# Patent

## Title
L-amino acid-producing bacterium and a method for producing an L-amino acid

## Abstract

An L-amino acid is produced by culturing a microorganism of the family Enterobacteriaceae which has the ability to produce an L-amino acid and which has been modified so as to increase the expression of the evgA gene, the gadE gene, and/or the ydeO gene. These genes encode a transcription factor involved in the EvgAS two-component system regulon. The culture takes place in a medium, and the L-amino acid is collected from the medium or cells.

## Background
The present application claims priority under 35 U.S.C. §119 to Japanese Patent Application No. 2005-279027, filed Sep. 27, 2005, U.S. Provisional Patent Application No. 60/723,936, filed Oct. 6, 2005, and Japanese Patent Application No. 2006-213584, filed Aug. 4, 2006, and is a continuation under 35 U.S.C. §120 of PCT/JP2006/319821, filed Sep. 27, 2006, the entirety of both of which is incorporated by reference. Also, the Sequence Listing on compact disk filed herewith is hereby incorporated by reference (File name: US-254 Seq List; File size: 132 KB; Date recorded: Mar. 26, 2008).


BACKGROUND OF THE INVENTION
Technical Field
The present invention relates to a method for producing an L-amino acid using a microorganism, and more specifically, to a method for producing an L-amino acid, such as L-lysine, L-threonine, L-tryptophan, etc. L-lysine, L-threonine, and L-tryptophan are used in industry as animal feed additives, health food ingredients, and for amino acid infusions.
Brief Description of the Related Art
To produce a target substance, such as an L-amino acid, etc., by fermentation using a microorganism, methods have been described which use a wild-type microorganism (wild-type strain), an auxotrophic strain derived from a wild-type strain, a metabolic regulation mutant strain as one of various types of drug-resistant mutant strains derived from a wild-type strain, a strain which has the characteristics of both the auxotrophic strain and metabolic regulation mutant strain, and so forth.
In recent years, recombinant DNA technology has been used to produce target substances by fermentation. For example, the ability of a microorganism to produce an L-amino acid has been improved by enhancing the expression of genes that encode L-amino acid biosynthesis enzymes (U.S. Pat. No. 5,168,056, U.S. Pat. No. 5,776,736), or by enhancing the influx of the carbon source to the L-amino acid biosynthesis system (U.S. Pat. No. 5,906,925).
The two-component system EvgAS has been identified in Enterobacteriaceae, such as Escherichia coli, etc. The function of the sensor kinase EvgS is unknown, but the response regulator transcription factor EvgA is known to regulate the transcription of many genes (Masuda, N. and Church, G. M., J. Bacteriol. 2002. 184(22):6225-6234. Escherichia coli gene expression responsive to levels of the response regulator EvgA.). EvgA is also known to positively regulate the transcription of the ydeO and gadE genes, which encode two transcription factors, YdeO and gadE, respectively. YdeO is also known to positively regulate the transcription of the gadE gene (Masuda, N. and Church, G. M., Mol. Microbiol. 2003. 48(3):699-712. Regulatory network of acid resistance genes in Escherichia coli.; Ma, Z., Masuda, N., and Foster, J. W., J. Bacteriol. 2004. 186(21):7378-7389. Characterization of EvgAS-YdeO-GadE branched regulatory circuit governing glutamate-dependent acid resistance in Escherichia coli.). However, the production of an amino acid using a microorganism with increased expression of the evgA, gadE, or ydeO genes has not been previously reported.
SUMMARY OF THE INVENTION
An aspect of the present invention is to provide a bacterial strain of the family Enterobacteriaceae which is capable of efficiently producing an L-amino acid, and to also provide a method for efficiently producing an L-amino acid using the bacterial strain.
The inventors of the present invention extensively studied in order to resolve the above-mentioned problems, resulting in the discovery that the L-amino acid-producing ability of a microorganism can be improved by modifying the microorganism to increase the expression of one or more of the evgA gene, gadE gene, or ydeO genes. These genes encode transcription factors involved in the EvgAS two-component system.
It is an aspect of the present invention to provide a method for producing an L-amino acid comprising culturing in a medium a bacterium of the family Enterobacteriaceae which has an ability to produce an L-amino acid and which has been modified to increase expression of a gene selected from the group consisting of evgA, gadE, ydeO, and combinations thereof, and collecting the L-amino acid from the medium and/or the bacterium.
It is a further aspect of the present invention to provide the method as described above, wherein the expression of said gene(s) is/are increased by a method selected from the group consisting of increasing the copy number of said gene(s), or modifying the expression regulatory sequence of said gene(s), and combinations thereof.
It is a further aspect of the present invention to provide the method as described above, wherein the expression of the evgS gene is increased.
It is a further aspect of the present invention to provide the method as described above, wherein the evgA gene is selected from the group consisting of:
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 23,
(b) a DNA which hybridizes with: i) a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 23, or ii) a probe prepared from said nucleotide sequence in i) under stringent conditions, and which encodes a protein having transcription factor activity.
It is a further aspect of the present invention to provide the method as described above, wherein the gadE gene is selected from the group consisting of:
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 27,
(b) a DNA which hybridizes with: i) a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 27, or ii) a probe that can be prepared from said nucleotide sequence in i) under stringent conditions, and which encodes a protein having transcription factor activity.
It is a further aspect of the present invention to provide the method as described above, wherein the ydeO gene is selected from the group consisting of:
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 29,
(b) a DNA which hybridizes with: i) a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 29, or ii) a probe prepared from said nucleotide sequence in i) under stringent conditions, and which encodes a protein having transcription factor activity.
It is a further aspect of the present invention to provide the method as described above, wherein the evgS gene is selected from the group consisting of:
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 25.
(b) a DNA which hybridizes with: i) a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 25, or ii) a probe prepared from said nucleotide sequence in i) under stringent conditions, and which encodes a protein having phosphotransfer activity.
It is a further aspect of the present invention to provide the method as described above, wherein the evgA gene encodes a protein selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 24,
(b) a protein comprising the amino acid sequence of SEQ ID NO: 24, but which includes one or more amino acid substitutions, deletions, insertions, additions, or inversions, and has transcription factor activity.
It is a further aspect of the present invention to provide the method as described above, wherein the gadE gene encodes a protein selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 28.
(b) a protein comprising the amino acid sequence of SEQ ID NO: 28, but which includes one or more amino acid substitutions, deletions, insertions, additions, or inversions, and has transcription factor activity.
It is a further aspect of the present invention to provide the method as described above, wherein the ydeO gene encodes a protein selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 30.
(b) a protein comprising the amino acid sequence of SEQ ID NO: 30, but which includes one or more amino acid substitutions, deletions, insertions, additions, or inversions, and has transcription factor activity.
It is a further aspect of the present invention to provide the method as described above, wherein the evgS gene encodes a protein selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 26,
(b) a protein comprising the amino acid sequence of SEQ ID NO: 26, but which includes one or more amino acid substitutions, deletions, insertions, additions, or inversions, and has phosphotransferase activity.
It is a further aspect of the present invention to provide the method as described above wherein the bacterium is a member of the genera selected from the group consisting of Escherichia, Enterobacter, Pantoea, Klebsiella, and Serratia. 
It is a further aspect 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, L-tryptophan, and combinations thereof.



BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 shows construction of plasmid vector pTS1, which has a temperature-sensitive replication origin.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereinafter, the present invention will be explained in detail.
1. The Microorganism of the Present Invention
The microorganism of the present invention is a member of the family Enterobacteriaceae, and has an ability to produce an L-amino acid. Furthermore, this microorganism has been modified so as to increase the expression of one or more of the evgA, gadE, or ydeO genes. These genes are known to encode the transcription factor involved in the EvgAS two-component system regulon. The expression “an ability to produce an L-amino acid” means an ability to produce an L-amino acid and cause accumulation of it to a level at which it can be collected from a medium or the microorganism's cells when the microorganism is cultured in the medium. The microorganism may be able to produce multiple L-amino acids. The microorganism may inherently be able to produce an L-amino acid, or may be modified by mutagenesis or recombinant DNA techniques so that it is able to produce an L-amino acid, as described below.
Also, the phrase “increase the expression of a gene” means that the transcription and/or translation of the gene is increased.
The type of the L-amino acid is not particularly limited. Examples include basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, and L-citrulline; aliphatic L-amino acids such as L-isoleucine, L-alanine, L-valine, L-leucine, and glycine; L-amino acids which are hydroxymonoaminocarboxylic acids such as L-threonine and L-serine; cyclic L-amino acids such as L-proline; aromatic L-amino acids such as L-phenyl-alanine, L-tyrosine, and L-tryptophan; sulfur-containing L-amino acids such as L-cysteine, L-cystine, and L-methionine; acidic L-amino acids such as L-glutamic acid, and L-aspartic acid; and amides of acidic L-amino acid such as L-glutamine, and L-asparagine, etc. In particular, L-lysine, L-threonine, and L-tryptophan are preferred. The microorganism of the present invention may be able to produce two or more amino acids.
1-1. Imparting L-Amino Acid-Producing Ability
The following includes a description of the method for imparting the ability to produce an L-amino acid to a microorganism, along with examples of microorganisms imparted with L-amino acid-producing ability, but any microorganism can be used as long as it has an L-amino acid-producing ability.
The microorganism is not particularly limited, as long as it belongs to the family Enterobacteriaceae, such as the genera Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Salmonella, Morganella, etc., and has an L-amino acid-producing ability. Specifically, any microorganism belonging to the family Enterobacteriaceae with its classification described in the NCBI (National Center for Biotechnology Information) database may be used. The bacterial strain, or parent strain, from the family Enterobacteriaceae which can be used to derive the microorganism of the present invention includes bacteria which belong to the genera Escherichia, Enterobacter, or Pantoea. 
There is no particular Escherichia bacterial strain which must be used, but those listed in Neidhardt et al., may be used (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.). One example is Escherichia coli. Specific examples of Escherichia coli are Escherichia coli W3110 (ATCC 27325) and Escherichia coli MG1655 (ATCC 47076), etc., which are prototypes derived from wild-type strains of K12.
These are available, for example, from the American Type Culture Collection (address: P.O. Box 1549, Manassas, Va. 20108, United States of America). They are available through this organization's web site via the use of accession numbers given to each bacterial strain. The accession numbers which correspond to each bacterial strain are given in the American Type Culture Collection's catalogue.
Examples of bacteria of the genus Enterobacter include Enterobacter agglomerans and Enterobacter aerogenes. An example of a bacterium of the genus Pantoea includes Pantoea ananatis. In recent years, based on 16S rRNA nucleotide sequence analysis, Enterobacter agglomerans has on occasion been reclassified as Pantoea agglomerans, Pantoea ananatis, and Pantoea stewartii. Any bacterium belonging to the genus Enterobacter or Pantoea may be used as long as the bacterium is classified in the family Enterobacteriaceae. When Pantoea ananatis is bred by genetic engineering, the strains Pantoea ananatis AJ13355 (FERM BP-6614), AJ13356 (FERM BP-6615), AJ13601 (FERM BP-7207), or any derivative thereof may be employed. When isolated, these strains were identified and deposited as Enterobacter agglomerans. As stated above, analysis by 16S rRNA nucleotide sequence has resulted in their being reclassified as Pantoea ananatis. 
The following is a description of methods for imparting L-amino acid-producing ability to microorganisms which belong to the family Enterobacteriaceae and methods for increasing the L-amino-acid-producing ability in these microorganisms.
To impart the ability to produce an L-amino acid, methods conventionally employed in the breeding of the coryneform bacteria or bacteria of the genus Escherichia (see “Amino Acid Fermentation”, Gakkai Shuppan Center (Ltd.), 1st Edition, published May 30, 1986, pp. 77-100) can be applied. Such methods include acquisition of an auxotrophic mutant, an analog-resistant strain, or a metabolic regulation mutant. Furthermore, a recombinant strain having enhanced expression of an L-amino acid biosynthesis enzyme can be constructed. When breeding the L-amino acid-producing bacteria, one or more properties, such as an auxotrophic or metabolic regulation mutation, and analog resistance may be imparted. The expression of one or more L-amino acid biosynthesis enzymes can be enhanced singly or in combinations of two or more. Furthermore, the technique of imparting these properties may be combined with the technique of enhancing the biosynthesis enzymes.
An auxotrophic mutant strain, L-amino acid analog-resistant strain, or metabolic regulation mutant strain with the ability to produce an L-amino acid can be obtained by subjecting a parent strain or wild-type strain to a conventional mutation treatment, such as exposure to X-rays or UV irradiation, or treatment with a mutagenic agent such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or ethyl methane sulfonate (EMS), etc., then selecting those which exhibit the desired properties and which also have the ability to produce an L-amino acid.
The following are examples of L-lysine-producing bacteria and method for construction.
For example, bacteria which have L-lysine-producing ability include an L-lysine analog-resistant strain or a metabolic regulation mutant strain. Examples of the L-lysine analogs include, but are not limited to, oxalysine, lysine hydroxamate, S-(2-aminoethyl)-cysteine (AEC), γ-methyl lysine, α-chlorocaprolactam, etc. Mutant strains which are resistant to these lysine analogs may be obtained by subjecting bacteria belonging to the genus Escherichia to a conventional artificial mutagenesis treatment. Examples of L-lysine-producing bacteria include Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; see JP56-18596A and U.S. Pat. No. 4,346,170), Escherichia coli VL611 (EP1016710), etc. The Escherichia coli WC1-96 strain (see WO96/17930) may also be used as an L-lysine producing bacterium. The WC1-96 strain was bred by imparting AEC resistance to the W3110 strain derived from Escherichia coli K-12. This strain was named Escherichia coli AJ13069, and was deposited on Dec. 6, 1994 at the National Institute of Bioscience and Human Technology of the Agency of Industrial Science and Technology (currently, International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology; Chuo 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305-8566, Japan) under Accession No. FERM P-14690. Then, it was converted to an international deposit under the Budapest Treaty on Sep. 29, 1995, and given Accession No. FERM BP-5252.
L-lysine-producing bacteria can also be constructed by increasing the activity of an L-lysine biosynthetic enzyme. The activity of these enzymes can be increased by increasing the copy number of the gene encoding the desired enzyme in the cells, or by modifying an expression regulatory sequence of the gene.
Examples of genes encoding L-lysine biosynthesis enzymes include, but are not limited to, genes encoding dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarbonylase (lysA), diaminopimelate dehydrogenase (ddh) (WO96/40934, U.S. Pat. No. 6,040,160), phosphoenolpyruvate carboxylase (ppc) (60-87788A), aspartate aminotransferase (aspC) (JP6-102028)B, diaminopimelate epimerase (dapF) (JP2003-135066A), aspartate semialdehyde dehydrogenase (asd) (WO00/61723), and other genes encoding enzymes of the diaminopimelate pathway, as well as a gene encoding homoaconitate hydratase (JP2000-157276A) and other genes encoding enzymes of the aminoadipate pathway.
Furthermore, wild-type dihydrodipicolinate synthase (DDPS) and aspartokinase (AK) are known to be suppressed by feedback inhibition by L-lysine; therefore, when dapA and lysC are used, it is preferable to use mutant genes encoding dihydrodipicolinate synthase and aspartokinase, respectively, that are resistant to the feedback inhibition by L-lysine (EP 0733710, U.S. Pat. No. 5,932,453).
Examples of the DNA encoding mutant dihydrodipicolinate synthase that is resistant to feedback inhibition by L-lysine include a DNA encoding DDPS having an amino acid sequence wherein the 118th histidine residue is substituted with tyrosine. (U.S. Pat. Nos. 5,661,012 and 6,040,160). Furthermore, examples of the DNA encoding a mutant AK that is resistant to feedback inhibition by L-lysine include a DNA encoding AK having the amino acid sequence wherein the 352-threonine residue is substituted with isoleucine (U.S. Pat. Nos. 5,661,012 and 6,040,160). These mutant DNAs can be obtained by site-directed mutagenesis using PCR, or the like.
The following is an example of a technique for imparting an L-lysine-producing ability by introducing a gene encoding an L-lysine biosynthesis enzyme into a host. That is, recombinant DNA is prepared by ligating a gene fragment that encodes the L-lysine biosynthesis gene with a vector that functions in the chosen host microorganism, preferably a multi copy vector, and transforming the host with the recombinant vector. Due to the transformation, the copy number of the gene encoding the L-lysine biosynthesis enzyme in the host cell increases, increasing the expression and consequently increasing the enzymatic activity.
The genes encoding the L-lysine biosynthesis enzymes are not particularly specified, as long as they can be expressed in the host microorganism. Examples include genes derived from Escherichia coli, and genes derived from the coryneform group of bacteria. Because the entire genome sequences of Escherichia coli and Corynebacterium glutamicum have been reported, it is possible to synthesize primers based on the nucleotide sequences of these genes and obtain these genes by PCR in which the genomic DNA of a microorganism, such as Escherichia coli K12, etc., is used as the template.
In order to clone the genes, plasmids that autonomously replicate in the family Enterobacteriaceae can be used. Examples include pBR322, pTWV228 (Takara Bio Inc.), pMW119 (Nippon Gene Co., Ltd.), pUC19, pSTV29 (Takara Bio Inc.), RSF1010 (Gene, 75:271-288 (1989)), etc. In addition to these, a phage vector may also be used.
To ligate the target gene to the above-mentioned vector, the vector is digested with a restriction enzyme matched to the end of the DNA fragment containing the target gene. The ligation is usually conducted with a ligase such as T4 DNA ligase. Target genes may be located on separate vectors, respectively, or located on the same vector. Typical methods known to those skilled in the art can be employed for digesting and ligating a DNA, as well as for preparing genomic DNA, performing PCR, preparing plasmid DNA, transformation, designing oligonucleotide primers, etc. These methods are described in Sambrook, J., and Russell, D. W. Molecular Cloning A Laboratory Manual/Third Edition. New York: Cold Spring Harbor Laboratory Press (2001), etc. Any method which achieves adequate transformation efficiency may be employed to introduce the recombinant DNA that has been prepared as described above into the host microorganism. An example of such a method is electroporation (Can. J. Microbiol. 43:197-201 (1997)). An example of a plasmid prepared using this method is the Lys-producing plasmid pCABD2 which contains the dapA, dapB, and LysC genes (WO 01/53459).
Enhancing the expression of genes encoding L-lysine biosynthesis enzymes can also be achieved by introducing multiple copies of the target gene into the genomic DNA of a microorganism. Multiple copies of the target gene can be introduced into the genomic DNA of the microorganism by using a sequence which is present in multiple copies on the genomic DNA as a target in homologous recombination. Such site-specific introduction of mutations based on gene substitution using homologous recombination has been described. Either a linear DNA or a plasmid containing a temperature-sensitive replication origin can be used in such methods (U.S. Pat. No. 6,303,383 and JP05-007491A). Repetitive DNA and inverted repeats present on the ends of transposable elements can be employed as sequences which are present in multiple copies on genomic DNA. An L-lysine biosynthesis gene may be ligated in tandem with a gene which is native to the genome, or it may be introduced into a non-essential region on the genome or a gene region in which the L-lysine yield will be improved by deletion. Or, as disclosed in U.S. Pat. No. 5,595,889, the target gene may also be located on a transposon, which is then transferred to introduce multiple copies to the genomic DNA. With either method, the number of copies of the target gene in the transformant increases, so that the enzymatic activity of L-lysine biosynthesis increases.
In addition to the above-described genetic amplification, an increase in the L-lysine biosynthesis enzyme activity can be achieved by replacing an expression regulatory sequence of the target gene, such as a promoter, etc., with a stronger one (see JP1-215280A). For example, the lac promoter, trp promoter, trc promoter, tac promoter, araBA promoter, lambda phage PR promoter, PL promoter, tet promoter, T7 promoter, φ10 promoter, etc., are known as strong promoters. Substitution of the native promoter with these promoters enhances expression of the target gene, thus increasing the enzymatic activity. Examples of strong promoters and methods for evaluating strength of promoters are described in Goldstein et al. (Biotechnol. Annu. Rev., 1, 105-128, (1995) Prokaryotic promoters in biotechnology.), etc.
An increase in the activity of the target enzyme can also be achieved by modifying an element which is involved in the regulation of expression of the gene that encodes the target enzyme, for example, an operator or repressor (Hamilton et al, J. Bacteriol. 171:4617-4622 (1989)). As disclosed in WO 00/18935, it is possible to substitute several bases in the promoter region of a target gene to modify and strengthen it. Furthermore, substituting several nucleotides in the spacer between the ribosome binding site (RBS) and the start codon, particularly in the sequence immediately upstream of the start codon, is known to have a strong effect on mRNA translation efficiency. Therefore, these regions may be modified to increase transcription efficiency. The expression regulatory regions of the target gene, such as a promoter, etc., can be determined by promoter probe vectors and gene analysis software such as GENETYX (GENETYX CORPORATION), etc. Expression of the target gene can be increased by substituting or modifying these promoters. Substitution of expression regulatory sequences can be conducted, for example, in the same manner as in the above-described gene substitution employing temperature-sensitive plasmids. The Red-driven integration method (WO2005/010175) may also be used.
Furthermore, in the L-amino-acid-producing bacteria, when the activity of an enzyme catalyzes a reaction to produce a compound other than the target L-amino acid, directs a reaction which branches off from the biosynthesis pathway of the target L-amino acid, or the activity of an enzyme has a negative effect on the synthesis or accumulation of the target L-amino acid, such activities may be reduced or deleted. In L-lysine production, such enzymes include homoserine dehydrogenase (thrA), lysine decarboxylase (cadA, ldcC), and malic enzyme (sfcA, b2463). Strains with reduced or deficient enzymatic activity are disclosed in WO 95/23864, WO96/17930, WO2005/010175, etc.
To reduce or delete the activities of these enzymes, a mutant which reduces or deletes the enzyme activity may be introduced into the gene of the above-mentioned enzyme on the genome, using a known mutatagenisis method or genetic recombination techniques. A mutant can be introduced, for example, by deleting the gene that encodes an enzyme on the genome by genetic recombination, or by modifying an expression regulatory sequence such as a promoter or a Shine-Dalgarno (SD) sequence, etc. This can also be achieved by introducing an amino acid substitution (missense mutation) or stop codon (nonsense mutation) in the region encoding the enzyme on the genome, by introducing a frameshift mutation to add or delete 1-2 bases, or by deleting a part of the gene or the entire region (J. Biol. Chem. 272:8611-8617 (1997)). Also, the enzyme activity can be reduced or deleted by deleting a part or the entire coding region of the gene that encodes the mutant enzyme, and then substituting the normal gene on the genome with the mutant gene by homologous recombination, etc., or introducing a transposon or IS element into said gene. The following methods may be used to introduce a mutation which reduces or deletes the above-mentioned enzyme activity by genetic recombination. An isolated DNA containing the target gene is mutated so that the resulting mutant gene does not produce an enzyme that functions normally. Then, a microorganism which belongs to the family Enterobacteriaceae is transformed with the DNA containing the mutated gene to cause recombination between the mutated gene and the target gene on the genome. Thus, the target gene on the genome may be substituted with the mutated gene. For gene substitution using this kind of homologous recombination, linear DNA can be used, such as in the method called “Red-driven integration” (Datsenko, K. A, and Wanner, B. L. Proc. Natl. Acad. Sci. USA. 97:6640-6645 (2000)), a method combining the Red-driven integration method and the λ phage excision system (Cho, E. H., Gumport, R. I., Gardner, J. F. J. Bacteriol. 184: 5200-5203 (2002)) (see WO2005/010175), etc. A temperature-sensitive replication origin could also be employed (U.S. Pat. No. 6,303,383; U.S. Pat. No. 5,616,480). Such site-specific introduction of mutations by gene substitution using homologous recombination as described above may also be performed using a plasmid which is not able to replicate in the chosen host.
The above-mentioned method for increasing the activity of the enzyme involving L-lysine biosynthesis and the method for lowering the enzyme activity may likewise be used in breeding other L-amino acid-producing bacteria. The following is a description of methods for breeding other L-amino acid bacteria.
The L-tryptophan-producing bacteria preferably used in the present invention include bacteria in which the activity of one or more of the following enzymes anthranilate synthase, phosphoglycerate dehydrogenase, or tryptophan synthase has been enhanced. Since anthranilate synthase and phosphoglycerate dehydrogenase both are subject to feedback inhibition by L-tryptophan and L-serine, the activities of these enzymes can be increased by retaining the desensitizing mutant enzyme. For instance, it is possible to obtain a bacterium which has a desensitizing enzyme by causing a mutation of the anthranilate synthase gene (trpE) and/or the phosphoglycerate dehydrogenase gene (serA) to prevent the feedback inhibition, then introducing the mutant gene into a microorganism belonging to the family Enterobacteriaceae. (U.S. Pat. No. 5,618,716, U.S. Pat. No. 6,180,373) A specific example of this kind of bacteria includes a transformant obtained by introducing plasmid pGH5 having a mutant serA that encodes desensitized phosphoglycerate dehydrogenase into Escherichia coli SV164 which retains desensitized anthranilate synthase (WO94/08301). Strain SV164 is obtained by introducing a gene that encodes desensitized anthranilate synthase into a trpE deficient strain of Escherichia coli KB862 (DSM7196) (See WO94/08031).
Bacteria transformed with recombinant DNA containing a tryptophan operon are also preferable. A specific example includes Escherichia coli transformed with a tryptophan operon containing a gene encoding desensitized anthranilate synthase (JP57-71397A, JP62-244382A, U.S. Pat. No. 4,371,614). Furthermore, it is possible to enhance or impart an ability to produce L-tryptophan by enhancing the expression of a gene encoding tryptophan synthase (trpBA). Tryptophan synthase consists of α and β subunits that are encoded by trpA and trpB, respectively. The nucleotide sequence of trpA is shown in SEQ ID NO: 13 and the nucleotide sequence of trpB is shown in SEQ ID NO: 15.
A strain with deficient trpR, the tryptophan operon repressor, and a strain with a mutant trpR are also preferable. (U.S. Pat. No. 4,371,614, WO2005/056776).
Another preferable L-tryptophan-producing bacterium is one in which malate synthase, isocitrate lyase, isocitrate dehydrogenase/phosphatase operon (ace operon) is structurally expressed or the expression of said operon has been enhanced. Specifically, it is preferable that the promoter of the ace operon is not suppressed by the repressor iclR or that the suppression by iclR has been removed. Such a bacterium can be obtained by disrupting the iclR gene or modifying the expression regulatory sequence of the ace operon. The iclR gene of Escherichia coli is shown in SEQ ID NO: 5. A bacterium with enhanced expression of the ace operon can be obtained by ligating a DNA containing the ace operon to a strong promoter and introducing the recombinant DNA into cells using a plasmid or by homologous recombination, or amplifying the copy number of the ace operon using a transposon. Genes contained in the ace operon include aceB, aceA, and aceK. The nucleotide sequences of aceB, aceA and aceK are shown in SEQ ID NOS: 9, 7, and 11, respectively.
Examples of L-tryptophan-producing bacteria include Escherichia coli AGX17 (pGX44) [NRRL B-12263], which is L-phenylalanine and L-tyrosine auxotrophic, and AGX6 (pGX50) aroP [NRRL B-12264], which contains the tryptophan operon-containing plasmid pGX50 (see U.S. Pat. No. 4,371,614, for both). These strains are available from the Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research (address: Peoria, Ill. 61604, USA).
L-tryptophan, L-phenylalanine, and L-tyrosine are all aromatic amino acids and share a common biosynthesis pathway. Examples of the genes encoding the biosynthesis enzymes for these aromatic amino acids include deoxyarabino-heptulosonate phosphate synthase (aroG), 3-dehydroquinate synthase (aroB), shikimate dehydratase, shikimate kinase (aroL), 5-enolpyruvylshikimate-3-phosphate synthase (aroA), and chorismate synthase (aroC) (EP763127). Therefore, by placing multiple copies of the genes encoding these enzymes on a plasmid or genome, the aromatic amino acid-producing ability can be improved. It is known that these genes are controlled by a tyrosine repressor (tyrR), so the enzyme activity of an aromatic amino acid biosynthesis may also be increased by deleting the tyrR gene (See EP763127).
Examples of L-phenylalanine-producing bacteria include Escherichia coli 
AJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197) which is tyrA, tyrR deficient, and strains with amplified genes encoding a phenylalanine excreting protein such as yddG and yedA (WO03/044192; US2003/0148473A1).
L-threonine-producing bacteria used in the present patent are preferably microorganisms belonging to the family Enterobacteriaceae wherein an L-threonine biosynthesis enzyme has been enhanced. Examples of such genes include genes encoding aspartokinase III (lysC), aspartate-semialdehyde dehydrogenase (asd), aspartokinase I (thrA included in thr operon), homoserine kinase (thrB), and threonine synthase (thrC). Two or more of these genes may be introduced. The L-threonine biosynthesis gene may be introduced into a bacterium of the genus Escherichia with suppressed threonine degradation. Examples such bacteria include the TDH6 strain wherein the threonine dehydrogenase activity has been deleted (U.S. Pat. No. 6,960,455), and so forth.
Activities of some of the enzymes of the L-threonine biosynthesis pathway are suppressed by L-threonine. Therefore, in order to construct an L-threonine-producing bacterium, it is preferable to modify the L-threonine biosynthesis enzyme so that the enzyme is not subject to feedback inhibition by L-threonine. The above-mentioned thrA, thrB, and thrC genes constitute the threonine operon which contains an attenuator structure. Their expression is suppressed by this attenuation. In addition, the expression of the threonine operon is inhibited by isoleucine and threonine present during the culture. The modification of the threonine operon can be achieved by removing the leader sequence in the attenuation region or the attenuator. (See Lynn, S. P., Burton, W. S., Donohue, T. J., Gould, R. M., Gumport, R. I., and Gardner, J. F. J. Mol. Biol. 194:59-69 (1987); WO02/26993; WO2005/049808).
A native promoter is present upstream of the threonine operon. The promoter may be substituted with a non-native promoter (see WO 98/04715). Alternatively, a threonine operon may be constructed so that the expression of the gene involved in threonine biosynthesis is controlled by the lambda phage repressor and promoter (See EP0593792). Also, to prevent feedback inhibition by L-threonine, an α-amino-β-hydroxyvaleric acid (AHV) resistant strain of Escherichia bacteria may be selected.
The copy number of the modified threonine operon as described above may be increased in the host, or may be ligated to a strong promoter to further enhance the expression of the operon. In addition to amplifying the copy number using a plasmid, the copy number can be increased by introducing the threonine operon on the genome using a transposon, Mu-phage, etc.
The gene encoding aspartokinase III gene (lysC) can be modified to prevent feedback inhibition by L-lysine. This modified lysC gene can be obtained using the method described in the U.S. Pat. No. 5,932,453.
Aside from the L-threonine biosynthesis enzymes, expression of genes related to the glycolytic system, TCA cycle, and respiratory chain can also be enhanced. Furthermore, the expression of any gene which controls expression of these genes, and/or a gene which induces the uptake of sugar can also be enhanced. Examples of genes which are effective in L-threonine production include genes encoding transhydrogenase (pntAB) (EP733712), phosphoenolpyruvate carboxylase (ppc) (WO95/06114), phosphoenolpyruvate synthase gene (pps) (EP877090), and a gene encoding pyruvate carboxylase of a coryneform bacterium or a bacterium of the genus Bacillus (WO99/18228, EP1092776).
The expression of genes that impart resistance to L-threonine, L-homoserine, and/or both in the host can also be enhanced. Examples of such genes include rhtA (Res. Microbiol. 154:123-135 (2003)), rhtB (EP0994190), rhtC (EP1013765), yfiK, and yeaS (EP1016710). For methods of imparting L-threonine resistance to a host, refer to EP0994190, WO90/04636.
Another example of an L-threonine-producing bacterium is Escherichia coli VKPM B-3996 (see U.S. Pat. No. 5,175,107). This VKPM B-3996 strain was deposited on Apr. 7, 1987, under Accession No. VKPM B-3996, at the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika (address: Russia, 117545 Moscow, 1 Dorozhny proezd. 1). In addition, the VKPM B-3996 strain contains the plasmid pVIC40 (WO90/04636), which was obtained by inserting a threonine biosynthesis gene (threonine operon: thrABC) into a wide host-range vector plasmid pAY32 which includes a streptomycin-resistant marker (see Chistorerdov, A. Y., and Tsygankov, Y. D. Plasmid, 16, 161-167 (1986)). The thrA gene on pVIC40, and the feedback inhibition by the L-threonine of aspartokinase I-homoserine dehydrogenase I has been desensitized.
Another example of an L-threonine-producing bacteria is Escherichia coli VKPM B-5318 (see EP0593792). The VKPM B-5318 strain was deposited under Accession No. VKPM B-5318 at the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika (address: Russia, 117545 Moscow, 1 Dorozhny proezd. 1) on May 3, 1990. This VKPM B-5318 strain is non-auxotrophic for isoleucine, and contains a recombinant plasmid constructed so that the gene involved in threonine biosynthesis, i.e., the threonine operon wherein the attenuator and inherent transcriptional regulatory regions have been deleted, is located downstream of the temperature-sensitive CI repressor, PR promoter, and the N-terminus of the Cro protein of the lambda phage. Therefore, expression of the gene involved in the threonine biosynthesis is controlled by the lambda phage repressor and promoter.
Examples of L-glutamic acid-producing bacteria include a microorganism belonging to the family Enterobacteriaceae which has been modified to increase the expression of the gene encoding an enzyme that is involved in L-glutamic acid biosynthesis. The enzymes involved in the L-glutamic acid biosynthesis include glutamate dehydrogenase (hereinafter, also referred to as “GDH”), glutamine synthetase, glutamate synthase, isocitrate dehydrogenase, aconitate hydratase, citrate synthase (hereinafter, also referred to as “CS”), phosphoenolpyruvate carboxylase (hereinafter, also referred to as “PEPC”), pyruvate carboxylase, pyruvate dehydrogenase, pyruvate kinase, phosphoenolpyruvate synthase, enolase, phosphoglycerolmutase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, fructose-bisphosphate aldolase, phosphofructokinase, glucosephosphate isomerase, etc. Of these enzymes, one or more of CS, PEPC, and GDH is preferable, and all three are more preferable.
Examples of microorganisms belonging to the family Enterobacteriaceae which have been modified to enhance the expression of the citrate synthase gene, phosphoenolpyruvate carboxylase gene, and/or glutamate dehydrogenase gene using the methods described above are disclosed in U.S. Pat. Nos. 6,197,559 & 6,331,419, EP0999282.
Furthermore, microorganisms belonging to the family Enterobacteriaceae which have been modified to increase the activity of either or both of 6-phosphogluconate dehydratase or 2-keto-3-deoxy-6-phosphogluconate aldolase may also be used (EP1352966).
For production of L-glutamic acid, Enterobacteriaceae may be used in which the activity of an enzyme that catalyzes a reaction to produce a compound other than L-glutamic acid, or which directs a reaction to branch off from the biosynthesis pathway of L-glutamic acid, has been deleted or reduced. Examples of such enzymes include 2-oxoglutarate dehydrogenase, isocitrate lyase, phosphate acetyltransferase, acetate kinase, acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase, etc. Of these, it is especially preferable to reduce or delete the activity of 2-oxoglutarate dehydrogenase.
Methods for deleting or reducing the activity of 2-oxoglutarate dehydrogenase in a microorganism belonging to the family Enterobacteriaceae are described in U.S. Pat. No. 5,573,945, U.S. Pat. No. 6,197,559, and U.S. Pat. No. 6,331,419. Specifically, examples of microorganisms belonging to the family Enterobacteriaceae wherein the activity of 2-oxoglutarate dehydrogenase has been deleted or reduced include the following:
Pantoea ananatis AJ13601 (FERM BP-7207)
Klebsiella planticola AJ13410 strain (FERM BP-6617)
Escherichia coli AJ12949 (FERM BP-4881)
Examples of an L-histidine-producing bacterium include Escherichia coli FERM P-5038 and FERM P-5048. These strains contain a vector which contains genetic information involved in L-histidine biosynthesis (JP56-005099A). Other examples includes bacteria which have been transformed with the amino acid export gene Rht (EP1016710), and Escherichia coli 80 which is resistant to sulfaguanidine, D, L-1,2,4-triazole-3-alanine, and streptomycin (VKPM B-7270, Russian Patent Publication No. 2119536), etc.
Microorganisms with enhanced expression of gene(s) encoding L-histidine biosynthesis pathway enzyme(s) may be used. Examples of such genes includes the genes encoding ATP phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase gene (hisI), phosphoribosyl-ATP pyrophosphohydrolase (hisIE), phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (hisA), amidotransferase gene (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), and histidinol dehydrogenase gene (hisD), etc.
The L-cysteine-producing bacteria include bacteria with reduced activity of cystathionine β-lyase (JP2003-169668A), and Escherichia bacteria that retain serine acetyltransferase, but with reduced or eliminated feedback inhibition by L-cysteine (JP11-155571A).
The L-proline-producing bacteria include Escherichia coli 702 (VKPMB-8011) which is resistant to 3,4-dehydroxyproline and azetidine-2-carboxylate, and the 702 ilvA strain (VKPMB-8012 strain) which is deficient in ilvA, and is derived from the strain 702 (JP2002-300874A).
Examples of L-arginine-producing bacteria include Escherichia coli mutant strains which are resistant to α-methylmethionine, p-fluorophenylalanine, D-arginine, arginine hydroxamic acid, S-(2-aminoethyl)-cysteine, α-methyleserine, β-2-thienylalanine, or sulfaguanidine (see JP56-106598A), etc. The Escherichia coli 237 strain is an L-arginine-producing bacterium that has a mutant which is resistant to feedback inhibition by L-arginine and retains highly active N-acetyl glutamate synthase (Russian Patent Application No. 2000117677). This strain, numbered VKPM B-7925, was deposited with the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika on Apr. 10, 2000, and converted to an international deposit under the Budapest Treaty on May 18, 2001. The Escherichia coli 382 strain, which is a derivative of the 237 strain and is an L-arginine-producing bacterium with improved acetic acid assimilating ability, may also be used (JP2002-017342A). The Escherichia coli 382 strain, numbered VKPM B-7926, was deposited with the Russian National Collection of Industrial Microorganisms (VKPM) on Apr. 10, 2000.
Also, for producing L-arginine, microorganisms with increased expression of gene(s) encoding enzyme(s) involved in L-arginine biosynthesis may be used. Examples of L-arginine biosynthesis enzymes are N-acetyl glutamate synthase (argA), N-acetyl-glutamyl-phosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetyl glutamate kinase (argB), acetyl ornithine transaminase (argD), acetyl ornithine deacetylase (argE), ornithine carbamoyl transferase (argF), argininosuccinate synthase (argG), argininosuccinate lyase (argH), and carbamoyl phosphate synthase (carAB). After each enzyme name, the name of the gene encoding it is given in parentheses. A mutation of the N-acetyl glutamate synthase gene (argA) to remove L-arginine feedback inhibition by substitution of the amino acid sequence corresponding to positions 15 to 19 in the wild-type may be used (EP1170361).
The L-leucine-producing bacteria include Escherichia coli with an inactive ilvE gene, which encodes the branched-chain amino-acid transaminase, and enhanced activity of the aromatic amino acid transaminase encoded by the tyrB gene (EP1375655A), Escherichia coli H-9068 (ATCC21530) which is resistant to 4-azaleucine or 5,5,5-trifluoroleucine, Escherichia coli H-9070 (FERM BP-4704), Escherichia coli H-9072 (FERM BP-4706) (U.S. Pat. No. 5,744,331), Escherichia coli with desensitized isopropylmalate synthase feedback inhibition by L-leucine (EP1067191), Escherichia coli AJ11478 which is resistant to β-2 thienylalanine and β-hydroxyleucine (U.S. Pat. No. 5,763,231), and so on.
L-isoleucine-producing bacteria include a 6-dimethyl aminopurine-resistant Escherichia coli mutant strain (JP5-304969A), L-isoleucine hydroxamate-resistant Escherichia coli mutant strain (JP5-130882A), thiaisoleucine-resistant Escherichia coli mutant strain (JP5-130882A), DL-ethionine-resistant Escherichia coli mutant strain (JP5-130882A), and arginine hydroxamate-resistant mutant strain (JP5-130882A). Examples of the recombinant Escherichia bacteria are those in which the genes encoding the L-isoleucine biosynthesis enzymes threonine deaminase or acetohydroxy acid synthase have been strengthened by transformation with a plasmid (JP2-458A, JP2-42988A, JP8-47397), etc.
L-valine-producing bacteria include Escherichia coli VL1970 (U.S. Pat. No. 5,658,766), etc. Examples of L-valine-producing bacteria further include a mutant which requires lipoic acid for its growth and/or lacks H+-ATPase (WO96/06926), and an Escherichia bacterium transformed with a DNA fragment containing the ilvGMEDA operon which expresses at least the ilvG, ilvM, ilvE, and ilvD genes. Since the expression of the ilvGMEDA operon is regulated (attenuated) by L-valine and/or L-isoleucine and/or L-leucine, the region required for attenuation should be removed or mutated in order to remove the suppression of the expression by L-valine (U.S. Pat. No. 5,998,178). The ilvGMEDA operon should not express the ilvA gene, which encodes threonine deaminase. Escherichia coli VL1970, in which the ileS17 mutation is present which removes the attenuation as described above, was deposited as Accession No. VKPM B-4411 at GNIIgenetika (Russian National Collection of Industrial Microorganisms (VKPM) Depositary, GNIIgenetika) (address: 1, Dorozhny Proezd., 1, 113545, Moscow, Russia).
Aside from a gene which encodes an inherent biosynthesis enzyme, a gene which is involved in sugar uptake, sugar metabolism (glycolytic system), and energy metabolism may be enhanced in the L-amino acid-producing bacteria used in the present invention.
Examples of the genes involved in sugar metabolism are genes which encode glycolytic enzymes or proteins which uptake sugar, such as genes encoding glucose-6-phosphate isomerase (pgi; WO01/02542), phosphoenolpyruvate synthase (pps; EP877090), phosphoglucomutase (pgm; WO03/04598), fructose-bisphosphate aldolase (fba; WO03/04664), pyruvate kinase (pykF; WO03/008609), transaldolase (talB; WO03/008611), fumarase (fum; WO01/02545), phosphoenolpyruvate synthase (pps; EP877090), the non-PTS sucrose uptake systems (csc; EP149911), and sucrose-assimilating genes (scrAB operon; WO90/04636).
Examples of the genes involved in energy metabolism include the transhydrogenase gene (pntAB; U.S. Pat. No. 5,830,716) and the cytochromoe bo type oxidase gene (cyoABCD; EP1070376).
A microorganism which has the ability to produce an L-amino acid may be modified, as described above, so as to increase the expression of one or more of the following genes: evgA, gadE, or ydeO, which are the genes encoding the transcription factor involved in the EvgAS two-component system. The ability to produce the target substance may be imparted after the modification to increase the expression of the evgA gene, gadE gene, or ydeO gene. Expression of the evgA gene, gadE gene, or ydeO gene may be increased by increasing the expression of the endogenous evgA gene, gadE gene, or ydeO gene through modification of the expression regulatory region, including promoter modification, as described later, or increasing the copy number of the evgA gene, gadE gene, or ydeO gene through introduction of a plasmid containing the evgA gene, gadE gene, or ydeO gene, or amplification of these genes on the chromosome of the bacterium, and so forth. Furthermore, a combination of these techniques may be employed.
The “EvgAS two-component system” means a pathway which activates transcription factors via EvgA-EvgA histidine-aspartate phosphorelation regulation. More specifically, the EvgS protein (SEQ ID NO: 26), which is a sensor kinase, autophosphorylates the histidine residue of this protein, and then transfers the phosphate group to aspartate residues specific to the EvgA protein (SEQ ID NO: 24), which is a response regulator and transcription factor. EvgA is activated by this phosphorylation and, as a result, regulates the transcription of many genes, as well as activating the transcription of the ydeO gene (SEQ ID NO: 29) that encodes the transcription factor YdeO protein (SEQ ID NO: 30) and/or the gadE gene (SEQ ID NO: 27) that encodes the transcription factor GadE protein (SEQ ID NO: 28) (J. Bacteriol. 184:6225-6234, 2002; Mol. Microbiol. 48:699-712, 2003). The YdeO protein also activates the transcription of the gadE gene. As a result of the EvgA and YdeO-activated transcription of the gadE gene, the expression of the GadE protein increases, and since it is a positive transcription factor, it amplifies the transcription of other genes (Microbiology. 150:61-72, 2004; J Bacteriol. 186:7378-89, 2004). Therefore, “the transcription factor involved in the EvgAS two-component regulator” as used herein refers to the EvgA protein, GadE protein, or YdeO protein.
The “sensor kinase” is a protein which senses an environmental factor, such as a ligand, phosphorylates its own histidine residues using ATP, and then delivers this phosphate group to a response regulator. The “response regulator” is a protein which conveys the information within cells after being activated by phosphorylating the specific aspartate from the sensor kinase. In many cases, it is a transcription factor.
The improvement in the expression of the gene encoding the EvgA protein, GadE protein, or YdeO protein (hereafter, the evgA, gadE, or ydeO genes) when comparing it to the parent strain, for example, a wild-type strain or non-modified strain, can be confirmed by comparing the amount of mRNA with that in the wild-type or non-modified strain. Northern hybridization and Reverse-Transcriptase PCR (RT-PCR) can be used to confirm the amount of expression. (Sambrook, J., and Russell, D. W. Molecular Cloning A Laboratory Manual/Third Edition. New York: Cold Spring Harbor Laboratory Press (2001)). The degree of the increase in enzymatic activity is not limited as long as the activity is increased as compared to that in the wild-type or non-modified strain, but it is advantageous, for example, for it to be 1.5 or more times, preferably 2 or more times, or more preferably 3 or more times that of the wild-type or non-modified strain. An increase in the enzymatic activity can be confirmed if the target protein amount is increased relative to that of the non-modified or wild-type strain. This can be detected, for instance, by Western blot using an antibody. (Sambrook, J., and Russell, D. W. Molecular Cloning A Laboratory Manual/Third Edition. New York: Cold Spring Harbor Laboratory Press (2001)).
The “transcription factor” indicates a transcription factor of a gene in which the expression is controlled by the EvgSA two-component regulator, and corresponds to the EvgA protein, GadE protein, and YdeO protein. These proteins positively regulate the transcription of the gene encoding various metabolic enzymes which are under the control of the EvgSA two-component regulator. For example, the transcription of the gnd gene that encodes 6-phosphogluconate dehydrogenase (GND) or the purA gene that encodes adenylosuccinate synthase is activated by the transcription factor of the EvgSA two-component regulator. That is, by improving the amount of the EvgA, GadE, or YdeO proteins that is produced compared to the wild-type strain or non-modified strain, the transcription amount of the EvgAS two-component regulator-controlled gene increases. The activity of the transcription factor can be measured using the electrophoretic mobility shift assay, which measures the ligation with DNA, or the transcription activity can be measured in vitro (Sambrook, J., and Russell, D. W. Molecular Cloning A Laboratory Manual/Third Edition. New York: Cold Spring Harbor Laboratory Press (2001)).
The evgA gene can be isolated or derived from (native to) an Escherichia bacterium and its homologs. For example, the evgA gene of Escherichia coli (SEQ ID NO: 23) encodes a protein having the amino acid sequence of SEQ ID NO: 24. (GenBank Accession No. NP_416870 [gi:16130301]).
Homologs of the evgA gene are derived from other microorganisms which have a high similarity in structure to the Escherichia evgA gene, improve the ability to produce an L-amino acid, and exhibit transcription factor activity when introduced into the host. Examples of evgA homologs are the evgA genes isolated or derived from (native to) the genera Salmonella, Shigella, and Yersinia, etc. These genes are registered at GenBank. Furthermore, based on the homology with the genes given in the above examples, evgA genes may be cloned from coryneform bacteria, such as Corynebacterium glutamicum, Brevibacterium lactofermentum, etc.; the bacteria of the genus Pseudomonas, such as Pseudomonas aeruginosa, etc.; bacteria of the genus Mycobacterium, such as Mycobacterium tuberculosis, etc.; and bacteria of the genus Bacillus. Genes having different names are acceptable as long as they are highly homologous with the Escherichia evgA gene. For example, evgA gene homologs include a gene cloned from the synthetic oligonucleotides of SEQ ID NOS: 17 and 18.
The nucleotide sequences of the evgA gene homologs can be obtained by searching for a gene with high homology from a known database, based on the above-mentioned sequence information. The homology of the amino acid sequences and nucleotide sequences can be determined using, for instance, Karlin and Altschul's BLAST algorithm (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) or FASTA (Methods Enzymol., 183, 63 (1990)). Based on this BLAST algorithm, programs called BLASTN or BLASTX have been developed (see http://www.ncbi.nih.gov/BLAST/).
The gadE gene can be isolated or derived from (native to) Escherichia bacteria and its homologs. An example of gadE gene of Escherichia coli includes the gene (SEQ ID NO: 27) which encodes the protein having the amino acid sequence of SEQ ID NO: 28. (GenBank Accession No. NP_417969 [gi:16131384]).
The homologs of the gadE gene are, as with the evgA gene homologs described above, genes which are derived from other microorganisms which have high similarity in structure to the Escherichia gadE gene, improve the ability to produce an L-amino acid, and exhibit transcription factor activity when introduced into the host. The gadE gene homologs include a gene cloned from the synthetic oligonucleotides of SEQ ID NOS: 19 and 20.
The ydeO gene can be isolated or derived from (native to) Escherichia bacterium and its homologs. An example of ydeO gene of Escherichia coli includes a gene (SEQ ID NO: 27) which encodes the protein having the amino acid sequence of SEQ ID NO: 28. (GenBank Accession No. NP_417969 [gi:16131384]).
The homologs of the ydeO gene are, as with the ydeO gene homologs described above, genes which are derived from other microorganisms which have high similarity in structure to the ydeO gene of a bacterium of the genus Escherichia, improve the ability to produce L-amino acid, and exhibit transcription factor activity when introduced into the host. The ydeO gene homologs include a gene cloned from the synthetic oligonucleotides of SEQ ID NOS: 21 and 22.
The evgA, gadE, or ydeO genes are not limited to the wild-type genes, and as long as the function of the encoded protein, i.e., the transcription factor activity, is not impaired, the proteins encoded by these genes can also include mutant or artificially modified products. Such products may have a sequence which includes one or several amino acid substitutions, deletions, insertions, additions, or the like, at one or more positions in the amino acid sequence of SEQ ID NO: 24, 28, or 30. Here, the term “several” differs depending on the position of the amino acid residues in the stereostructure of the protein or the type of the amino acid. Specifically, this number can be 1 to 20, preferably 1 to 10, and more preferably 1 to 5. The above substitutions, deletions, insertions, or additions are conservative mutations that preserve transcription factor activity. A conservative mutation is a mutation wherein substitution takes place mutually among Phe, Trp, Tyr, if the substitution site is an aromatic amino acid; among Leu, Ile, Val, if the substitution site is a hydrophobic amino acid; between Gln, Asn, if it is a polar amino acid; among Lys, Arg, His, if it is a basic amino acid; between Asp, Glu, if it is an acidic amino acid; and between Ser, Thr, if it is an amino acid having a hydroxyl group. Typical conservative mutations are conservative substitutions. Specific examples of substitutions that are considered to be conservative include: substitution of Ala with Ser or Thr; substitution of Arg with Gln, His, or Lys; substitution of Asn with Glu, Gln, Lys, His, or Asp; substitution of Asp with Asn, Glu, or Gln; substitution of Cys with Ser or Ala; substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg; substitution of Glu with Gly, Asn, Gln, Lys, or Asp; substitution of Gly with Pro; substitution of His with Asn, Lys, Gln, Arg, or Tyr; substitution of Ile with Leu, Met, Val, or Phe; substitution of Leu with Ile, Met, Val, or Phe; substitution of Lys with Asn, Glu, Gln, His, or Arg; substitution of Met with Ile, Leu, Val, or Phe; substitution of Phe with Trp, Tyr, Met, Ile, or Leu; substitution of Ser with Thr or Ala; substitution of Thr with Ser or Ala; substitution of Trp with Phe or Tyr; substitution of Tyr with His, Phe, or Trp; and substitution of Val with Met, Ile, or Leu. Substitutions, deletions, insertions, additions, or inversions and the like of the amino acids described above include naturally occurring mutations (mutant or variant) as a result in differences in species, or individual differences of microorganisms that express the evgA, gadE, and ydeO genes. Such genes can be obtained by modifying the nucleotide sequences shown in SEQ ID NOS: 23, 25, and 29 using, for example, site-directed mutagenesis, so that the site-specific amino acid residue in the protein encoded includes substitutions, deletions, insertions, or additions.
Moreover, the evgA, gadE, or ydeO genes include sequences which encode a transcription factor that has at least 80%, preferably at least 90%, more preferably at least 95% or even more preferably at least 97% homology with the entire amino acid sequences of SEQ NOS. 26, 30, or 32, respectively. Also, the evgA, gadE, or ydeO genes also include sequences in which codons are substituted with equivalent codons that are more readily utilized by the host into which these genes are respectively introduced. Likewise, as long as the evgA, gadE, or ydeO gene product maintains the function of the transcription factor, its N terminal or C terminal may be extended or removed. For example, 50 or less, preferably 20 or less, more preferably 10 or less, and even more preferably 5 or less amino acid residues may be extended or removed. More specifically, a gene which encodes a protein having from 5 to 50 amino acids of SEQ ID NOS:24, 18, or 30 extended or removed from the N terminal, and/or the C terminal may be used.
Also, variants of the genes can be obtained by conventional mutation treatments of the microorganism, such as in vitro mutation of the evgA, gadE, or ydeO genes using hydroxylamine, or the use of ultraviolet light or a known mutation agent, such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or Ethyl Methyl Sulfonate (EMS). Whether or not these genes encode a protein that has transcription factor activity can be confirmed, for example, by expressing these genes in the appropriate cells, and investigating if transcription factor activity is present.
The evgA, gadE, or ydeO genes can also hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 23, 27, or 29, respectively, or with a probe prepared from these sequences, and which encodes proteins having transcription activity. Here, the term “stringent conditions” refers to conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. Although it is difficult to clearly express such conditions in numbers, these conditions include when highly homologous fragments of DNA, for example, DNAs having homology no less than 70%, hybridizes each other and DNAs having homology lower than the above do not hybridize each other. Alternatively, stringent conditions are exemplified by washing once or preferably two to three times, at a temperature and salt concentration of, for example, 60° C., 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS, and more preferably, 68° C., 0.1×SSC, 0.1% SDS.
As a probe, a nucleotide sequence of SEQ ID NO:23, 27 or 29 or a part of these sequences may also be used. Such a probe can be prepared employing PCR in which a DNA fragment containing one of these nucleotide sequences is used as the template, with an oligonucleotide prepared based on the nucleotide sequence of SEQ ID NO: 23, 27, or 29 as the primer. For example, when a DNA fragment of a length of about 300 bp is used as the probe, the washing conditions for the hybridization are 50° C., 2×SSC, and 0.1% SDS.
The expression of the evgA, gadE, or ydeO gene can be enhanced by employing, for example, genetic recombination techniques to increase the number of copies of the gene(s) in the cell. For example, a DNA fragment containing the evgA, gadE, or ydeO gene is ligated with a vector, preferably a multicopy type vector, which is able to function in the host microorganism. The microorganism is then transformed with this recombinant DNA.
When the evgA, gadE, or ydeO gene of Escherichia coli is used, these genes can be obtained by PCR (PCR: polymerase chain reaction; see White, T. J. et al., Trends Genet. 5, 185 (1989)) in which the genomic DNA of Escherichia coli is used as the template, and a primer is prepared based on the nucleotide sequence of SEQ ID NO: 22, 27, or 29, for example, the primers shown in SEQ ID NOS: 17 and 18 for the evgA gene, the primers shown in SEQ ID NOS: 19 and 20 for the gadE gene, or the primers shown in SEQ ID NOS: 21 and 22 for the ydeO gene. The evgA, gadE, and ydeO genes of other microorganisms belonging to the family Enterobacteriaceae can also be obtained from known evgA, gadE, and ydeO genes in those microorganisms or evgA, gadE, and ydeO genes in microorganisms of other species, or the genomic DNA or genomic DNA library of microorganisms, using PCR wherein primers are prepared based on other sequence information of the transcription factor, or the hybridization method wherein the probe is prepared based on the above-mentioned sequence information. Incidentally, the genomic DNA can be prepared from DNA donor microorganisms. For example, Saito and Miura's method, etc., (see H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Seibutsu Kogaku Jikkensho [Bioengineering Experiments], edited by The Society of Biotechnology, Japan, pp. 97-98, Baifukan, 1992), may be used.
Next, the recombinant DNA is prepared by ligating the PCR-amplified evgA, gadE, or ydeO gene with a DNA vector capable of functioning in the cells of the host microorganism. The chosen vector should be autonomously replicable in the cells of the host microorganism. Examples of autonomously replicable vectors for Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pACYC184, (pHSG and pACYC are available from Takara Bio Inc.), RSF1010, pBR322, and pMW219 (pMW is available from Nippon Gene Co., Ltd.), pSTV29 (available from Takara Bio Inc.), etc.
Recombinant DNA prepared as described above may be introduced to a microorganism in accordance with known transformation methods. For example, the permeability of the DNA may be increased by treating the recipient bacteria with calcium chloride, as reported with regards to Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)). Another method is to introduce the DNA after preparing competent cells from the cells at the growth phase, as reported with regards to Bacillus subtilis (Duncan, C. H., Wilson, G. A. and Young, F. E., Gene, 1, 153 (1977)). Also, another method is to change the host microorganism, as known for Bacillus subtilis, actinomycetes and yeast, into the protoplast or spheroplast state. Cells in this state can easily take up the recombinant DNA (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. Acad. Sci. USA, 75 1929 (1978)).
The copy number of the evgA, gadE, or ydeO gene can be increased by integrating multiple copies of the evgA, gadE, or ydeO gene as described above onto the genomic DNA of the microorganism. This can be performed by homologous recombination using a target sequence which is present in multiple copies on the genomic DNA, such as repetitive DNA and inverted repeats present on the ends of transposable elements. Also, these genes may be ligated in tandem, with the evgA, gadE, or ydeO gene existing on the genome or incorporated in multiple copies into unnecessary genes on the genome. These genes can be introduced using a temperature-sensitive vector or integration vector.
As disclosed in JP2-109985A, it is also possible to incorporate the evgA, gadE, or ydeO gene into a transposon, and transfer it so that multiple copies of the gene are integrated into the chromosomal DNA. Integration of the evgA, gadE, or ydeO gene into the genome can be confirmed by Southern hybridization using a probe having a part of the evgA, gadE, or ydeO gene.
Aside from increasing the copy number of the gene described above, expression of the evgA, gadE, or ydeO gene can also be enhanced employing the methods described in WO00/18935. These method include, for example, substituting the expression regulatory sequence such as a promoter of the evgA, gadE, or ydeO gene, etc., on the genomic DNA or plasmid with a stronger one, creating a consensus sequence of the −35, −10 regions of each gene, amplifying a regulator which can enhance the expression of the evgA, gadE, or ydeO gene, and deleting or weakening a regulator which would decrease the expression of the evgA, gadE, or ydeO gene. For example, the lac promoter, trp promoter, trc promoter, tac promoter, araBA promoter, lambda phage PR promoter, PL promoter, tet promoter, T7 promoter, φ10 promoter, etc., are all known as strong promoters. It is also possible to introduce a nucleotide substitution, etc., into the promoter region or SD region of the evgA, gadE, or ydeO gene so to increase the promoter strength. Examples of methods for evaluating the strength of promoters and examples of strong promoters are described in articles by Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)), etc. Furthermore, substitution of several nucleotides in the spacer region between the ribosome binding site (RBS) and the start codon, particularly in the sequence immediately upstream of the start codon, is known to have a strong effect on mRNA translation efficiency. The expression regulatory region such as a promoter etc. of the evgA, gadE, or ydeO gene, can be identified by a promoter-probe vector and gene analysis software such as GENETYX, etc. Expression of the evgA, gadE, or ydeO gene can be enhanced by substitutions or modifications of these promoters. Expression regulatory sequences can be substituted, for example, by employing temperature-sensitive plasmids or the Red-driven integration method (WO2005/010175).
A mutation which increases transcription of a gene whose expression is regulated by the evgA, gadE, or ydeO gene may also be introduced into the evgA, gadE, or ydeO gene. Examples of mutations which increase the activity of the protein encoded by the evgA, gadE, or ydeO gene are a mutation of the promoter sequence, which increases the transcription amount of the evgA, gadE, or ydeO gene, and a mutation within the encoded region of the gene, which increases the specific activity of the transcription.
The protein encoded by the evgA, gadE, or ydeO gene is activated by histidine kinase EvgS. Therefore, in expression of the evgA, gadE, or ydeO gene may be increased in the host microorganism by increasing the expression of the evgS gene that encodes EvgS. The evgS gene includes the evgS gene derived or isolated from an Escherichia bacterium and its homologs. For example, the evgS gene of Escherichia coli is exemplified by a gene (SEQ ID NO: 25) that encodes a protein having the amino acid sequence of SEQ ID NO: 26. (GenBank Accession No. NP_417969 [gi:16131384]).
The homologs of the evgS gene include those which are derived from other microorganisms, have high similarity in structure to the Escherichia evgS gene, improve the ability to produce an L-amino acid, and exhibit transcription factor activity when introduced into the host. Examples of evgS homologs include evgS genes of the genera Salmonella, Shigella, and Yersinia registered at GenBank. Furthermore, based on the homology with the genes given in the above examples, evgS genes may be cloned from coryneform bacteria, such as Corynebacterium glutamicum, Brevibacterium lactofermentum, etc.; bacteria of the genus Pseudomonas, such as Pseudomonas aeruginosa, etc.; bacteria of the genus Mycobacterium, such as Mycobacterium tuberculosis, etc.; and bacteria of the genus Bacillus. Ones with different gene names are acceptable as long as they are highly homologous with the evgS of the bacteria of the genus Escherichia. For example, in Escherichia coli, the evgS gene forms an operon with the evgA gene, and can be obtained along with the evgA gene by PCR using the synthetic oligonucleotides of SEQ ID NOS: 17 and 18. With other microorganisms, it is expected that these genes can be obtained likewise using the oligonucleotides of SEQ ID NOS: 17 and 18.
The evgS gene is not limited to a wild-type gene and may include a variant evgS gene as long as the function of the protein, i.e., the transcription factor activity, is not impaired. Such a variant may include a mutant or artificially modified protein having a sequence including one or several amino acid substitutions, deletions, insertions, additions, or the like at one or more positions in the amino acid sequence of SEQ ID NO: 26, i.e., a protein that has conservative mutation. The terms “several” and “conservative mutation” are the same as those described above regarding the evgA gene, gadE gene, and ydeO gene.
The expression of evgS can be increased using the same method as used for the evgA, gadE, and ydeO genes as described above.
The expression of the evgA, gadE, or ydeO gene can be increased using one of the same method or different methods may be used for each gene.
2. Method for Producing L-Amino Acid
The method for producing L-amino acid of the present invention includes the steps of culturing the microorganism in a medium, producing and accumulating the L-amino acid in said medium or in the cells of the microorganism, and collecting the L-amino acid from said medium or the cells.
Conventional mediums typically used in the fermentation and production of L-amino acids using microorganisms may be used. That is, an ordinary medium containing a carbon source, nitrogen source, inorganic ions, and other organic components as needed may be used. Carbon sources include a sugar such as glucose, sucrose, lactose, galactose, fructose, a starch hydrolysase, etc., an alcohol such as glycerol, solbitol, etc., an organic acid such as fumaric acid, citric acid, succinic acid, etc. Of these, glucose, fructose, and sucrose may be advantageously used. Nitrogen sources include an inorganic ammonium salt such as ammonium sulfate, ammonium chloride, ammonium phosphate, etc., an organic nitrogen such as a soybean hydrolysate, etc., ammonia gas, ammonia water, etc. As the organic micronutrient source, it is advantageous that the medium include an appropriate amount of the required substances, such as vitamin B1, L-homoserine, etc., or yeast extract, etc. In addition to these, according to necessity, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions, etc., can be added. The medium may be either natural or synthetic as long as it contains a carbon source, nitrogen source, inorganic ions, and, as needed, other organic micronutrients.
An amino acid which improves the growth of a microorganism or productivity of target substance may be added to the medium. For example, it is advantageous that L-threonine, L-homoserine or L-isoleucine is added for L-lysine fermentation. L-isoleucine, L-lysine, L-glutamic acid or L-homoserine is added for L-threonine fermentation, and L-phenylalanine or L-tyrosine or the like is added for L-tryptophan fermentation. The concentration of the L-amino acid is around 0.01-10 g/L.
The culture should be performed under aerobic conditions for 1-7 days at a temperature of 24° C.-37° C., with a pH of 5-9. To adjust the pH, an inorganic or organic acidic or alkali substance, and ammonia gas and the like may be used. L-amino acids can be collected from the fermentation broth using a combination of a conventional ion-exchange resin method, precipitation method, and other known methods. If the L-amino acid accumulates inside the cells, the cells can be disrupted by ultrasonication, etc. Then, cell debris can be removed by centrifugal separation to obtain the supernatant, from which the L-amino acid can be recollected using an ion-exchange resin method, etc.
EXAMPLES
The present invention will be explained more specifically below with reference to the following non limiting Examples.
Reference Example 1
Construction of L-Tryptophan-Producing Bacteria
1-1. Introduction of the serA Gene
The phosphoglycerate dehydrogenase gene (serA) is present on the plasmid pGH5 (WO9408031) and was inserted into the genome of a bacterium using the transposon Mud. Plasmid pCE1134 (JP2-109985A) which contains MudII1734 was digested with BamHI to remove a DNA fragment containing the lac operon, the termini were blunt-ended, and a SmaI linker was inserted. This plasmid was redigested with SmaI, and self-closed to form a circular plasmid named pMu1134. A DNA fragment containing serA was excised from plasmid pGH5 by cleaving with ScaI and SalI, and the termini of the fragment were blunt-ended. Then the fragment was inserted into the SmaI site of the above described pMu1134. Thus, the plasmid pMudserA with Mud containing the pGH5-derived serA gene (named MudserA) was constructed.
The L-tryptophan-producing bacterial strain SV164 (WO9408031), which is desensitized to anthranilate synthase was used to obtain the bacterial strain L1 in which MudserA was transferred onto the genome using acquisition of kanamycin resistance as an index. From the results of a Southern hybridization experiment, it was determined that there was only one location where the MudserA was inserted in the L1 strain. Also, with the cloning of a genomic DNA fragment containing MudserA by PCR and the determination of its nucleotide sequence, it was determined that the insertion was at the position No. 240, 950 on the E. coli K-12 genome (GenBank Accession No. U00096).
1-2. Introduction of the Trp Operon
Next, the copy number of the trp operon was increased by inserting the trp operon into the genome using a transposon. The trp operon genes were excised from the plasmid pGX100. pGX100 was constructed by inserting a DNA fragment derived from E. coli MTR#2 (U.S. Pat. No. 4,371,614) into the plasmid pBR313. E. coli MTR#2 has a desensitized trpE gene. A DNA fragment containing the approximately 7.6 kb trp operon can be excised from the plasmid by cleaving with XhoI and SmaI. A DNA fragment containing the trp operon was excised from pGX100 by cleaving with XhoI and SmaI, and the termini of the fragment were blunt-ended. Then, the fragment was inserted into the SmaI site of the above-described pCE1134. The same DNA fragment containing the trp operon can also be cloned directly from the E. coli MTR#2 strain genomic DNA, employing PCR, using the primers SEQ ID NOS: 1 and 2. As described above, plasmid pMudtrpG′ lac was constructed. This plasmid contains Mud into which the trp operon genes derived from the MTR#2 strain had been inserted (named MudtrpG′ lac).
Prior to increasing the copy number of MudtrpG′ lac by inserting MudtrpG′ lac into the genome, the host strain was made lactose-assimilation deficient for the purpose of using complementation of lactose-assimilating ability as a selection marker for the strain. The ilvG gene of the L-threonine-producing bacterium VKPM B-3996 (U.S. Pat. No. 5,175,107) was P1-transduced into the L1 strain to render it L-valine resistant (See WO2005/103228). The P1 transduction was conducted and transductants were spread on an M9 minimum medium (4 g/L glucose, 12.8 g/L Na2HPO4.7H2O, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 5 mM MgSO4, 0.1 mM CaCl2, 1 mg/L thiamine, 20 mg/l L-Phe, 20 mg/L L-Tyr, 20 mg/L L-Met, 3 mg/L pyridoxine, 20 mg/L L-Val, 20 mg/L tetracycline), and a colonies resistant to valine were obtained, and named L1ValR.
lacZ98::Tn10 was P1-transduced into L1ValR from the ME8581 strain (HfrH (valS←uxuAB):lacZ98::Tn10 relA1 thi-1, deposited at the National Institute of Genetics), with Tn10-derived tetracycline resistance as the indicator. The strain obtained was lactose-assimilation deficient, as expected. Next, in order to obtain a strain with deficient lactose-assimilating ability, but without Tn10, a tetracycline-sensitive strain 14-1-lac-tets was obtained from the transduced strains through the replica method. The lactose-assimilating ability was still deficient in the 14-1-lac-tets strain. When confirming the Tn10 status of the strain by Southern hybridization, no band hybridizing with the tet gene was detected, but a band hybridizing with the IS10 region of Tn10 was detected. Therefore, it was determined that IS10 remains on the lacZ gene.
Using pMudtrpG′ lac and the 14-1-lac-tets strain, bacterial strain No. 202 in which MudtrpG′ lac was transferred onto the genome was obtained, with complementation of lactose-assimilating ability as the indicator. If the inserted transposon, or the gene on transposon, tends to drop off from the transposon-inserted strain, a subculture may be performed in a nutrient-rich medium to select a bacterial strain which stably retains kanamycin resistance, lactose-assimilating ability, etc. As a result of Southern hybridization, it was determined that there was only one location where MudtrpG′ lac was inserted into the No. 202 strain. Also, with the cloning of a genomic DNA fragment containing MudtrpG′ lac by PCR and the determination of its nucleotide sequence, it was determined that the insertion was at the position No. 530,249 on the E. coli K-12 genome (GenBank Accession No. U00096).
Next, the scrK, scrY, scrA, scrB, and scrR genes, which are involved in sucrose assimilation, were introduced into No. 202 by P1 transduction, and the resulting bacterial strain was designated No. 202 scr (see WO90/04636).
1-3. Construction of a Plasmid for Disrupting iclR
The iclR fragment was amplified by PCR using Pyrobest DNA Polymerase (Takara Shuzo) according to the attached instruction manual. For the PCR reaction, oligonucleotides of SEQ ID NOS: 3 and 4 were used as the primers, with the W3110 genome extracted using an RNA/DNA maxi Kit (QIAGEN) as the template. After the PCR, the amplified DNA fragment was purified using Wizard PCR Preps (Promega). The purified DNA fragment was digested with restriction endonucleases EcoRI and Hind III (Takara Shuzo), then underwent phenol chloroform treatment and ethanol precipitation to obtain purified DNA. This fragment and pUC18 (Takara Shuzo), which was digested with the same enzymes and then purified, were ligated using DNA ligation Kit Ver. 2 (Takara Shuzo). With this ligation reaction solution, the JM109 competent cells (Takara Shuzo) were transformed, and then spread onto an LB agar plate containing 50 μg/mL ampicillin (Amp) (Meiji Seika) (LB+Amp plate), and colonies were selected at 37° C. The colonies were cultured in a test tube using an LB medium containing 50 μg/mL Amp at 37° C., and the plasmid was extracted using the automatic plasmid extractor PI-50 (Kurabo).
The obtained plasmid pUCiclR was digested with restriction endonuclease EcoO65I (Takara Shuzo), blunt-ended, and ligated using a BKL kit (Takara Shuzo). With the ligation reaction solution, JM109 was transformed, and colonies were selected as described above, and the plasmid was extracted. The obtained plasmid was digested with EcoRI and HindIII, purified, and ligated with the temperature-sensitive plasmid pTS1, which had been digested with the same enzymes and purified. pTS1 was obtained by replacing a PstI-HindIII fragment of pMAN031 (see J. Bacteriol. 162, 1196-1202 (1985), FIG. 1) with a PstI-HindIII fragment of pBR322 (Takara Shuzo). JM109 was transformed with the ligation reaction solution, and colonies were selected on the LB+Amp plate at 30° C. The colonies were cultured at 30° C. in a test tube using LB medium containing 50 μg/mL Amp, and the plasmid was extracted as described above. The plasmid which generates a fragment with an expected length by digestion with EcoRI and HindIII was designated as the iclR disrupting plasmid pTSΔiclR.
1-4. Obtaining an iclR-Disrupted Strain
No. 202 scr was transformed with pTSΔiclR, and colonies were selected on the LB+Amp plate at 30° C. After liquid culture was performed overnight at 30° C., the culture was diluted by 10−3 and spread on the LB+Amp plate, and the colonies were selected at 42° C. Specifically, the culture was spread on the LB+Amp plate and cultured at 30° C., cells which covered 1⁄8 of the plate were suspended in a 2 mL LB medium, and cultured with shaking for 4-5 hours at 42° C. The cells diluted by 10−5 were spread on the LB plate and colonies were obtained. Of these, one hundred colonies were spread on the LB plate and LB+Amp plate, respectively, and their growth was confirmed, and by which the Amp sensitivity and resistance were confirmed. Amp-sensitive strains were examined by colony PCR using the oligonucleotides of SEQ ID NOS: 3 and 4 as the primers. As a result, an iclR-deficient strain (No. 202ΔiclR) in which an amplified fragment could not be cut with EcoO65I was obtained.
Example 1
Construction of a Plasmid for Enhancing the Expression of the evgAS, gadE, and ydeO Genes
1-1. Construction of a Plasmid for Enhancing the Expression of the evgA and evgS Genes
The entire nucleotide sequence of the genome of Escherichia coli (Escherichia coli K-12 strain) has been reported (Science, 277, 1453-1474 (1997)), and it is known that evgAS forms an operon structure. Based on the nucleotide sequences of the evgAS genes (GenBank Accession Nos. AAC75428 and AAC75429) which were reported in the above report, the 5′ primer SEQ ID NO:17 having a HindIII site, and the 3′ primer SEQ ID NO:18 having an EcoRI site were synthesized. Using these primers, PCR was performed with the genomic DNA of the Escherichia coli W3110 strain as the template, and the amplified product was treated with restriction endonucleases HindIII and EcoRI to obtain a fragment containing the evgAS genes. The purified PCR product was ligated to vector pMW118 (Nippon Gene Co., Ltd.) which had been digested with HindIII and EcoRI, thus constructing the plasmid pMW-evgAS for amplifying the evgAS operon.
1-2. Construction of a Plasmid for Enhancing the Expression of gadE
The entire nucleotide sequence of the genome of Escherichia coli (Escherichia coli K-12 strain) has been reported (Science, 277, 1453-1474 (1997)). Based on the nucleotide sequence of the gadE gene (GenBank Accession No. AAC76537) which was reported in the above report, the 5′ primer SEQ ID NO:19 having a BamHI site, and the 3′ primer SEQ ID NO:20 having a HindII site were synthesized. Using these primers, PCR was performed with the genomic DNA of the Escherichia coli W3110 strain as the template, and the amplified product was treated with restriction endonucleases HindIII and BamHI to obtain a fragment containing the gadE gene. The purified PCR product was ligated to vector pMW118 (Nippon Gene Co., Ltd.), which had been digested with HindIII and BamHI, thus constructing the plasmid pMW-gadE for amplifying gadE.
1-3. Construction of a Plasmid for Enhancing ydeO
The entire nucleotide sequence of the genome of Escherichia coli (Escherichia coli K-12 strain) has been reported (Science, 277, 1453-1474 (1997)). Based on the nucleotide sequence of the ydeO gene (GenBank Accession No. AAC74572) which was reported in the above report, in the 5′ primer SEQ ID NO:21 having a HindIII site, and the 3′ primer SEQ ID NO:22 having a EcoRI site were synthesized. Using these primers, PCR was performed with the genomic DNA of the Escherichia coli W3110 strain as the template, and the amplified product was treated with restriction endonucleases HindIII and EcoRI to obtain a fragment containing the ydeO gene. The purified PCR product was ligated to vector pMW118 (Nippon Gene Co., Ltd.), which had been digested with HindIII and EcoRI, thus constructing the plasmid pMW-ydeO for amplifying ydeO.
Example 2
Effect of evgAS, ydeO, and gadE Gene Amplification on an L-Tryptophan-Producing Strain of a Bacterium of the Genus Escherichia 
The L-tryptophan-producing strain No. 202ΔiclR constructed in Reference Example 1 was transformed with each of the gadE-amplifying plasmid pMW-gadE, evgAS-amplifying plasmid pMW-evgAS, and the ydeO-amplifying plasmid pMW-ydeO created in Example 1, thus obtaining ampicillin-resistant strains. After confirming that these plasmids had been introduced, the strain with the gadE-amplifying plasmid pMW-gadE was designated No. 202ΔiclR/gadE, the strain with the evgAS-amplifying plasmid pMW-evgAS was designated No. 202ΔiclR/evgAS, and the strain with the ydeO-amplifying plasmid pMW-ydeO was designated No. 202ΔiclR/ydeO.
evgAS, ydeO, or gadE may be amplified in the same manner using known L-tryptophan-producing bacteria other than the No. 202ΔiclR strain.
The above-described strains were cultured in LB medium containing 50 mg/L ampicillin at 37° C. until the OD600 became approx. 0.6. An equal volume of 40% glycerol solution was added to the culture broth, stirred, and then appropriate amounts were dispensed and stored at −80° C. to make the glycerol stock.
After melting the glycerol stocks of these strains, 100 mL of each was homogeneously spread onto an L plate containing 50 mg/L ampicillin, and the plate was incubated at 37° C. for 24 hours. Approx. 1⁄8 of the cells on the plate were inoculated into a 20 mL fermentation medium with 50 mL ampicillin in a 500 mL Sakaguchi flask, and cultured at 37° C. for 48 hours using a reciprocal shaking incubator. After the culture, the amount of L-tryptophan which had accumulated in the medium was measured using an amino acid-analyzer L-8500 (Hitachi). The medium composition used for the culture was as follows.
L-Tryptophan-Producing Medium:

















Glucose
40
g/L



(NH4)2SO4
15
g/L



KH2PO4
1.5
g/L



MgSO4•7H2O
0.3
g/L



FeSO4•7H2O
0.01
g/L



MnSO4•7H2O
0.01
g/L



Yeast Extract
2.0
g/L



ThiaminHCl
0.005
g/L



Pyridoxine
0.03
g/L



L-Met
0.05
g/L



L-Phe
0.1
g/L



L-Tyr
0.1
g/L



CaCO3
30
g/L










Adjusted to pH 7.0 with KOH, autoclaved at 120° C. for 20 min. Glucose and MgSO4.7H2O were mixed and sterilized separately from other components. CaCO3 was added after dry-heat sterilization.
The OD600 and L-tryptophan accumulation at the 48th hour are shown in Table 1.







TABLE 1







Effect of evgAS, ydeO, and gadE amplification


on Trp-producing bacteria No.202ΔiclR











Strain
OD (600 nm)
Trp concentration (g/l)







No.202ΔiclR
7.0
7.2



No.202ΔiclR/evgAS
7.2
7.6



No.202ΔiclR/ydeO
7.0
7.4



No.202ΔiclR/gadE
7.4
7.5










No. 202ΔiclR/evgAS, No. 202ΔiclR/ydeO, and No. 202ΔiclR/gadE, which are the evgAS, ydeO, and gadE gene-amplified strains, respectively, each indicated an increase in the accumulation of L-tryptophan as compared to the control No. 202ΔiclR.
Example 3
Effect of evgAS, ydeO, and gadE Amplification on an L-Threonine-Producing Strain of a Bacterium of the Genus Escherichia 
Escherichia coli VKPM B-5318 strain (see EP0593792) was used as an L-threonine-producing strain of a bacterium of the genus Escherichia 
The VKPM B-5318 strain was transformed with each of the gadE-amplifying plasmid pMW-gadE, evgAS-amplifying plasmid pMW-evgAS, and the ydeO-amplifying plasmid pMW-ydeO constructed in Example 1, thus obtaining ampicillin-resistant strains. After confirming that these plasmids had been introduced, the strain with the gadE-amplifying plasmid pMW-gadE was designated B5318/gadE, the strain with the evgAS-amplifying plasmid pMW-evgAS was designated B5318/evgAS, and the strain with the ydeO-amplifying plasmid pMW-ydeO was designated B5318/ydeO.
evgAS, ydeO, or gadE may be amplified in the same manner using other known L-threonine-producing bacteria.
The above-described strains were cultured in LB medium containing 50 mg/L ampicillin at 37° C. until the OD600 became approx. 0.6. An equal volume of 40% glycerol solution was added to the culture broth, stirred, and then appropriate amounts were dispensed and stored at −80° C. to make the glycerol stock.
After melting the glycerol stocks of these strains, 100 mL of each was homogeneously spread onto an L plate containing 50 mg/L ampicillin, and the plate was incubated at 37° C. for 24 hours. Approx. 1⁄8 of the cells on the plate were inoculated into a 20 mL fermentation medium described below with 50 mL ampicillin in a 500 mL Sakaguchi flask, and cultured at 37° C. for 48 hours using a reciprocal shaking incubator. After the culture, the amount of L-threonine which had accumulated in the medium was measured using an amino acid-analyzer L-8500 (Hitachi). The medium composition used for the culture was as follows.
L-Threonine-Producing Medium:

















Glucose
40
g/L



(NH4)2SO4
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 (Japanese Pharmacopoeia)
30
g/L










Adjusted to pH 7.0 with KOH, autoclaved at 120° C. for 20 min. Glucose and MgSO4.7H2O were mixed and sterilized separately. CaCO3 was added after dry-heat sterilization.
The OD600 and L-threonine accumulation at the 48th hour are shown in Table 2.







TABLE 2







Effect of evgAS, ydeO, and gadE amplification on











Strain
OD (600 nm)
Thr concentration (g/l)







B5318
8.1
6.7



B5318/evgAS
8.1
6.9



B5318/ydeO
7.9
7.0



B5318/gadE
8.4
7.2










B5318/evgAS, B5318/ydeO, and B5318/gadE, which are the evgAS, ydeO, and gadE gene-amplified strains, each indicated an increase in the accumulation of L-threonine compared to the control VKPM B-5318.
Example 4
Construction of an L-Lysine-Producing Bacterium
4-1. Construction of a Strain in which the cadA and ldcC Genes that Encode Lysine Decarboxylase are Disrupted
First, a strain which does not produce lysine decarboxylase was constructed. Lysine decarboxylase isozymes are encoded by the cadA gene (GenBank Accession No. NP_418555. SEQ ID NO: 31) and the ldcC gene (GenBank Accession No. NP_414728. SEQ ID NO: 33) (see WO96/17930). The WC196 strain (see WO96/17930), which is an AEC (S-(2-aminoethyl)-cysteine)-resistant strain, was used as the parent strain to construct the Escherichia coli L-lysine-producing strain.
The cadA and ldcC genes encoding lysine decarboxylase were deleted using a method called “Red-driven integration”, which was initially developed by Datsenko and Wanner (Proc. Natl. Acad. Sci. USA. 97. 6640-6645 (2000)), and a λ phage excision system (J. Bacteriol. 184. 5200-5203 (2002)). According to the “Red-driven integration” method, a gene-disrupted strain can be constructed in a single step by employing a PCR product obtained by using primers for which a part of the target gene was designed at the 5′ terminus and a part of the antibiotic-resistant gene was designed at the 3′terminus. Furthermore, in combination with the λ phage excision system, the antibiotic-resistant gene integrated into the gene-disrupted strain can be removed (JP2005-058227A).
4-2. Disruption of the cadA Gene
Plasmid pMW118-attL-Cm-attR (WO2005/010175) was used as the PCR template. pMW118-attL-Cm-attR is a plasmid wherein the attL and attR genes, which are the attachment sites of the λ phage, and the cat gene, which is an antibiotic-resistant gene, have been inserted into pMW118 (Takara Bio Inc.) in the following order: attL-cat-attR.
PCR was conducted using the primers SEQ ID NOS: 35 and 36, wherein each of the primers has a sequence corresponding to each ends of attL and attR at the 3′ end, and has a sequence corresponding to a part of the target cadA gene at the 5′ end.
The PCR product was purified using agarose gel, then introduced by electroporation to an Escherichia coli WC1-96 strain containing the plasmid pKD46, which has a temperature-sensitive replication origin. Plasmid pKD46 (Proc. Natl. Acad. Sci. USA. 97. 6640-6645 (2000)) contains the λ phage DNA fragments, which total 2154 bases, and include the genes (γ, β, exo genes) that encode Red recombinase in the λ Red homologous recombination system controlled by the arabinose-induced ParaB promoter (GenBank/EMBL Accession No. J02459, 31088th-33241st).
Competent cells for electroporation were prepared as follows. That is, an Escherichia coli WC1-96 strain cultured overnight at 30° C. in an LB medium containing 100 mg/L ampicillin was diluted 100 times in a 5 mL SOB medium containing ampicillin (20 mg/L) and L-arabinose (1 mM) (Molecular Cloning: Lab Manual 2nd edition, Sambrook, J., et al., Cold Spring Harbor Laboratory Press (1989)). The diluted suspension was aerated at 30° C. until the OD600 reached approx. 0.6, and then the suspension was concentrated 100 times and washed three times with 10% glycerol to prepare it for electroporation. Electroporation was performed using 70 μL competent cells and approx. 100 ng PCR product. 1 mL SOC medium (Molecular Cloning: Lab Manual 2nd edition, Sambrook, J., et al., Cold Spring Harbor Laboratory Press (1989)) was added to the post-electroporation cells, and cultured at 37° C. for 2.5 hours, then cultured on a plate medium of L-agar containing Cm (chloramphenicol) (25 mg/L) at 37° C., thus selecting the Cm-resistant recombinant. Next, to remove the pKD46 plasmid, the recombinant was subcultured twice on an L-agar medium containing Cm at 42° C., the ampicillin resistance of the colony obtained was tested, and an ampicillin-sensitive strain from which the pKD46 was omitted was obtained.
Deletion of the cadA gene in the mutant identified by the chloramphenicol-resistant gene was confirmed using the PCR. The cadA deficient strain obtained was designated WC196ΔcadA::att-cat.
Next, to remove the att-cat gene in the cadA gene, a helper plasmid, pMW-intxis-ts (WO2005/010175) was used. pMW-intxis-ts is a plasmid which contains a gene that encodes λ phage integrase (Int) and a gene that encodes excisionase (Xis).
The competent cells of the WC196ΔcadA::att-cat strain obtained as described above were prepared, transformed with helper plasmid pMW-intxis-ts, and cultured on a plate medium of L-agar containing 50 mg/L ampicillin at 30° C., thus selecting the ampicillin-resistant strain.
Next, to remove the pMW-intxis-ts plasmid, the ampicillin-resistant transformant was subcultured twice on an L-agar medium at 42° C. The ampicillin and chloramphenicol resistance of the colony were tested, and a chloramphenicol and ampicillin-sensitive strain from which the att-cat and pMW-intxis-ts were removed was obtained. This strain was designated WC196ΔcadA.
4-3. Disruption of the ldcC Gene in the WC196ΔcadA Strain
The ldcC gene in the WC196ΔcadA strain was deleted by the technique described above, using primers SEQ ID NOS: 37 and 38. Thus, WC196ΔcadAΔldcC, which is a cadA, ldcC-disrupted strain, was obtained.
Example 5
Effect of evgAS Amplification on an L-Lysine-Producing Strain of an Escherichia Bacterium
5-1. Introduction of Lysine-Producing Plasmid into the WC196ΔcadAΔldcC Strain
The WC196ΔcadAΔldcC strain was transformed with the lysine-producing plasmid pCAB1 which contains the dapA, dapB, and lysC genes (WO01/53459), to obtain the WC196ΔcadAΔldcC/pCAB1 strain (WC196LC/pCAB1).
The WC196LC/pCAB1 strain was transformed with the evgAS-amplifying plasmid pMW-evgAS, created in Example 1, thus obtaining an ampicillin-resistant strain. After confirming that the desired plasmid had been introduced, the evgAS-amplifying plasmid pMW-evgAS-introduced strain was designated WC196LC/pCAB1/evgAS.
evgAS may be amplified in the same manner using other known L-lysine-producing bacteria.
The strains created as above were cultured in LB medium containing 25 mL streptomycin and 50 mg/L ampicillin at 37° C. until the OD600 became approx. 0.6. An equal volume of 40% glycerol solution was added to the culture broth, stirred, and then appropriate amounts were dispensed and stored at −80° C. to make the glycerol stock.
5-2. Lysine-Producing Culture
After melting the glycerol stocks of these strains, 100 mL of each was homogeneously spread onto an L plate containing 50 mg/L ampicillin, and the plate was incubated at 37° C. for 24 hours. Approx. 1⁄8 of the cells on the plate obtained were inoculated into a 20 mL fermentation medium described below with 50 mL ampicillin in a 500 mL Sakaguchi flask, and cultured at 37° C. for 48 hours using reciprocal shaking. After the culture, the amount of L-lysine which had accumulated in the medium was measured using a Biotech-analyzer AS210 (Sakura Seiki). The medium composition used for the culture was as follows.
L-Lysine-Producing Medium:

















Glucose
40
g/L



(NH4)2SO4
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 (Japanese Pharmacopoeia)
30
g/L










Adjusted to pH 7.0 with KOH, autoclaved at 120° C. for 20 min. Glucose and MgSO4.7H2O were mixed and sterilized separately. CaCO3 was added after dry-heat sterilization.
The OD600 and L-lysine accumulation at the 48th hour are shown in Table 3.







TABLE 3







Effect of evgAS amplification on lysine-producing


bacterium WC196LC/pCAB1









strains
OD (600 nm)
Lys concentration (g/l)





WC196LC/pCAB1
7.3
14.3


WC196LC/pCAB1/evgAS
7.5
15.3









WC196LC/pCAB1/evgAS, which is the evgAS gene-amplified strain, indicated a significant increase in the accumulation of L-lysine compared to the control WC196LC/pCAB1.
Example 6
Effect of gadE Amplification on an L-Lysine-Producing Strain of an Escherichia Bacterium
6-1. Construction of an L-Lysine-Producing Strain in which the gadE Gene has been Enhanced
A gadE gene fragment was excised from the plasmid pMW-gadE, which contains the gadE gene of the Escherichia coli W3110 strain created in Example 1, and the fragment was ligated with vector pUC18 (Takara Shuzo) and digested with HindIII and BamHI, thus constructing the gadE-amplifying plasmid pUC-gadE.
The WC196LC/pCAB1 strain was transformed with the above-mentioned pUC-gadE, thus obtaining an ampicillin-resistant strain. After confirming that the desired plasmid had been introduced, the strain with the gadE-amplifying plasmid pUC-gadE was designated WC196LC/pCAB1/pUC-gadE.
evgAS, ydeO, or gadE may be amplified in the same manner using other known L-lysine-producing bacteria. Amplification of these genes may be performed in any order or combination.
The strains created as above were cultured in LB medium containing 25 mg/L streptomycin and 50 mg/L ampicillin at 37° C. until the OD600 reached approx. 0.6. An equal volume of a 40% glycerol solution was added to the culture broth, stirred, and then appropriate amounts were dispensed and stored at −80° C. to make the glycerol stock.
6-2. Lysine-Producing Culture
After melting the glycerol stocks of these strains, 100 mL of each was homogeneously spread onto an L plate containing 25 mg/L streptomycin and 50 mg/L ampicillin, and the plate was incubated at 37° C. for 24 hours. Approx. 1⁄8 of the cells on the plate were inoculated into a 20 mL fermentation medium described below with 25 mg/L streptomycin and 50 mL ampicillin in a 500 mL Sakaguchi flask, and cultured at 37° C. for 48 hours using a reciprocal shaking incubator. After the culture, the amount of L-lysine which had accumulated in the medium was measured using a Biotech-analyzer AS210 (Sakura Seiki). The medium composition used for the culture was as follows.
L-Lysine-Producing Medium:

















Glucose or Fructose
40
g/L



(NH4)2SO4
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



Isoleucine
0.1
g/L



Yeast Extract
2.0
g/L



CaCO3 (Japanese Pharmacopoeia)
30
g/L










Adjusted to pH 7.0 with KOH, autoclaved at 120° C. for 20 min. Glucose and MgSO4.7H2O were mixed and sterilized separately. CaCO3 was added after dry-heat sterilization.
The OD600 and L-lysine accumulated at the 48th hour are shown in Table 4.







TABLE 4







Effect of gadE amplification on lysine-producing bacterium


WC196LC/pCAB1 in glucose culture and fructose culture













L-lysine



Carbon
OD
concentration


Strain
source
(600 nm)
(g/l)













WC196LC/pCAB1
Glucose
8.8
8.9


WC196LC/pCAB1/pUCgadE
Glucose
8.6
9.2


WC196LC/pCAB1
Fructose
7.4
9.1


WC196LC/pCAB1/pUCgadE
Fructose
7.3
10.1









The gadE gene-amplified strain WC196LC/pCAB1/pUCgadE produced an increase in the accumulation of L-lysine in both glucose and fructose cultures compared to the control WC196LC/pCAB1, and in particular, a significant increase in the accumulation of L-lysine in the fructose culture was observed.
INDUSTRIAL APPLICABILITY
By using the microorganism of the present invention, L-amino acids can be efficiently produced by fermentation, specifically, L-lysine, L-threonine.
EXPLANATION OF SEQUENCE LISTING
SEQ ID NO: 1: primer for amplifying trp operon
SEQ ID NO: 2: primer for amplifying trp operon
SEQ ID NO: 3: primer for amplifying iclR gene
SEQ ID NO: 4: primer for amplifying iclR gene
SEQ ID NO: 5: nucleotide sequence of the iclR gene
SEQ ID NO: 6: sequence of an amino acid encoded by the iclR gene
SEQ ID NO: 7: nucleotide sequence of the aceA gene
SEQ ID NO: 8: sequence of an amino acid encoded by the aceA gene
SEQ ID NO: 9: nucleotide sequence of the aceB gene
SEQ ID NO: 10: sequence of an amino acid encoded by the aceB gene
SEQ ID NO: 11: nucleotide sequence of the aceK gene
SEQ ID NO: 12: sequence of an amino acid encoded by the aceK gene
SEQ ID NO: 13: nucleotide sequence of the trpA gene
SEQ ID NO: 14: sequence of an amino acid encoded by the trpA gene
SEQ ID NO: 15: nucleotide sequence of the trpB gene
SEQ ID NO: 16: sequence of an amino acid encoded by the trpB gene
SEQ ID NO: 17: primer for amplifying evgAS operon
SEQ ID NO: 18: primer for amplifying evgAS operon
SEQ ID NO: 19: primer for amplifying gadE gene
SEQ ID NO: 20: primer for amplifying gadE gene
SEQ ID NO: 21: primer for amplifying ydeO gene
SEQ ID NO: 22: primer for amplifying ydeO gene
SEQ ID NO: 23: nucleotide sequence of the evgA gene
SEQ ID NO: 24: amino acid sequence of EvgA
SEQ ID NO: 25: nucleotide sequence of the evgS gene
SEQ ID NO: 26: amino acid sequence of EvgS
SEQ ID NO: 27: nucleotide sequence of the gadE gene
SEQ ID NO: 28: amino acid sequence of GadE
SEQ ID NO: 29: nucleotide sequence of the ydeO gene
SEQ ID NO: 30: amino acid sequence of YdeO
SEQ ID NO: 31: nucleotide sequence of the cadA gene
SEQ ID NO: 32: sequence of an amino acid encoded by the cadA gene
SEQ ID NO: 33: nucleotide sequence of the ldcC gene
SEQ ID NO: 34: sequence of an amino acid encoded by the ldcC gene
SEQ ID NO: 35: PCR primer for disrupting the cadA gene
SEQ ID NO: 36: PCR primer for disrupting the cadA gene
SEQ ID NO: 37: PCR primer for disrupting the ldc gene
SEQ ID NO: 38: PCR primer for disrupting the ldc 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. Each of the aforementioned documents is incorporated by reference herein in its entirety.

## Claims

1. A method for producing an L-amino acid comprising:
A) culturing in a medium a bacterium of the family Enterobacteriaceae which has an ability to produce an L-amino acid and which has been modified to increase the expression of a gene selected from the group consisting of evgA, gadE, ydeO, and combinations thereof,
B) producing and accumulating the L-amino acid in the medium, and
C) collecting the L-amino acid from the medium,
wherein expression of said gene(s) is/are increased by a method selected from the group consisting of:
a) increasing the copy number of said gene(s),
b) modifying an expression regulatory sequence of said gene(s),
c) modifying the bacterium so that the expression of an evgS gene that encodes a sensor kinase is increased by a method selected from the group consisting of:
i) increasing the copy number of the evgS gene,
ii) modifying the expression regulatory sequence of the evgS gene, and
iii) combinations thereof; and

d) combinations thereof, and
wherein said L-amino acid is selected from the group consisting of a basic amino acid, a hydroxymonoaminocarboxylic acid, and an aromatic amino acid,
wherein the basic amino acid is selected from the group consisting of L-lysine, L-ornithine, L-arginine, L-histidine, and L-citrulline;
wherein the hydroxymonoaminocarboxylic acid is selected from the group consisting of L-threonine and L-serine; and
wherein the aromatic amino acid is selected from the group consisting of L-tryptophan, L-phenylalanine, and L-tyrosine.

2. The method according to claim 1, wherein the evgA gene is selected from the group consisting of:
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 23, and
(b) a DNA which hybridizes with:
i) a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 23, or
ii) a probe prepared from the nucleotide sequence in i) under stringent conditions, and which encodes a protein having transcription factor activity.

3. The method according to claim 1, wherein the gadE gene is selected from the group consisting of:
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 27, and
(b) a DNA which hybridizes with:
i) a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 27 or
ii) a probe prepared from the nucleotide sequence in i) under stringent conditions, and which encodes a protein having transcription factor activity.

4. The method according to claim 1, wherein the ydeO gene is selected from the group consisting of:
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 29, and
(b) a DNA which hybridizes with:
i) a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 29 or
ii) a probe prepared from the nucleotide sequence in i) under stringent conditions, and which encodes a protein having transcription factor activity.

5. The method according to claim 1, wherein the evgS gene is selected from the group consisting of:
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 25, and
(b) a DNA which hybridizes with:
i) a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 25, or
ii) a probe prepared from the nucleotide sequence in i) under stringent conditions, and which encodes a protein having phosphotransfer activity.

6. The method according to claim 1, wherein the evgA gene encodes a protein selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 24, and
(b) a protein comprising the amino acid sequence of SEQ ID NO: 24, but which includes one or more amino acid substitutions, deletions, insertions, additions, or inversions and has transcription factor activity.

7. The method according to claim 1, wherein the gadE gene encodes a protein selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 28, and
(b) a protein comprising the amino acid sequence of SEQ ID NO: 28, but which includes one or more amino acid substitutions, deletions, insertions, additions, or inversions and has transcription factor activity.

8. The method according to claim 1, wherein the ydeO gene encodes a protein selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 30,
(b) a protein comprising the amino acid sequence of SEQ ID NO: 30, but which includes one or more amino acid substitutions, deletions, insertions, additions, or inversions and has transcription factor activity.

9. The method according to claim 1, wherein the evgS gene encodes a protein selected from the group consisting of:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 26,
(b) a protein comprising the amino acid sequence of SEQ ID NO: 26, but which includes one or more substitutions, deletions, insertions, additions, or inversions and has phosphotransfer activity.

10. The method according to claim 1 wherein the bacterium is a member of the genera selected from the group consisting of Escherichia, Enterobacter, Pantoea, Klebsiella, and Serratia.

11. The method according to claim 1 wherein the L-amino acid is selected from the group consisting of L-lysine, L-threonine, L-tryptophan, and combinations thereof.