-threonine production medium for Escherichia bacteria] Glucose 40 g/L

(NH₄)_ZSO₄ 16 g/L

KH₂PO₄ 1.0 g/L

MgSO₄ - 7H₂O 1.0 g/L

FeSO₄ - 7H₂O 0.01 g/L

MnSO₄ - 7H₂O 0.01 g/L

Yeast extract 2.0 g/L

CaCO₃ (JP grade) 30 g/L

The medium was adjusted to pH 7.0 with KOH and sterilized using an autoclave at 12O⁰C for 20 minutes. Glucose and MgSO₄-7H₂O were mixed and sterilized independently from the other components. CaCO₃ was added after dry heat sterilization.

Table 1 shows the OD and the amounts of L-threonine accumulated after 18 hours. [0094]

Table 1: Effect of amplification of the mdtEF operon in L-threonine producing B-5318 strain

Strain name OD (600 nm) Concentration of L-threonine (g/1)

B-5318/pMW118 9.2 7.3

B-5318/mdtEF 9.2 7.7

[0095]

In the case of the strain in which the mdtEF opeτon was amplified (B5318/mdtEF), the amount of accumulated L-threonine was significantly higher than the control strain

(B-5318/pMW118).

[0096]

[Explanation of the Sequence Listing]

SEQ ID NO: 1: nucleotide sequence of the mdtE gene

SEQ ID NO: 2: amino acid sequence encoded by the mdtE gene

SEQ ID NO: 3: nucleotide sequence of the mdtF gene

SEQ ID NO: 4: amino acid sequence encoded by the mdtF gene

SEQ ID NO: 5: nucleotide sequence of threonine operon promoter

SEQ ID NO: 6: 5'-primer for amplifying the mdtEF operon

SEQ ID NO: 7: 3'-primer for amplifying the mdtEF operon

SEQ ID NO: 8: primer for amplifying the mdtE gene

SEQ ID NO: 9: primer for amplifying the mdtF gene

Industrial Applicability

[0097]

By the method of the present invention, L-amino acids including L-lysine, L-threonine and L-tryptophan can be efficiently produced by fermentation.

Claims

1. A method for producing an L-amino acid comprising cultivating a bacterium having an L-amino acid-producing ability in a medium, and collecting the L-amino acid from the medium, wherein said bacterium belongs to the Enterobacteriaceae family, and is modified so that expression of the mdtE and mdtF genes is enhanced.

2. The method according to claim 1, wherein the mdtE gene is a DNA selected from the group consisting:

(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1; and

(b) a DNA that is able to hybridize with the nucleotide sequence complementary to SEQ ID NO: 1, or with a probe which can be prepared from the nucleotide sequence of SEQ ID NO: 1, under stringent conditions, and encodes a protein that exhibits multidrug resistance transporter activity in the presence of the MdtF protein.

3. The method according to claim 1, wherein the mdtF gene is a DNA selected from the group consisting of:

(c) a DNA comprising the nucleotide sequence of SEQ ID NO: 3; and

(d) a DNA that is able to hybridize with the nucleotide sequence complementary to SEQ ID NO: 3, or with a probe which can be prepared from the nucleotide sequence of SEQ ID NO: 3, under stringent conditions, and encodes a protein that exhibits a multidrug resistance transporter activity in the presence of the MdtE protein.

4. The method according to claim 1, wherein the mdtE gene encodes a protein selected from the group consisting of:

(A) a protein comprising the amino acid sequence of SEQ ID NO: 2; and

(B) a protein comprising an amino acid sequence which includes substitutions, deletions, insertions, additions, or inversions of one or several amino acids in SEQ ID NO: 2 and exhibits multidrug resistance transporter activity in the presence of the MdtF protein.

5. The method according to claim 1, wherein the mdtF gene encodes a protein selected from the group consisting of:

(C) a protein comprising the amino acid sequence of SEQ ID NO: 4; and (D) a protein comprising an amino acid sequence which includes substitutions, deletions, insertions, additions, or inversions of one or several amino acids in SEQ ID NO: 4 and exhibits multidrug resistance transporter activity in the presence of the MdtE protein.

6. The method according to any one of claims 1 to 5, wherein expression of the mdtE and mdtF genes is enhanced by increasing the copy numbers of the genes or by modifying expression regulatory sequences of the genes.

7. The method according to any one of claims 1 to 6, wherein the bacterium belongs to a genus selected from the group consisting of Escherichia, Enter obacter, Pantoea, Klebsiella, and Serratia.

8. The method according to any one of claims 1 to 7, wherein the L-amino acid is selected from the group consisting of L-lysine, L-threonine, and L-tryptophan.