WO2013129432A1 - A MUTANT PROTEIN ENCODED BY THE yddG GENE, AND A METHOD FOR PRODUCING AROMATIC L-AMINO ACIDS USING A BACTERIUM OF THE GENUS ESCHERICHIA - Google Patents

Abstract

The present invention relates to the biotechnology industry and provides a bacterium of the genus Escherichia harboring a DNA which encodes mutant amino acid exporter protein YddG. In addition, the method for producing aromatic L-amino acids such as L-phenylalanine and L-tryptophan using said bacterium is provided.

Description

DESCRIPTION

A MUTANT PROTEIN ENCODED BY THE yddG GENE, AND A METHOD FOR PRODUCING AROMATIC L-AMINO ACIDS USING A BACTERIUM OF THE GENUS ESCHERICHIA

Technical Field

The present invention relates to the microbiological industry, and specifically to a method for producing L-amino acids, and more specifically aromatic L-amino acids such as L-phenylalanine, L-tyrosine, and L-tryptophan, by fermentation using a bacterium of the genus Escherichia that has been modified to obtain the mutant amino acid exporter protein YddG. The mutant protein and the method using thereof are advantageous to improvement of aromatic L-amino acids productivity.

Background Art

Conventionally L-amino acids have been industrially produced by method of fermentation utilizing strains of microorganisms obtained from natural sources or mutants of the same especially modified to enhance L-amino acid productivity.

There have been disclosed many techniques to enhance L-amino acid productivity, for example, by transformation of microorganism by recombinant DNA (see, for example, U.S. Patent No. 4,278,765). These techniques are based on the increasing of activities of the enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes from the feedback inhibition by produced L-amino acid (see, for example, Japanese Patent Publication No. 56-18596 (1981), WO 95/16042 or U.S. Patent Nos. 5,661,012 and 6,040,160).

On the other hand, the enhancement of amino acid excretion activity may improve the productivity of L-amino acid producing strain. Lysine-producing strain of a bacterium belonging to the genus Corynebacterium having increased expression of L-lysine excretion gene (tysE) is disclosed (WO 9723597 A2). In addition, genes encoding efflux proteins suitable for secretion of L-cysteine, L-cystine, N-acetylserine or thiazolidine derivatives are also disclosed (U.S. Patent No. 5,972,663).

At present, several Escherichia coli (E. coli) genes encoding putative membrane proteins enhancing L-amino acid production are disclosed. Additional copies oirhtB gene make a bacterium more resistant to L-homoserine and enhance the production of L- homoserine, L-threonine, L-alanine, L-valine and L-isoleucine (European Patent application No. 994190 A2). Additional copies of the rhtC gene make a bacterium more resistant to L-homoserine and L-threonine and enhance production of L-homoserine, L- threonine and L-leucine (European Patent application No. 1013765 Bl). Additional copies of yahN, yeaS, yfiK, and yggA genes enhance production of L-glutamic acid, L- lysine, L-threonine, L-alanine, L-histidine, L-proline, L-arginine, L-valine and L- isoleucine (European Patent application No. 1016710 Bl).

It has been revealed that the rhtA gene exists on E. coli chromosome close to the glnHPQ operon that encodes components of the glutamine transport system, and that the rhtA gene is identical to ybiF open reading frame (ORF) between pexB and ompX genes. The unit expressing a protein encoded by the ORF has been designated as rhtA gene (rht is abbreviated from resistance to homoserine and threonine).

In addition, it has been found that the rhtA gene amplification also conferred resistance to homoserine and threonine. The rhtA23 mutation is an A-for-G substitution at position -1 with respect to the ATG start codon (ABSTRACTS of 17^th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, Calif, Aug. 24-29, 1997, abstract No. 457). It is known that the nucleotide composition of the spacer between the Shine-Dalgarno (SD) sequence and start codon and especially the sequences immediately upstream of the start codon profoundly affect mRNA

translatability. A 20-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon (Gold et al., Annu. Rev.

Microbiol, 1981, 35:365-403; Hui et al., EMBO J., 1984, 3:623-629). Therefore, it may be predicted that rhtA23 mutation increases expression of the rhtA gene.

The rhtA gene encodes a protein that consists of 295 amino acid residues and has calculated molecular weight of 31.3 kDa. The analysis of the RhtA sequence revealed that it is a highly hydrophobic protein containing 10 predicted transmembrane segments. A PSI-BLAST search of the nucleotide sequence of E. coli strain K-12 belonging to the genus Escherichia (Blattner F.R. et al., Science, 1997, 277:1453-1474) has revealed at least 10 proteins paralogous to RhtA. Among them there are proteins encoded by the ydeD and yddG genes. It has been shown that the ydeD gene is involved into efflux of the cysteine pathway metabolites (Dassler T. et al., Mol. Microbiol, 2000, 36: 1101-11 12; U.S. Patent No. 5,972,663).

The yddG gene has been identified by the present inventors as a 10

transmembrane helices-based membrane protein (Airich L.G. et al., J. Mol. Microbiol. Biotechnol, 2010, 19: 189-197) capable of exporting aromatic amino acids in E. coli (Doroshenko V.G. et al., FEMS Microbiol Lett., 2007, 275:312-318). A mutant E. coli strain having enhanced activity of the YddG protein has been found to affect positively L- phenylalanine and L-tryptophan productivity (Russian Patent No. 2222596).

To date, no data has been reported demonstrating the effect from the mutant YddG protein according to the present invention on production of aromatic L-amino acids, more specifically, L-phenylalanine and L-tryptophan, using a bacterium belonging to the genus Escherichia.

Summary of the Invention

The present invention provides an aromatic L-amino acid- producing strain having enhanced productivity and a method for producing the aromatic L-amino acid such as L- phenylalanine, L-tyrosine, and L-tryptophan using the strain.

These were achieved by the finding that mutant yddG gene, which is not directly involved in biosynthetic pathway of target L-amino acid, conferred on a microorganism higher productivity of aromatic L-amino acids when the amino acid residue in the position 26, or 24 and 26 in the amino acid sequence of the wild-type allele of the yddG gene was/were replaced with another L-amino acid residue(s). Thus the present invention has been completed.

It is an aspect of the present invention to provide a mutant bacterial amino acid exporter protein which is selected from the group consisting of:

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

B) a variant protein comprising the amino acid sequence of SEQ ID NO: 2, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues and has amino acid exporter activity,

wherein the protein has a substitution selected from the group consisting of:

(C) L-isoleucine residue in the position number 24 is replaced by L-phenylalanine residue, or L-tryptophan residue, or L-tyrosine residue, or L-methionine residue, or L-leucine residue;

(D) glycine residue in the position number 26 is replaced by L-phenylalanine residue, or L-isoleucine residue, or L-glutamic acid residue, or L-leucine residue, L-methionine residue, or L-valine residue, or L-tryptophan residue, or L-tyrosine residue; and

(E) a combination of thereof,

wherein the position number is counted with SEQ ID NO: 2. It is a further aspect of the present invention to provide the mutant protein as described above, wherein the L-isoleucine residue in the position number 24 is replaced by for L-phenylalanine residue.

It is a further aspect of the present invention to provide the mutant protein as described above, wherein the glycine residue in the position number 26 is replaced by L- phenylalanine residue.

It is a further aspect of the present invention to provide the mutant protein as described above, wherein the glycine residue in the position number 26 is replaced by L- isoleucine residue.

It is a further aspect of the present invention to provide the mutant protein as described above, wherein the glycine residue in the position number 26 is replaced by L- glutamic acid residue.

It is a further aspect of the present invention to provide the mutant protein as described above, wherein the mutant protein is derived from a bacterium belonging to the genus Escherichia.

It is a further aspect of the present invention to provide the mutant protein as described above, wherein the mutant protein is derived from bacterium belonging to the species Escherichia coli.

It is an aspect of the present invention to provide a DNA encoding the mutant bacterial amino acid exporter protein as described above.

It is an aspect of the present invention to provide an aromatic L-amino acid- producing bacterium belonging to the genus Escherichia harboring the DNA as described above.

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

It is a further aspect of the present invention to provide the bacterium as described above, wherein the aromatic L-amino acid is selected from the group consisting of L- phenylalanine, L-tyrosine, and L-tryptophan.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the aromatic L-amino acid is L-phenylalanine.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the aromatic L-amino acid is L-tryptophan.

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

(F) cultivating the bacterium as described above in a culture medium; (G) accumulating the aromatic L-amino acid in the culture medium; and

(H) collecting the aromatic L-amino acid from the culture medium.

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

It is a further aspect of the present invention to provide the method as described above, wherein the aromatic L-amino acid is L-phenylalanine.

It is a further aspect of the present invention to provide the method as described above, wherein the aromatic L-amino acid is L-tryptophan.

It is an aspect of the present invention to provide a method for producing lower alkyl ester of N-( -L-aspartyl)-L-phenylalanine comprising:

(I) cultivating the bacterium as described above in a culture medium to produce and accumulate L-phenylalanine in the medium, wherein the bacterium has L- phenylalanine productivity;

(J) esterifying the L-phenylalanine to generate a lower alkyl ester of the L- phenylalanine; and

(K) generating a lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine from L- aspartic acid or its derivative and the lower alkyl ester of the L-phenylalanine. It is a further aspect of the present invention to provide the method as described above further comprising:

(L) condensing the lower alkyl ester of L-phenylalanine with a derivative of L- aspartic acid, wherein the derivative is N-acyl-L-aspartic anhydride;

(M) separating the lower alkyl ester of N-(N-acyl-a-L-aspartyl)-L-phenylalanine from the reaction mixture; and

(N) deprotecting the lower alkyl ester of N-(N-acyl-a-L-aspartyl)-L-phenylalanine to generate the lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine.

It is an aspect of the present invention to provide the method for producing lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine comprising:

(O) cultivating the bacterium as above described in a culture medium to produce and accumulate L-phenylalanine in the medium, wherein the bacterium has L- phenylalanine productivity;

(P) esterifying L-aspartic acid to generate a lower alkyl α,β-diester of the L- aspartic acid; and

(Q) generating a lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine from the lower alkyl α,β-diester of the L-aspartic acid and the L-phenylalanine. In the present invention, an amino acid is of L-configuration unless otherwise noted.

Brief Description of Drawings

FIG. 1 shows the scheme for introduction of the mutations into the yddG gene.

FIG. 2 shows the time-course of Phe accumulation by E. coli DV157 derivative strains.

W\57htrE P_L-yddG^G26F Cm^R (■);

DV157 htrE :?_L-yddG^G26E Cm^R (0);

DV 157 htrE: :?_L-yddG^{l2 F/G261} Cm^R (Δ);

OVl57htrE P_≠0-yddG Cm^R ( A);

OV\57htrE::?_L-yddG Cm^R (·).

Description of the Invention

The present invention is described in details below.

1. Bacterium

The bacterium as described herein is an L-amino acid producing bacterium belonging to the genus Escherichia, wherein the L-amino acid production by the bacterium is enhanced by modifying the amino acid sequence of the amino acid exporter protein of the present invention in a cell of the bacterium. The "amino acid exporter protein" is also referred to as "transmembrane protein" in the present specification.

The phrase "L-amino acid producing bacterium" as described herein can mean a bacterium which has an ability to produce and accumulate L-amino acid in a medium, exemplary aromatic L-amino acid, when the bacterium is cultured in the medium. The L- amino acid producing ability may be possessed by the bacterium as a property of a wild- type strain of the bacterium or may be imparted or enhanced by breeding.

The phrase "aromatic L-amino acid" includes L-phenylalanine, L-tyrosine, and L- tryptophan.

The phrase "to produce and accumulate L-amino acid in a medium" or

"accumulating L-amino acid in a medium" can mean an ability to produce, excrete or secrete, and cause accumulation of an L-amino acid in a medium.

Preferred embodiment of the bacterium as described herein is L-phenylalanine- producing bacterium belonging to the genus Escherichia which has the transmembrane protein of the present invention, amino acid sequence of which is modified. Another preferred embodiment of the bacterium as described herein is L-tryptophan-producing bacterium belonging to the genus Escherichia which has the transmembrane protein of the present invention, amino acid sequence of which is modified. In addition, the bacterium as described herein may be L-tyrosine-producing bacterium belonging to the genus Escherichia which has the transmembrane protein of the present invention, amino acid sequence of which is modified.

The bacterium belonging to the genus Escherichia is not particularly limited, however for example, bacteria described by Neidhardt, F.C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1) can be used.

The bacterium as described herein can harbor the DNA having the yddG gene modified to provide mutation in the position number 24 or 26, or both corresponding to that one(s) in the wild-type amino acid sequence encoded by the unmodified yddG gene of SEQ ID NO: 1. Exemplary substitution of the amino acid residue in the position number 24, which is the L-isoleucine residue as follows from the wild-type YddG amino acid sequence of SEQ ID NO: 2, can be the replacement by L-tryptophan residue, or L- tyrosine residue, or L-methionine residue, or L-leucine residue. Exemplary substitution of the amino acid residue in the position number 26, which is the glycine residue as follows from the wild-type YddG amino acid sequence of SEQ ID NO: 2, can be the replacement by L-phenylalanine residue, or L-isoleucine residue, or L-glutamic acid residue, or L- leucine residue, L-methionine residue, or L-valine residue, or L-tryptophan residue, or L- tyrosine residue. The exemplary substitutions in the position number 24 or 26, or both in the amino acid sequence encoded by the wild-type yddG gene, are not limited to the substitutions specified above. This is possible because amino acids, both proteinogenic and non-proteinogenic, or others, can be divided into families according to properties of their side-chain group at C2-atom such as space geometry, polarity (charge),

hydrophobicity or hydrophilicity, and so forth as well as the three dimensional structure of an amino acid sequence or a protein is not affected by such a change.

The bacterium as described herein can have the mutant yddG gene incorporated into the chromosome of the bacterium to replace the wild-type allele of the yddG gene, or in addition to the wild-type allele of the yddG gene. In case that two or more mutant yddG genes are harbored by the bacterium as described herein, the genes may be harbored together on the same plasmid or separately on different plasmids. It is also acceptable that one of the genes is harbored on a chromosome, and the other gene is harbored on a plasmid. The mutant yddG can be incorporated into the bacterium as described herein in such a way that the expression level of the mutant yddG gene is enhanced.

Enhancing of the mutant yddG gene expression as described herein may be achieved by placing the DNA harboring the mutant yddG gene under the control of a potent promoter. For example, the lac promoter, the trp promoter, the trc promoter, the tac promoter, the PR or the PL promoters of lambda phage are all known to be potent promoters. Potent promoters providing a high level of gene expression in a bacterium belonging to the family Enterobacteriaceae can be used. Alternatively, the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter region of the gene(s) or operon genes on the bacterial chromosome to obtain a stronger promoter function, thus resulting in the increased transcription level of the gene located downstream of the promoter. Furthermore, it is known that substitution of several nucleotides in the spacer between ribosome binding site (RBS) and the start codon, especially the sequences immediately upstream of the start codon, profoundly affect the mRNA translatability. For example, a 20-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon (Gold L. et al., Annu. Rev. Microbiol, 1981, 35:365-403; Hui A. et al, EMBO J., 1984, 3:623-629). The use of a potent promoter can be combined with multiplication of gene copies.

Methods for preparation of plasmid DNA, digestion, ligation and transformation of DNA, selection of an oligonucleotide as a primer, incorporation of mutations, and the like may be ordinary methods well-known to the person skilled in the art. These methods are described, for instance, in Sambrook J., Fritsch E.F. and Maniatis T., "Molecular Cloning: A Laboratory Manual", 2^nd ed., Cold Spring Harbor Laboratory Press (1989). Methods for molecular cloning and heterologous gene expression are described in Bernard R. Glick, Jack J. Pasternak and Cheryl L. Patten, "Molecular Biotechnology: principles and applications of recombinant DNA", 4^th ed., Washington, D.C: ASM Press (2009); Evans Jr., T.C. and Xu M.-Q., "Heterologous gene expression in E. colF, 1^st ed., Humana Press (2011). Methods for recombinant gene expression are described in Balbas P. and Lorence A., "Recombinant gene expression: reviews and protocols", 2^nd ed., Totowa N.J., Humana Press (2004).

The level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene using various known methods including Northern blotting, quantitative RT-PCR, and the like. The amount of the protein coded by the gene can be measured by known methods including SDS-PAGE followed by immunoblotting assay (Western blotting analysis), and the like. The DNA, which is used for modification of the bacterium as described herein, may encode a transmembrane protein having L-amino acid exporter activity. The phrase "L-amino acid exporter activity" can mean an activity of the transmembrane protein to excrete or secrete L-amino acid, especially, L-aromatic amino acid such as L- phenylalanine, L-tyrosine, and L-tryptophan from inside of a bacterial cell harboring the protein to outside of the cell.

More concretely, the DNA is represented by the mutant yddG gene which is a mutant nucleotide sequence of the wild-type allele of the yddG gene shown in SEQ ID NO: 1.

The wild-type yddG gene encodes aromatic amino acid exporter YddG (KEGG, Kyoto Encyclopedia of Genes and Genomes, entry No. bl473). The yddG gene (GenBank accession No. NC_000913.2; nucleotide positions: 1544312 to 1545193, complement; Gene ID: 945942) is located between the fdnG gene on the opposite strand and yddL gene on the same strand of the chromosome of E. coli K-12. The nucleotide sequence of the yddG gene and the amino acid sequence of the YddG protein encoded by the yddG gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

Since there may be some differences in DNA sequences between the genera or strains of the genus Escherichia, the yddG gene to be modified as described herein is not limited to the gene shown in SEQ ID NO: 1, but may include genes which are variant nucleotide sequences of or homologous to SEQ ID NO: 1, and which encode variants of the wild-type protein. The substitution of amino acid residue(s) in the position number 24 or 26, or both in the wild-type YddG protein of SEQ ID NO: 2 or its variant protein can be achieved by modifying a wild-type nucleotide sequence shown in SEQ ID NO: 1, or a variant nucleotide sequence thereof.

As the inventors have predicted in silico that the wild-type YddG protein may contain a cleavage site between G26 and V27 of SEQ ID NO: 2 to provide a putative signal peptide, it is obvious that another substitution(s), which are different from those in the position 124 and G26 of SEQ ID NO: 2, may be used in accordance with present invention. Such substitution(s) may be located within 3 amino acid residues, in another example within 5 amino acid residues towards C- and/or N-end of YddG protein relatively to preferable cleavage site as specified above.

The phrase "a variant protein" can mean a protein which has one or several changes in the sequence compared with wild-type sequence shown in SEQ ID NO: 2 in the position(s) other than 24 and 26 , whether they are substitutions, deletions, insertions, and/or additions of amino acid residues, but still maintain an activity similar to that of the wild-type or mutant YddG protein. The number of one or several changes in the variant protein depends on the position in the three dimensional structure of the protein or the type of amino acid residues. It can be, but is not strictly limited to, 1 to 30, in another example 1 to 20, in another example 1 to 15, in another example 1 to 10, and in another example 1 to 5, in SEQ ID NO: 2 or its mutant sequence corresponding to the mutant YddG protein as described herein. These changes in the variant protein can occur in regions of the protein that are not critical for the function of the protein. This is because some amino acids have high homology to one another so that the three dimensional structure or activity is not affected by such a change. Therefore, the protein variants encoded by the yddG gene may have an identity of not less than 90%, not less than 95%, not less than 97%, not less than 98%, or not less than 99% with respect to the entire amino acid sequence shown in SEQ ID NO: 2, as long as the functionality of the wild- type and mutant YddG proteins is maintained. The exemplary mutant YddG proteins includes a protein having a wild-type sequence of YddG protein but has the L-amino acid substitution (s) at the position(s) 26, or 24 and 26, and a protein having the L-amino acid substitution (s) at the position(s) 26, or 24 and 26 and further amino acid changes that are not critical for the function of the protein as described above.

The substitution, deletion, insertion, and/or addition of one or several amino acid residues can be a conservative mutation(s) so that the activity and features of the variant protein are maintained, and are similar to those of the wild-type and mutant YddG proteins. The representative conservative mutation is a conservative substitution.

Examples of conservative substitutions include substitution of Ser or Thr for Ala, substitution of Gin, His or Lys for Arg, substitution of Glu, Gin, Lys, His or Asp for Asn, substitution of Asn, Glu or Gin for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp or Arg for Gin, substitution of Asn, Gin, Lys or Asp for Glu, substitution of Pro for Gly, substitution of Asn, Lys, Gin, Arg or Tyr for His, substitution of Leu, Met, Val or Phe for He, substitution of He, Met, Val or Phe for Leu, substitution of Asn, Glu, Gin, His or Arg for Lys, substitution of He, Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, He or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe or Trp for Tyr, and substitution of Met, He or Leu for Val.

To evaluate the degree of protein or DNA homology or identity, several calculation methods can be used, such as BLAST search, FASTA search and ClustalW method. The BLAST (Basic Local Alignment Search Tool,

www.ncbi.nlm.nih.gov/BLAST/) search is the heuristic search algorithm employed by the programs blastp, blastn, blastx, megablast, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Samuel K. and Altschul S.F. ("Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc. Natl. Acad. Sci. USA, 1990, 87:2264-2268;

"Applications and statistics for multiple high-scoring segments in molecular sequences". Proc. Natl. Acad. Sci. USA, 1993, 90:5873-5877). The computer program BLAST calculates three parameters: score, identity and similarity. The FAST A search method is described by Pearson W.R. ("Rapid and sensitive sequence comparison with FASTP and FASTA", Methods Enzymol., 1990, 183:63-98). The ClustalW method is described by Thompson J.D. et al. ("CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice", Nucleic Acids Res., 1994, 22:4673-4680).

Moreover, the wild-type yddG gene can be variant nucleotide sequences. The phrase "a variant nucleotide sequence" can mean a nucleotide sequence which codes "a variant protein". The phrase "a variant nucleotide sequence" can also mean a nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence complementary to the sequence shown in SEQ ID NO: 1, , or a probe which can be prepared from the nucleotide sequence under stringent conditions. "Stringent conditions" include those under which a specific hybrid, for example, a hybrid having identity of not less than 90%, not less than 95%, not less than 97%, not less than 98%, or not less than 99% is formed, and a non-specific hybrid, for example, a hybrid having homology lower than the above is not formed. For example, stringent conditions can be exemplified by washing one time or more, or in another example, two or three times, at a salt

concentration of I xSSC, 0.1% SDS, or in another example, O.l x SSC, 0.1% SDS at 60°C, or in another example at 65°C. Duration of washing depends on the type of membrane used for blotting and, as a rule, can be what is recommended by the manufacturer. For example, the recommended duration of washing for the Amersham Hybond™-N+ positively charged nylon membrane (GE Healthcare) under stringent conditions is 15 minutes. The washing step can be performed 2 to 3 times. As the probe, a part of the sequence complementary to the sequence shown in SEQ ID NO; 1 may also be used. Such a probe can be produced by PCR using oligonucleotides as primers prepared on the basis of the sequence shown in SEQ ID NO: 1, and a DNA fragment containing the nucleotide sequence as a template. The length of the probe is recommended to be >50 bp; it can be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp. For example, when a DNA fragment having a length of about 300 bp is used as the probe, the washing conditions after hybridization can be exemplified by 2^χ SSC, 0.1% SDS at 50°C, or at 60°C, or in another example at 65°C.

As the gene encoding the YddG protein of the genus Escherichia has already been elucidated (see above), the yddG gene or variant nucleotide sequences encoding variant proteins of the YddG protein can be obtained by PCR (polymerase chain reaction; refer to White T.J. et al., Trends Genet., 1989, 5:185- 89) utilizing primers prepared based on the nucleotide sequence of the yddG gene from a bacterium belonging to the genus

Escherichia or other microorganisms. .

The positions 24 and 26 as described above are not necessarily absolute positions from the N-terminus of the YddG protein, but can indicate relative positions with respect to the amino acid sequence of SEQ ID NO: 2. For example, if one amino acid residue is deleted from the YddG protein having the amino acid sequence shown in SEQ ID NO: 2 at a position upstream of position 24, the position 24 then becomes position 23. Even in such a case, the amino acid residue of the position 23 is still regarded as an amino acid residue of the "position 24". Absolute position of amino acid substitution can be determined by alignment of amino acid sequence of an objective YddG protein and the amino acid sequence of SEQ ID NO: 2.

Aromatic L-amino acid-producing bacteria

As a bacterium of the present invention which is modified to produce the mutant YddG protein, bacteria which are able to produce an aromatic L-amino acid may be used.

L-phenylalanine, L-tyrosine, and L-tryptophan are all aromatic amino acids and share the common biosynthesis pathway. Examples of the genes encoding 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 multi-copying the gene encoding these enzymes on a plasmid or genome, the aromatic amino acid-producing ability can be improved. It is known that these genes can be controlled by a tyrosine repressor (tyrR), so the enzyme activity of an aromatic L-amino acid biosynthesis may also be increased by deleting the tyrR gene (EP763127).

In order to enhance an aromatic L-amino acid productivity of a bacterium, biosynthesis of an amino acid other than the target aromatic amino acid may be attenuated. For example, when the target amino acid is L-tryptophan, biosynthetic pathways of L- phenylalanine and/or L-tyrosine may be attenuated (U.S. Patent No. 4,371,614). Furthermore, 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase (DS) encoded by the aroF or aroG gene is subject to feedback inhibition by aromatic amino acids.

Therefore, a bacterium may be modified so that the bacterium contains mutant DS which is not subject to the feedback inhibition. Such a mutant DS can be obtained, for example, by replacement of L-aspartic acid residue at position 147 or L-serine residue at position 181 by other amino acid residue in AroF. In case of AroG, the mutant DS can be obtained, for example, by replacement of L-asprtic acid residue at position 146, L-methionine residue at position 147, L-proline residue at position 150, or L-alanine residue at position 202 by other L-amino acid residue; or replacement of L-methionine residue at position 157 and L-alanine residue at position 219 by other L-amino acid residues. An aromatic L- amino acid producing bacterium can be obtained by introducing into the bacterium a mutant gene which encodes such a mutant DS (EP0488424).

L-phenylalanine-producing bacteria

Examples of parent strains for deriving L-phenylalanine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus

Escherichia, such as E. coli AJ12739 (tyrA::TnlO, tyrR) (VKPM B-8197); E. coli

HW1089 (ATCC 55371) harboring the mutant pheA34 gene (U.S. Patent No. 5,354,672); E. coli MWECl Ol-b (KR8903681); E. coli NRRL B-12141, NRRL B-12145, NRRL B- 12146 and NRRL B-12147 (U.S. Patent No. 4,407,952). Also, as a parent strain, E. coli K-12 [W31 10 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used (EP 488424 Bl). Furthermore, L-phenylalanine producing bacteria belonging to the genus Escherichia with an enhanced activity of the protein encoded by the yedA gene or the yddG gene may also be used (Russian Patent application Nos.

2001131570 and 2001 131571). Also, L-phenylalanine producing bacteria belonging to the genus Escherichia which is resistant to L-phenylalanine and/or an amino acid analog such as -fluoro-phenylalanine, 5-fluoro-DL-tryptophane, or the like may be used (U.S. Patent No. 7,666,655).

Examples of L-phenylalanine-producing bacteria or parent strains for deriving L- phenylalanine-producing bacteria include E. coli strains DV157 (MG1655AtyrRAtyrA) and

(see Example 1), DV1060 (MG1655AtyrRAtyrA- pheAAtrpRAtnaA). The strain DV157 was obtained by disrupting the tyrR and tyrA genes of E. coli strain MG1655, and the strain DV1060 was obtained by disrupting the tyrR, t rA-pheA, and trpR genes of E. coli strain MG1655 (Doroshenko V.G. et al., FEMS Microbiol. Lett., 2007, 275:312-318). The strain MG1655 (ATCC47076, ATCC 700926) are available from the American Type Culture Collection (ATCC, Address: P.O. Box 1549, Manassas, VA 20108, United States of America).

L-trvptophan-producing bacteria

Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus

Escherichia, such as E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Patent No. 5,756,345); E. coli SV164 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine and a trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan (U.S. Patent No. 6,180,373); E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient in the enzyme tryptophanase (U.S. Patent No. 4,371,614); E. coli AGX17/pGX50,pACKG4-pps in which a phosphoenolpyruvate-producing ability is enhanced (WO9708333, U.S. Patent No. 6,319,696), and the like may be used. L- tryptophan-producing bacteria belonging to the genus Escherichia with an enhanced activity of the identified protein encoded by the yedA gene or the yddG gene may also be used (Russian Patent application Nos. 2001 131570 and 2001131571). Also, L- phenylalanine producing bacteria belonging to the genus Escherichia which is resistant to L-phenylalanine and/or an amino acid analog such as -fluoro-phenylalanine, 5-fluoro- DL-tryptophane, or the like may be used (U.S. Patent No. 7,666,655).

Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention also include strains in which one or more activities of the enzymes selected from anthranilate synthase, phosphoglycerate dehydrogenase, and tryptophan synthase are enhanced. The anthranilate synthase and phosphoglycerate dehydrogenase are both subject to feedback inhibition by L-tryptophan and L-serine, so that a mutation desensitizing the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such a mutation include an E. coli SV164 which harbors desensitized anthranilate synthase and a transformant strain obtained by introducing into the E. coli SV164 the plasmid pGH5 (WO 94/08031), which contains a mutant serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase.

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

The bacterium as described herein can be obtained by introduction of the aforementioned DNA into a bacterium inherently having an ability to produce an aromatic L-amino acid. Alternatively, the bacterium as described herein can be obtained by imparting an ability to produce an aromatic L-amino acid to a bacterium already harboring the aforementioned DNA.

L-tyrosine-producing bacteria

Examples of tyrosine-producing bacteria include Escherichia bacteria with a desensitized prephenate dehydratase gene (tyrA) desensitized to the inhibition by tyrosine (European Patent Laid-open No. 1616940).

2. Method for producing L-amino acids

The method for producing an L-amino acid, specifically aromatic L-amino acids such as L-phenylalanine, L-tryptophan and L-tyrosine, includes the steps of cultivating the bacterium as described herein in a culture medium, to allow the L-amino acid to be produced and accumulated in the culture medium, and collecting the L-amino acid from the culture medium.

In the present invention, the cultivation, collection, and purification of L-amino acids from the medium and the like may be performed in a manner similar to the conventional fermentation method wherein an amino acid is produced using a

microorganism. A medium used for culture may be either a synthetic medium or a natural medium so long as the medium includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the microorganism requires for growth. As the carbon source, saccharides such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, and hydrolyzates of starches;

alcohols such as glycerol, mannitol, and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid, malic acid, and succinic acid; and the like can be used.

Depending on the mode of assimilation of the used microorganism, alcohol including ethanol and glycerol can be used. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen such as amines, peptone, digested fermentative microorganisms, and soybean- hydrolyzates; ammonia gas; aqueous ammonia; and the like can be used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used. If necessary, vitamins such as vitamin Bl and some additional nutrient may be added to the medium.

The cultivation can be performed under aerobic conditions such as a shaking or stirring culture with aeration, at a temperature of 20 to 42°C, preferably 35 to 40°C. The pH of the culture is adjusted within 5 and 9, preferably within 6.8 and 7.0. The pH of the culture can be adjusted by using ammonia, calcium carbonate, various acids, various bases, various salts, and buffers. Usually, a 1 to 3-day cultivation may lead to the accumulation of the target L-amino acid in the culture medium.

After cultivation, solids such as cells and cell debris can be removed from the liquid medium by centrifugation or membrane filtration, and then the target L-amino acid can be collected and purified by conventional techniques such as ion-exchange, concentration and crystallization methods.

Phenylalanine produced by the method of the present invention may be used for, for example, producing lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine (also referred to as "aspartame"). That is, the method of the present invention includes method for producing lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine by using L- phenylalanine as a raw material. The method comprises esterifying L-phenylalanine produced by the method of the present invention as described above to generate a lower alkyl ester of L-phenylalanine and synthesizing lower alkyl ester of N-(a-L-aspartyl)-L- phenylalanine from obtained a lower alkyl ester of L-phenylalanine and L-aspartic acid. If a derivative of L-aspartic acid is used for coupling with a lower alkyl ester of L- phenylalanine, the deprotecting step may be required to generate the lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine. As lower alkyl ester, methyl ester, ethyl ester, and propyl ester, or the like can be mentioned.

In the method of the present invention, a process for synthesizing lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine from a lower alkyl ester of L-phenylalanine and aspartic acid or its derivative is not particularly limited and any conventional method can be applied so long as L-phenylalanine or its derivative can be used for synthesis of lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine. Concretely, for example, lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine may be produced by the following process (U.S. Pat. No. 3,786,039). L-phenylalanine is esterified to obtain a lower alkyl ester of L- phenylalanine. The L-phenylalanine alkyl ester is reacted with L-aspartic acid derivative of which amino group and β-carboxyl group are protected and cc-carboxyl group is esterified or transformed into halogenocarbonyl group, or the like, to activate. The exemplary derivative includes N-acyl-L-aspartic anhydride such as N-formyl-, N- carbobenzoxy-, or N-p-methoxycarbobenzoxy-L-aspartic anhydride. By the condensation reaction, mixture of N-(N-acyl-a-L-aspartyl)-L-phenylalanine and N-(N-acyl- -L- aspartyl)-L-phenylalanine may be obtained. If the condensation reaction is performed under existence of an organic acid of which acid dissociation constant at 37°C is 10^"4 or less, ratio of a form to β form in the mixture is increased (Japanese Patent Publication No. 51-113841 (1976)). Then the N-(N-acyl-a-L-aspartyl)-L-phenylalanine is separated from the mixture, followed by hydrogenating to obtain N-(ot-L-aspartyl)-L-phenylalanine.

Hydrogenating, or deprotecting of amino group of a-L-aspartyl residue, may be performed under acid hydrolysis conditions.

A lower alkyl ester of N-( -L-aspartyl)-L -phenylalanine may produced by cultivating the bacterium of the present invention in a culture medium to produce and accumulate L- phenylalanine in the medium, wherein the bacterium has L-phenylalanine productivity; esterifying L-aspartic acid to generate a lower alkyl ,β-diester of the L-aspartic acid; and generating a lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine from the lower alkyl α,β-diester of the L-aspartic acid and the L-phenylalanine (European Patent application No. 1587941 Al).

Examples

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

Example 1. Construction of E. coli DV157 strains having mutant yddG gene

The E. coli L-phenylalanine-producing strains DV157 tfrE::P_plo-j¾WG and

DV157 'htrE PL-yddG have been described previously (Doroshenko V.G. et al., FEMS Microbiol. Lett., 2007, 275:312-318). Both strains contain additional copy of the yddG gene integrated into the htrE gene. In the E. coli ΌΥ 157 htrE .V^o-yddG strain the copy of yddG is silent due to the T7 phage promoter (Ρ_φι₀) located upstream of the gene.

The mutations were introduced into the chromosome of the E. coli

DV 157 htrE F io-yddG strain using modified Red-mediated integration method

(Doroshenko V.G. et al, FEMS Microbiol. Lett., 2007, 275:312-318; Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). The polymerase chain reaction (PCR) was performed using forward primers PI (SEQ ID NO: 3) for introducing the mutation replacing glycine at position 26 by phenylalanine (G26F mutation), or P2 (SEQ ID NO: 4) for introducing the mutation replacing glycine at position 26 by glutamic acid (G26E mutation), or P3 (SEQ ID NO: 5) for introducing the mutation replacing isoleucine at position 24 and glycine at position 26 by phenylalanine and isoleucine, respectively (I24F/G26I mutation) and backward primer P4 (SEQ ID NO: 6), and chromosomal DNA as the template. The temperature profile was the following: initial DNA denaturation for 5 min at 94°C followed by 30 cycles of: denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, and elongation at 72°C for 1 min; and the final elongation for 7 min at 72°C. The primers have been designed in such a way that using PCR the silent promoter Ρ_φ10 can be replaced by the active promoter PL marked with chloramphenicol-resistance marker (Cm^R) encoded by the cat gene. The structure of the

fragment and the scheme of integration of the fragment is shown in Figure 1. Thus, E. coli strains OY\57htrE: :PL-yddGCm^R, DV157/ztrE: :PL- yddG^G26FCm^R, OVl57htrE: P_L-yddG^G26ECm^R, and DV157/ rE::P_L-j^G^I24F/G26ICm^R were obtained. The mutations in E. coli OV\57htrE::V_h-yddG^G26VCm^R, DV 157 htrE ?_L- yddG^G26ECm^R, and DV157/ztrE::P_L- d G^{I24F G26I}Cm^R strains were confirmed by sequencing analysis.

Example 2. Production of L-phenylalanine by the modified E. coli DV157 strains

The modified E. coli DV 5 htrE::F_L-yddG^G26FCm^R, DV 157 htrE ?_L- yddG^G26ECm^R, OVl57htrE::P_L-yddG ^F/G26lCm^R and the control DV157/ztrE::P_L- yddGCm^R, OV 157 htrE::P_≠0-yddGCm^R strains were each cultivated at 37°C and vigorous shaking (250 rpm) to OD₆₀₀ nm 2 (logarithmic phase of growth) in M9 medium (Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual (3^rd ed.). Cold Spring Harbor Laboratory Press) supplemented with Tyr (125 mg/L). Then, cells were washed with 0.9% (w/v) NaCl and concentrated twice in fresh M9 medium (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor) in total volume of 10 mL to obtain cell density of OD₆₀o _nm 4 (~2xl 0⁹ CFU/mL, CFU means colony-forming unit). The cells were cultivated in 20 x 200 mm test-tubes at 37°C for 2 h on a rotary shaker at 250 rpm as described in Doroshenko V.G. et al., FEMS Microbiol. Lett., 2007, 275:312-318 (the short-term cultivation). Aliquots (1.5 mL) of culture broth were taken off after 0, 1 , and 2 h from cultivation start, cells were precipitated at 10000 rpm, and supernatant was analyzed using high-performance liquid chromatography (HPLC).

The conditions were as follows:

Column: Eclipse XDB-C 18, 4.6 x 75 mm, 3.5 μιη (Agilent Technologies).

Temperature: 23°C.

Flow rate: 1 mL/min.

Injection volume: 10 μΐ_^.

Detection: UV 220 nm.

Buffers:

A: 25 mM NaH₂P0₄, pH 2.5 (adjusted by H₃P0₄),

B: H₂0,

C: 70% aqueous CH₃CN.

Gradient profile:

From 0 min to 12 min: A- 100%,

From 12 min to 32 min: A-85.7% and C-14.3%,

From 32 min to 34 min: A-42.8% and C-57.2%,

From 34 min to 36 min: C- 100%,

From 36 min to 42 min: B-100%,

From 42 to 44 min: A- 100%,

From 44 to 66 min: A- 100%.

The results of 3 independent fermentations (as average values) are shown in Figure 2 and Table 1. As it can be seen from the Figure 2 and Table 1 , the modified E. coli

DV157/ztrE::P_L-j« 7^G26ECm^R, and DV157/2/rE::P_L- yddG^l24f/G26lCm^R strains caused a higher amount of accumulation of L-phenylalanine as compared with the control strains. Table 1.

Example 3. Construction of E. coli DV1060 strains having mutant yddG gene

The modifications ?_L-yddG^G26F,

?_L-yddG^G26E, and ?_L-yddG marked by Cm^R marker were each transformed using PI -transduction into the E. coli L- tryptophan-producing strain DV1060 (Doroshenko V.G. et al., FEMS Microbiol. Lett., 2007, 275:312-318; Miller J.H. (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor). The strains described in Example 1 were used as donor strains. As a result, the E. coli OVl060htrE::?_L-yddG^G26FCm^R, DV1060/ztr£::P_L- ¾ ?^{I24F G26I}Cm^R, DV1060/ztr£::P_L- ^G^G26ECm^R, and DV1060/ztrE::P_L-j^GCm^R strains were obtained. The construction of the

strain is described in Doroshenko V.G. et al., FEMS Microbiol. Lett., 2007, 275:312-318.

Example 4. Production of L-tryptophan by the modified E. coli DV1060 strains

The modified E. coli OW\060htrE::?_L-yddG^G26FCm^R, OVl060htrE::?_L- yddG ^F/G26lCm^R, O\l060htrE::?_L-yddG^G26ECm^R and the control DV1060/ztrE::P_L- yddGCm^R, OVl060htrE::?_≠0-yddG strains were each cultivated at 37°C and vigorous shaking (250 rpm) to OO^oo nm 2 (logarithmic phase of growth) in M9 medium (see Example 2) supplemented with Phe (125 mg/L) and Tyr (125 mg/L). Then, cells were washed with 0.9% (w/v) NaCl and concentrated twice in fresh M9 medium (see Example 2) in total volume of 10 mL to obtain cell density of OD₆₀₀ nm 4 (~2xl 0⁹ CFU/mL, CFU means colony-forming unit). The cells were cultivated in 20 ^χ 200 mm test-tubes at 37°C for 3 h on a rotary shaker at 250 rpm as described in Doroshenko V.G. et al., FEMS Microbiol. Lett., 2007, 275:312-318 (the short-term cultivation). Aliquots (1.5 mL) of culture broth were taken off after 0, 1, 2, and 3 h from cultivation start, cells were precipitated at 10000 rpm, and supernatant was analyzed using HPLC as described in Example 2.

The results of 3 independent fermentations (as average values) are shown in Table 2. As it can be seen from the Table 2, the modified E. coli OVl060htrE::P_L-yddG^G26rC ^R, OVl060htrE ?_L-yddG^G26ECm^R, and DV1060/ztrE::P_L-^G^I24F/G26ICm^R strains caused a higher amount of accumulation of L-tryptophan as compared with the control strains.

Table 2.

Production of L-tryptophan by the modified E. coli DV1060 strains

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

Industrial Applicability

According to the present invention, production of aromatic L-amino acids by bacteria belonging to the genus Escherichia improved.

Claims

1. A mutant bacterial amino acid exporter protein which is selected from the group consisting of:

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

B) a variant protein comprising the amino acid sequence of SEQ ID NO: 2, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues and has amino acid exporter activity,

wherein the protein has a substitution selected from the group consisting of:

(C) L-isoleucine residue in the position number 24 is replaced by L-phenylalanine residue, or L-tryptophan residue, or L-tyrosine residue, or L-methionine residue, or L-leucine residue;

(D) glycine residue in the position number 26 is replaced by L-phenylalanine residue, or L-isoleucine residue, or L-glutamic acid residue, or L-leucine residue, L- methionine residue, or L-valine residue, or L-tryptophan residue, or L-tyrosine residue; and

(E) a combination of thereof,

wherein the position number is counted with SEQ ID NO: 2.

2. The mutant protein according to claim 1, wherein said L-isoleucine residue in the position number 24 is replaced by L-phenylalanine residue.

3. The mutant protein according to claim 1, wherein said glycine residue in the

position number 26 is replaced by L-phenylalanine residue.

4. The mutant protein according to claim 1, wherein said glycine residue in the

position number 26 is replaced by L-isoleucine residue.

5. The mutant protein according to claim 1, wherein said glycine residue in the

position number 26 is replaced by L-glutamic acid residue.

6. The mutant protein according to claim 1, wherein said mutant protein is derived from a bacterium belonging to the genus Escherichia.

7. The mutant protein according to claim 1, wherein said mutant protein is derived from bacterium belonging to the species Escherichia coli.

8. A DNA encoding the mutant bacterial amino acid exporter protein according to any of claims 1 to 7.

9. An aromatic L-amino acid-producing bacterium belonging to the genus Escherichia harboring the DNA according to claim 8.

10. The bacterium according to claim 9, wherein said bacterium belongs to the species Escherichia coli.

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

12. The bacterium according to claim 1 1, wherein said aromatic L-amino acid is L- phenylalanine.

13. The bacterium according to claim 11, wherein said aromatic L-amino acid is L- tryptophan.

14. A method for producing aromatic L-amino acid comprising:

(F) cultivating the bacterium according to any of claims 9 to 13 in a culture medium;

(G) accumulating said aromatic L-amino acid in the culture medium; and

(H) collecting said aromatic L-amino acid from the culture medium.

15. The method according to claim 14, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.

16. The method according to claim 15, wherein said aromatic L-amino acid is L- phenylalanine.

17. The method according to claim 15, wherein said aromatic L-amino acid is L- tryptophan.

18. A method for producing lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine

comprising:

(I) cultivating the bacterium according to any of claims 9 to 13 in a culture medium to produce and accumulate L-phenylalanine in the medium, wherein the bacterium has L-phenylalanine productivity;

(J) esterifying the L-phenylalanine to generate a lower alkyl ester of the L- phenylalanine; and

(K) generating a lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine from L- aspartic acid or its derivative and the lower alkyl ester of the L-phenylalanine.

19. The method according to claim 18 comprising:

(L) condensing the lower alkyl ester of L-phenylalanine with a derivative of L- aspartic acid, wherein the derivative is N-acyl-L-aspartic anhydride;

(M) separating the lower alkyl ester of N-(N-acyl-a-L-aspartyl)-L-phenylalanine from the reaction mixture; and

(N) deprotecting the lower alkyl ester of N-(N-acyl-a-L-aspartyl)-L-phenylalanine to generate the lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine. A method for producing lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine comprising:

(O) cultivating the bacterium according to claims 9 to 13 in a culture medium to produce and accumulate L-phenylalanine in the medium, wherein the bacterium has L-phenylalanine productivity;

(P) esterifying L-aspartic acid to generate a lower alkyl α,β-diester of the L-aspartic acid; and

(Q) generating a lower alkyl ester of N-(a-L-aspartyl)-L-phenylalanine from the lower alkyl ^-diester of the L-aspartic acid and the L-phenylalanine.