WO2007119576A9 - A method for producing an l-amino acid using a bacterium of the enterobacteriaceae family - Google Patents

Abstract

A method for producing an L-amino acid, for example L-threonine, using a bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified to enhance an activity of mutant glucose-specific PTS permease is described.

Description

DESCRIPTION

A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE ENTEROBACTERIACEAE FAMILY

Technical field

The present invention relates to a method for producing an L-amino acid by fermentation, and more specifically to genes which aid in this fermentation. These genes are useful for improving L-amino acid production, particularly L-threonine.

Background art

Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.

Many techniques to enhance L-amino acid production yields have been reported, including transforming microorganisms with recombinant DNA (see, for example, US patent 4,278,765). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes of the feedback inhibition by the resulting L-amino acid (see, for example, WO 95/16042 or US patents 4,346,170; 5,661,012 and 6,040,160).

The bacterial phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) catalyzes uptake and concomitant phosphorylation of carbohydrates (Postma, P. W., J. W. Lengeler, and G. R. Jacobson. 1996. Phosphoenolpyruvate: carbohydrate phosphotransferase systems, p. 1149-1174. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology, Washington, D.C.)

In Escherichia coli K- 12, D-glucose (GIc) is taken up and concomitantly phosphorylated either by the glucose-specific enzyme II (EII) transporter (II^Glc) (glucose- specific PTS permease) or the mannose-specific EII transporter (II^Man) (manXYZ genes) of PTS. During glucose transport, the phosphoryl groups are sequentially transferred from PEP through two common intermediates, enzyme I (EI; gene: ptsT) and the phosphohistidine carrier protein (HPr; gene: ptsH), to sugar-specific protein EII (IICB^G1°) and then to glucose. II^Glc consists of two subunits, IIA^Glc (encoded by err gene (catabolite repression resistance)) and membrane-bound IICB^Glc (encoded hyptsG gene). The err gene is part of the ptsHI err operon, and is separated from the ptsG gene, which maps at 25.0 min. The IICB^Glc subunit is composed of an amino-terminal, hydrophobic IIC^Glc domain, which largely determines substrate specificity, and a carboxy-terminal, hydrophilic IIB^Glc domain, which is phosphorylated at the Cys421 residue.

The EIICB^GlQ has a limited range of substrates; it is the major transporter for GIc but it is also capable of transporting mannose and the non-metabolizable GIc analogue a- methylglucoside. However, EIICB^Glcis normally incapable of transporting the amino sugars. The expression of ptsG is controlled by the mlc-encoded transcription factor. Growth on GIc induces ptsG expression presumably by relieving MIc repression (Plumbridge, J., Microbiology, 146, 2655-2663 (2000); Seitz, S. et al, J. Biol. Chem., 278, 12, pp. 10744-10751 (2003)). Some mutants of the II^Glc with broadened apparent substrate specificities and altered catalytic properties have been characterized (Table 1).

Mutations that occur in the N-terminal amphipathic leader sequence show that fructose could enter the cell via the genetically altered II^Glc by facilitated diffusion. Translocation of other sugars (mannose, methyl a-glucoside, 2-deoxyglucose) also appears to be altered. For example, mutations V12F, V12G, and Gl 3 C enhance transport of glucose and methyl α-glucoside as well as increase growth rates on mannose and glucosamine. Mutations in the loop and transmembrane regions of II^GIc appear to broaden its substrate specificity. Ribose, fructose, and mannitol were shown to be taken up at increased rates by representative mutant cells in various genetic backgrounds. It was proposed that the mutations might in some way affect the protein conformation so as to promote non-specific sugar accessibility to the II^Glc channel (Notley-McRobb, L. & Ferenci, T., J. Bacteriol., 182, 4437-4442 (2000)). At least some of the II^Glc mutations exerted secondary effects on gene expression (Notley-McRobb, L. & Ferenci, T., J. Bacteriol., 182, 16, 4437-4442 (2000); Zeppenfeld, T. et al, J. Bacteriol., 182, 16, 4443- 4452 (2000); Plumbridge, J., Microbiology, 146, 2655-2663 (2000); Seitz, S. et al, J. Biol. Chem., 278, 12, 10744-10751 (2003)), probably by altering the direct interaction of the global transcriptional repressor, MIc, with the free (dephosphorylated) form of II^Glc _. A method for producing succinic acid from industrial-grade hydrolysates uses an organism that contains unidentified mutations in the genes ptsG,pflB, and idhA, and has been disclosed (PCT application WO2005116227).

Also, the process for producing L-amino acid, especially L-threonine, using microorganisms of the Enter obacteriaceae family, in which one or more of the genes selected from the group containing ptsG gene are overexpressed, is disclosed (WO03/004670).

Table 1

References to Table 1:

1. Ruijter, G.J.G. et al, J. Bacterid., 174, 9, 1843-2850 (1992). 2. Buhr, A. et al, J. Biol. Chem., 267, 6, 3847-3851 (1992).

3. Begley, G. S. et al, J. Bacterid., 178, 3, 940-942 (1996).

4. Lanz, R. and Erni, B., J. Biol. Chem., 273, 20, 12239-12243 (1998).

5. Manche, K. et al, Genetics, 153, 5-12 (1999).

6. Oh₅ H. et al, J. Biol. Chem., 274, 14006-14011 (1999).

7. Plumbridge, J., Microbiology, 146, 2655-2663 (2000).

8. Zeppenfeld, T. et al, J. Bacterid., 182, 16, 4443-4452 (2000).

9. Notley-McRobb, L. & Ferenci, T., J. Bacteriol., 182, 16, 4437-4442 (2000).

10. Seitz, S. et al, J. Biol. Chem., 278, 12, pp. 10744-10751 (2003)).

11. Aboulwafa, M. et al, Microbiology, 149, 763-771 (2003). ^"

SUMMARY OF THE INVENTION Objects of the present invention include enhancing the productivity of L-amino acid-producing strains and providing a method for producing L-amino acids using these strains.

This aim was achieved by finding a novel mutant glucose-specific enzyme II (EII) transporter (II^Glc) (glucose-specific PTS permease), the expression of which enhances L- amino acid production, particularly L-threonine.

It is an object of the present invention to provide a mutant IIB/IIC subunit of glucose-specific PTS permease selected from the group consisting of:

(A) a protein comprising the L-amino acid sequence of SEQ ID NO: 2, except that amino acids at positions 263 and/or 359 are replaced with other L- amino acids; and

(B) a variant of protein (A), which has glucose-specific PTS permease activity when combined with the HA (Crr) subunit.

It is a further object of the present invention to provide the mutant IIB/IIC subunit of glucose-specific PTS permease described above, wherein the alanine at position 263 is replaced with valine and/or the isoleucine at positions 359 is replaced with valine or leucine.

It is a further object of the present invention to provide a DNA coding for mutant HB/ IIC subunit of glucose-specific PTS permease described above. It is a further object of the present invention to provide a mutant glucose-specific PTS permease comprising the mutant IIB/IIC subunit described above.

It is a further object of the present invention to provide an L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein said bacterium has been modified to contain the mutant glucose-specific PTS permease as described above.

It is a further object of the present invention to provide the bacterium described above, wherein the bacterium has been transformed with the DNA described above.

It is a further object of the present invention to provide the bacterium described above, wherein expression of the gene encoding the mutant IIB/IIC subunit of glucose- specific PTS permease described above is enhanced by increasing the copy number of said gene or by modifying an expression regulating sequence of said gene.

It is a further object of the present invention to provide the bacterium described above, wherein said bacterium is selected from the group consisting of Escherichia, Enterohacter, Erwinia, Klebsiella, Pantoea, Providencia, Salmonella, Serratia, Shigella, and Morganella.

It is a further objecto of the present invention to provide the bacterium described above, wherein said L-amino acid is selected from the group consisting of an aromatic L- amino acid and a non-aromatic L-amino acid.

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

It is a further object of the present invention to provide the bacterium described above, wherein said non-aromatic L-amino acid is selected from the group consisting of L- threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L- valine, L- histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartate, L-glutamine, L-glutamic acid, L-proline, and L-arginine.

It is a further object of the present invention to provide the bacterium described above, wherein said bacterium has been further modified to enhance expression of a gene selected from the group consisting of the mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I and is resistant to feedback inhibition by threonine,

- the thrB gene which codes for homoserine kinase,

- the thrC gene which codes for threonine synthase, - the rhtA gene which codes for a putative transmembrane protein,

- the asd gene which codes for aspartate-β-semialdehyde dehydrogenase,

- the aspC gene which codes for aspartate aminotransferase (aspartate transaminase), and

- combinations thereof.

It is a further object of the present invention to provide the bacterium described above, wherein said bacterium has been modified to increase expression of said mutant thrA gene, said thrB gene, said thrC gene, and said rhtA gene.

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

A) cultivating the bacterium described above in a culture medium, and

B) collecting the L-amino acid from the culture medium.

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

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

It is a further object of the present invention to provide the method described above,

It is a further object of the present invention to provide the method described above, wherein said L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L- arginine.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the alignment of the primary sequences of IIB/IIC subunit of glucose-specific PTS permease from Escherichia coli (Ec) (SEQ ID NO: 2), Salmonella typhimurium (5W)(SEQ ID NO: 18), Salmonella choleraesuis (Sc) (SEQ ID NO: 19), Salmonella paratyphi (Spt) (SEQ ID NO: 20), Salmonella typhi (St) (SEQ ID NO: 21), Yersinis pestis (Yp) (SEQ ID NO: 22), Yersinis pseudotuberculosis (Ypt) (SEQ ID NO: 23), Shigella sonnei (Ss) (SEQ ID NO: 24), Shigella flexneri (Sf) (SEQ ID NO: 25). The alignment was done by using the PIR Multiple Alignment program (http://pir.georgetown.edu). The identical amino acids are marked by an asterisk (*), similar amino acids are marked by colon (:).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS PtsG/Crr, the glucose-specific PTS permease, belongs to the functional superfamily of the phosphoenolpyruvate (PEP)-dependent, sugar transporting phosphotransferase system (PTS). The PTS transports and simultaneously phosphorylates its sugar substrates in a process called group translocation. PtsG/Crr takes up exogenous glucose, releasing the phosphate ester into the cell cytoplasm in preparation for metabolism, primarily via glycolysis.

The mutant IIB/IIC subunit of the glucose-specific PTS permease according to the present invention is one of the following:

(A) the protein of SEQ ID NO: 2, except that the L-amino acids at positions 263 and/or 359 are replaced with other L-amino acids; and

(B) a variant of protein (A), which has an activity of glucose-specific PTS permease when combined with the HA (Crr) subunit.

The phrase "activity of glucose-specific PTS permease" means the activity of transporting and simultaneously phosphorylating its sugar substrates. The activity of glucose-specific PTS permease can be detected by, for example, the method described by Postma, P. W. et al (Microbiol. Rev., 57, 3, 543-94 (1993)). It is estimated that the mutant IIB/IIC subunit of the present invention has an activity of glucose-specific PTS permease higher than that of the wild-type IIB/IIC subunit when combined with the HA subunit.

In the present invention, the phrase "amino acid residues at positions 263 and/or 359" refers to the positions in the amino acid sequence of wild-type PtsG from E. coli, which is shown in SEQ ID NO: 2. However, these position may change. For example, if an amino acid residue is inserted in the N-terminus portion, the amino acid residue inherently located at position 263 becomes position 264. In such a case, the amino acid residue at original position 263 is the amino acid residue at position 263 in the present invention.

The mutant PtsG may include deletion, substitution, insertion, or addition of one or several amino acids at one or a plurality of positions other than at positions 263 and 359, provided that the glucose-specific PTS permease activity is not lost or reduced. Replacing the alanine at position 263 and the isoleucine at position 359 in SEQ ID NO: 2 with another amino acids is preferable.

Amino acids that may replace the Ala at position 263 include Arg, Asp, Asn, Cys, GIu, GIn₅ GIy, His, lie, Met, Leu, Lys, Phe, Ser, Trp, Tyr, VaI, Pro and Thr. Hydrophobic amino acids such as GIy, VaI, Leu, and He are preferable, and amino acids which have a branched chain, such as VaI, are more preferable.

Amino acids that may replace the He at position 359 include Ala, Arg, Asp, Asn, Cys, GIu, GIn₅ GIy₅ His, Met, Leu, Lys, Phe, Ser, Trp, Tyr, VaI, Pro, and Thr. Hydrophobic amino acids such as Ala, GIy, VaI, and Leu are preferable, and amino acids which have a branched chain such as Leu, VaI are more preferable.

The mutant PtsG and mutant ptsG gene according to the present invention can be obtained from the wild-type ptsG gene by site-specific mutagenesis using known methods, such as PCR (polymerase chain reaction; refer to White, TJ. et al., Trends Genet., 5, 185 (1989)) utilizing primers based on the nucleotide sequence of the gene.

The wild-type ptsG gene which encodes the IIB/IIC subunit of glucose-specific PTS permease (synonyms - Bl 101, TgI, Umg, UmgC, Gpt, Cat, Car, GIcA, PtsG) from Escherichia coli has been elucidated (nucleotide numbers from 1157092 to 1158525 in the sequence of GenBank accession NC_000913.2, gi: 49175990). The ptsG gene is located between the >>c/?7 ORF aadβiuE gene on the chromosome of E. coli K-12.

The wild-type err gene which encodes the HA subunit of glucose-specific PTS permease (synonyms - B2417, TreD, Tgs, lex, Gsr, Crr) from Escherichia coli has been elucidated (nucleotide numbers from 2533856 to 2534365 in the sequence of GenBank accession NC_000913.2, gi: 49175990). The crr gene is located between the ptsl and pdxK genes on the chromosome of E. coli K-12. The nucleotide sequence of the crr gene is shown in SEQ ID NO: 13. The amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO: 14.

Therefore, the ptsG and crr genes can be obtained by PCR (polymerase chain reaction; refer to White, TJ. et al, Trends Genet, 5, 185 (1989)) utilizing primers based on the known nucleotide sequence of the gene. Genes coding for the HA, HB, HC subunits, and combinations thereof of the glucose-specific PTS permease from other microorganisms can be obtained in a similar manner.

The ptsG gene derived from Escherichia coli is exemplified by a DNA which encodes the following protein (A) or (B): (A) a protein which has the amino acid sequence shown in SEQ ID NO: 2; or

(B) a variant protein of the amino acid sequence shown in SEQ ID NO: 2, which has an activity of glucose-specific PTS permease when combined with the HA (Crr) subunit.

The phrase "variant protein" as used in the present invention means a protein which has changes in the sequence, whether they are deletions, insertions, additions, or substitutions of amino acids, but still maintains the desired activity at a useful level, for example, useful for the enhanced production of an L-amino acid. The number of changes in the variant protein depends on the position in the three dimensional structure of the protein or the type of amino acid residue. The number of changes may be 1 to 30, preferably 1 to 15, and more preferably 1 to 5 for the protein (A). These changes can occur in regions of the protein which are not critical for the function of the protein. This is because some amino acids have high homology to one another so the three dimensional structure or activity is not affected by such a change.

Therefore, the protein variant (B) may be one which has a homology of not less than 70%, preferably not less than 80%, and more preferably not less than 90%, and most preferably not less than 95% with respect to the entire amino acid sequence of the IIB/IIC subunit of glucose-specific PTS permease shown in SEQ ID NO. 2, as long as the activity of glucose-specific PTS permease is maintained. Since glucose-specific PTS permease consists of IIB/IIC subunit encoded by ptsG gene and HA subunit encoded by crr gene, functional variant proteins (B) can be selected when Crr protein (HA subunit) is present and a complex of glucose-specific enzyme II (EII) transporter (II^Glc) is formed.

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

The substitution, deletion, insertion, or addition of one or several amino acid residues should be conservative mutation(s) so that the activity is maintained. 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 GIu, GIn, Lys, His or Asp for Asn, substitution of Asn, GIu or GIn for Asp, substitution of Ser or Ala for Cys, substitution of Asn, GIu, Lys, His, Asp or Arg for GIn, substitution of Asn, GIn, Lys or Asp for GIu, substitution of Pro for GIy, substitution of Asn, Lys, GIn, Arg or Tyr for His, substitution of Leu, Met, VaI or Phe for He, substitution of He, Met, VaI or Phe for Leu, substitution of Asn, GIu, GIn, His or Arg for Lys, substitution of He, Leu, VaI or Phe for Met, substitution of Trp, Tyr, Met, He or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe or Trp for Tyr, and substitution of Met, He or Leu for VaL

Data comparing the primary sequences of the IIB/IIC subunit of glucose-specific PTS permease from Escherichia coli (Ec), Salmonella typhimurium (Stm), Salmonella choleraesuis (Sc), Salmonella paratyphi (Spf), Salmonella typhi (St), Yersinis pestis (Yp), Yersinis pseudotuberculosis (Ypt), Shigella sonnei (Ss), Shigella flexneri (Sf) show a high level of homology among these proteins (see Figure 1). From this point of view, substitutions or deletions of the amino acid residues which are identical (marked by asterisk) in all the above-mentioned proteins could be crucial for their function. It is possible to substitute similar (marked by colon) and nonsimilar (marked by dot or space) amino acids residues by the similar amino acid residues without deterioration of the protein activity. But modifications of other non-conserved amino acid residues may not lead to alteration of the activity of the IIB/IIC subunit of glucose-specific PTS permease.

The DNA which encodes substantially the same protein as the IIB/IIC subunit of glucose-specific PTS permease described above may be obtained, for example, by modifying the nucleotide sequence of the DNA encoding IIB/IIC subunit of glucose- specific PTS permease (SEQ ID NO: 1), for example, by means of site-directed mutagenesis so that one or more amino acid residues at a specified site is deleted, substituted, inserted, or added. DNA modified as described above may be obtained by conventionally known mutation treatments. Such treatments include hydroxylamine treatment of the DNA encoding proteins of present invention, or treatment of the bacterium containing the DNA with UV irradiation or a reagent such as N-methyl-N'-nitro-N- nitrosoguanidine or nitrous acid.

A DNA encoding substantially the same protein as IIB/IIC subunit of glucose- specific PTS permease can be obtained by expressing DNA having a mutation as described above in an appropriate cell, and investigating the activity of any expressed product. A DNA encoding substantially the same protein as IIB/IIC subunit of glucose-specific PTS permease can also be obtained by isolating a DNA that is able to hybridize with a probe having a nucleotide sequence which contains, for example, the nucleotide sequence shown as SEQ ID NO: 1, under stringent conditions, and encodes a protein having glucose- specific PTS permease activity when combined with the HA (Crr) subunit. The "stringent conditions" referred to herein are conditions under which so-called specific hybrids, for example, a hybrid having homology of not less than 60%, more preferably not less than 70%, further preferably not less than 80%, and still more preferably not less than 90%, and most preferably not less than 95% are formed, and non-specific hybrids, for example, a hybrid having homology lower than the above are not formed. Alternatively, stringent conditions may be exemplified by conditions under which DNA is able to hybridize at a salt concentration equivalent to ordinary washing conditions in Southern hybridization, i.e., 1 x SSC, 0.1% SDS, preferably 0.1 x SSC, 0.1% SDS, at 60°C. Duration of washing depends on the type of membrane used for blotting and, as a rule, what is recommended by the manufacturer. For example, recommended duration of washing for the Hybond N+ nylon membrane (Amersham) under stringent conditions is 15 minutes. Preferably, washing may be performed 2 to 3 times.

A partial sequence of the nucleotide sequence of SEQ ID NO: 1 can also be used as a probe. Probes may be prepared by PCR using primers based on the nucleotide sequence of SEQ ID NO: 1, and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 1 as a template. When a DNA fragment having a length of about 300 bp is used as the probe, the hybridization conditions for washing include, for example, 50°C, 2 x SSC and 0.1% SDS.

The substitution, deletion, insertion, or addition of nucleotides as described above also includes mutations which naturally occur (mutant or variant), for example, due to variety in the species or genus of bacterium, and which contains the glucose-specific PTS permease.

In the present invention, "L-amino acid-producing bacterium" means a bacterium which has an ability to cause accumulation of an L-amino acid in a medium when the bacterium is cultured in the medium. The L-amino acid-producing ability may be imparted or enhanced by breeding. The phrase "L-amino acid-producing bacterium" as used herein also means a bacterium which is able to produce and cause accumulation of an L-amino acid in a culture medium in amount larger than a wild-type or parental strain of the bacterium, for example, E. coli, such as E. coli K- 12, and preferably means that the bacterium is able to cause accumulation in a medium of an amount not less than 0.5 g/L, more preferably not less than 1.0 g/L of the target L-amino acid. "L-amino acids" include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L- glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L- phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. L- threonine is particularly preferred.

.The Enterohacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Providencia, Salmonella, Serratia, Shigella, Morganella, etc.. Specifically, bacteria classified into the Enterobacteriaceae family according to the taxonomy used by the NCBI (National Center for Biotechnology Information) database

(http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) can be used. A bacterium belonging to the genus Escherichia or Pantoea is preferred. The phrase "a bacterium belonging to the genus Escherichia" means that the bacterium is classified in the genus Escherichia according to the classification known to a person skilled in the art of microbiology. Examples of a bacterium belonging to the genus Escherichia as used in the present invention include, but are not limited to, Escherichia coli (E. coli).

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

The term "a bacterium belonging to the genus Pantoea" means that the bacterium is classified as the genus Pantoea according to the classification known to a person skilled in the art of microbiology. Some species of Enterobacter agglomerans have been recently re- classified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, or the like, based on the nucleotide sequence analysis of 16S rRNA etc. (Int. J. Syst. Bacteriol., 43, 162-173 (1993)).

The bacterium of the present invention encompasses a strain of the Enterobacteriaceae family which has an ability to produce an L-amino acid and has been modified to contain mutant IIB/IIC subunit of glucose-specific PTS permease.The strain which contains the mutant IIB/IIC subunit of glucose-specific PTS permease can be constructed by transformation with DNA encoding the mutant protein or introduction of the mutation into the native ptsG gene by homologous recombination. In addition, the bacterium of the present invention encompasses a strain of the Enterobacteriaceae family wherein the glucose-specific PTS permease activity is enhanced by introduction of the mutant IIB/IIC subunit of glucose specific PTS permease. The bacterium of the present invention encompasses a strain of the Enterobacteriaceae family wherein expression of the gene encoding the mutant IIB/IIC subunit is enhanced. As a result, the strain contains the mutant glucose-specific PTS permease including the mutant IIB/IIC subunit and the wild- type IIA subunit. The mutant glucose specific PTS permease has a higher activity than that of the wild type glucose specific PTS permease. Therefore, the activity of glucose-specific PTS permease activity of the bacterium of the present invention is higher when compared to that of a non-modified strain, for example, a wild-type strain. Additionally, expression of the gene encoding the mutant IIB/IIC subunit may be enhanced by increasing a copy number of the gene or modifying an expression regulating sequence of the gene. A wild- type strain that may be used for comparison purposes includes, for example, Escherichia coli K- 12. In the present invention, the amount of accumulated L-amino acid, for example, L-threonine, can be increased in a culture medium as a result of enhancing the intracellular activity of glucose-specific PTS permease.

Enhancing glucose-specific PTS permease activity in a bacterial cell can be attained by introduction of the mutant ptsG gene encoding mutant IIB/IIC subunit of glucose- specific PTS permease.

The phrase "expression of the gene is enhanced" means that the expression of the gene is higher than that of a non-modified strain, for example, a wild-type strain. Examples of such modifications include increasing the copy number of expressed gene(s) per cell, increasing the expression level of the gene(s), and so forth. The quantity of the copy number of an expressed gene is measured, for example, by restricting the chromosomal DNA followed by Southern blotting using a probe based on the gene sequence, fluorescence in situ hybridization (FISH), and the like. The level of gene expression can be measured by various known methods including Northern blotting, quantitative RT-PCR, and the like. The amount of the protein expressed by the gene can be measured by known methods including SDS-PAGE followed by immunoblotting assay (Western blotting analysis), and the like. Furthermore, wild-type strains that can be used as a control include, for example, Escherichia coli K-12 or Pantoea ananatis FERM BP-6614. As a result of enhancing the intracellular activity of mutant glucose-specific PTS permease, L-amino acid accumulation, for example L-threonine, accumulation in a medium is observed.

"Transformation of a bacterium with DNA encoding a protein" means introduction of the DNA into a bacterium, for example, by conventional methods. Transformation of this DNA will result in an increase in expression of the gene encoding the protein of present invention, and will enhance the activity of the protein in the bacterial cell. Methods of transformation include any known methods that have hitherto been reported. For example, a method of treating recipient cells with calcium chloride so as to increase permeability of the cells to DNA has been reported for Escherichia coli K- 12 (Mandel, M. and Higa, A., J. MoI. Biol, 53, 159 (1970)) and the like may be used.

Methods of gene expression enhancement include increasing the gene copy number. Introducing a gene into a vector that is able to function in a bacterium of the Enterobacteriaceae family increases the copy number of the gene. Preferably, low-copy vectors are used. Examples of low-copy vectors include but are not limited to pSClOl, pMWl 18, pMWl 19, and the like. The term "low copy vector" is used for vectors, the copy number of which is up to 5 copies per cell.

Enhancement of gene expression may also be achieved by introduction of multiple copies of the gene into a bacterial chromosome by, for example, homologous recombination, such as by employing a linear DNA, which is known as "Red-driven integration" (Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)), Mu integration, or the like. For example, one act of Mu integration allows for introduction of up to 3 copies of the gene into a bacterial chromosome.

Increasing the copy number of the mutant glucose-specific PTS permease gene can also be achieved by introducing multiple copies of the mutant glucose-specific PTS permease gene into the chromosomal DNA of the bacterium. In order to introduce multiple copies of the gene into a bacterial chromosome, homologous recombination is carried out using a sequence which exists in multiple copies as targets in the chromosomal DNA. Sequences having multiple copies in the chromosomal DNA include, but are not limited to repetitive DNA, or inverted repeats existing at the end of a transposable element. Also, as disclosed in US patent No. 5,595,889, it is possible to incorporate the glucose-specific PTS permease gene into a transposon, and allow it to be transferred to introduce multiple copies of the gene into the chromosomal DNA.

Enhancing gene expression may also be achieved by placing the DNA of the present invention under the control of an expression control sequence, such as a promoter, which is stronger than the native expression control sequence. For example, the P_tac promoter, the lac promoter, the trp promoter, the trc promoter, the PR, or the PL promoters of lambda phage are all known to be potent promoters. The use of a potent promoter can be combined with multiplication of gene copies. Alternatively, the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter to increase the transcription level of a 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 et al, Annu. Rev. Microbiol., 35, 365-403, 1981; Hui et al, EMBO J., 3, 623-629, 1984). Previously, it was shown that the rhtA23 mutation is an A-for-G substitution at the -1 position relative 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, California August 24-29, 1997, abstract No. 457). Therefore, it may be suggested that the rhtA23 mutation enhances rhtA gene expression and, as a consequence, increases resistance to threonine, homoserine and some other substances which are transported out of the cells.

The alteration of the expression control sequence can be performed, for example, in the same manner as the gene substitution using a temperature-sensitive plasmid, as disclosed in International Patent Publication WO 00/18935 and Japanese Patent Application Laid-OpenNo. 1-215280.

Methods for preparation of plasmid DNA include, but are not limited to digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer and the like, or other methods well known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).

The bacterium of the present invention can be obtained by introduction of the aforementioned DNAs into a bacterium which inherently has the ability to produce L- amino acid. Alternatively, the bacterium of the present invention can be obtained by imparting an ability to produce L-amino acid to the bacterium already containing the DNAs.

The mutant IIB/IIC subunit produced in the bacterium which has the mutant ptsG gene forms the mutant glucose-specific PTS permease with the IIB/IIC subunit expressed from the wild type err gene which is inherently present in the bacterium. When expression of the mutant IIB/IIC subunit is enhanced, it is preferable that expression of the err gene encoding HA subunit is also enhanced. When both of the mutant ptsG gene and the err gene are introduced into a bacterium using a vector, introduction of both genes may be carried out separately by using different vectors or both of the genes may be carried on the same vector by using a single vector.

The above description for the variant IIB/IIC subunit having the conservative mutations and mutant ptsG gene encoding the variant, and method for enhancing expression of the mutant IIB/IIC subunit can be applied to HA subunit and err gene in the same way.

It is preffered that expression of the native ptsG gene is attenuated in a bacterium so that the wild-type_IIB/IIC subunit does not compete with the mutant IIB/IIC subunit to form the glucose-specific PTS permease with the HA subunit.

Examples of methods of attenuating expression of the native ptsG gene include mutating or deleting the gene. For example, this can be achieved by using recombination to inactivate the gene on the chromosome, or to modify an expression regulating sequence such as a promoter or the Shine-Dalgarno (SD) sequence (WO95/34672; Carrier, T.A. and Keasling, J.D., Biotechnol Prog 15, 58-64 (1999)). This can also be achieved by introducing an amino acid substitution (missense mutation) into the region encoding the enzyme on the chromosome, introducing a stop codon (nonsense mutation), introducing or deleting one or two bases to create a frame shift mutation, or partially deleting a portion or a region of the gene, or the entire gene (Qiu, Z. and Goodman, M.F., J. Biol. Chem., 272, 8611-8617 (1997); Kwon, D. H. Et al, J. Antimicrob. Chemother., 46, 793-796 (2000)).

L-amino acid-producing bacteria

As a bacterium of the present invention which is modified to contain the mutant glucose-specific PTS permease, bacteria which are able to produce either an aromatic or a non-aromatic L-amino acids may be used.

The bacterium of the present invention can be obtained by modifying a bacterium which inherently has the ability to produce L-amino acids so that the bacterium contains the mutant glucose-specific PTS permease. Alternatively, the bacterium of present invention can be obtained by imparting the ability to produce L-amino acids to a bacterium already containing the mutant glucose-specific PTS permease.

L-threonine-producing bacteria Examples of parent strains for deriving the L-threonine-producing bacteria of the present invention include, but are not limited to, L-threonine-producing bacteria belonging to the genus Escherichia, such as E. coli TDH-6/pVIC40 (VKPM B-3996) (US Patent 5,175,107 and US Patent 5,705,371), E. co/z NRRL-21593 (US Patent 5,939,307), E. coli FERM BP-3756 (US Patent 5,474,918), E. coli FERM BP-3519 and FERM BP-3520 (US Patent 5,376,538), E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 1978, 14: 947- 956), E. coli VL643 and VL2055 (EP 1149911 A), and the like.

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

Preferably, the bacterium of the present invention is further modified to enhance expression of one or more of the following genes:

- the mutant thrA gene which encodes aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine; the thrB gene which encodes homoserine kinase;

- the thrC gene which encodes threonine synthase;

- the rhtA gene which encodes a putative transmembrane protein;

- the asd gene which encodes aspartate-a-semialdehyde dehydrogenase; and

- the aspC gene which encodes aspartate aminotransferase (aspartate transaminase).

The thrA gene which encodes aspartokinase homoserine dehydrogenase I of Escherichia coli has been elucidated (nucleotide positions 337 to 2799, GenBank accession no. NC_000913.2, gi: 49175990). The thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K- 12. The thrB gene which encodes homoserine kinase of Escherichia coli has been elucidated (nucleotide positions 2801 to 3733, GenBank accession no. NC_000913.2, gi: 49175990). The thrB gene is located between thrA and thrC genes on the chromosome of E. coli K-12. The thrC gene which encodes threonine synthase of Escherichia coli has been elucidated (nucleotide positions 3734 to 5020, GenBank accession no. NC_000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaX open reading frame on the chromosome of E. coli K- 12. All three genes function as a single threonine operon.

A mutant thrA gene which encodes aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine, as well as the thrB and thrC genes, can be obtained as one operon from the well-known plasmid pVIC40 which is present in the threonine producing E. coli VKPM B-3996. Plasmid pVIC40 is described in detail in US Patent 5,705,371.

The rhtA gene exists at 18 min on the E. coli chromosome close to the glnHPQ operon, which encodes components of the glutamine transport system. The rhtA gene is identical to ORFl (ybiF gene, positions 764 to 1651, GenBank accession no. AAA218541, gi:440181) and located between the pexB and ompX genes. The unit expressing a protein encoded by the ORFl has been designated the rhtA gene (rht: resistance to homoserine and threonine). Also, it was revealed that the rhtA23 mutation is an A-for-G substitution at position -1 with respect to the ATG start codon (ABSTRACTS of the 17^th International Congress of Biochemistry and Molecular Biology in conjugation with the Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California, August 24-29, 1997, abstract No. 457, EP 1013765 A).

The asd gene of E. coli has already been elucidated (nucleotide positions 3572511 to 3571408, GenBank accession no. NC_000913.1, gi:16131307), and can be obtained by PCR (polymerase chain reaction; refer to White, TJ. et al., Trends Genet., 1989, 5:185), utilizing primers based on the nucleotide sequence of the gene. The asd genes of other microorganisms can be obtained in a similar manner.

Also, the aspC gene of E. coli has already been elucidated (nucleotide positions 983742 to 984932, GenBank accession no. NC_Q00913.1, gi:16128895), and can be obtained by PCR. The aspC genes of other microorganisms can be obtained in a similar manner.

L-lysine-producing bacteria Examples of L-lysine-producing bacteria belonging to the genus Escherichia include mutants having resistance to an L-lysine analogue. The L-lysine analogue inhibits growth of bacteria belonging to the genus Escherichia, but this inhibition is fully or partially desensitized when L-lysine is present in a medium. Examples of the L-lysine analogue include, but are not limited to, oxalysine, lysine hydroxamate, S-(2-aminoethyl)- L-cysteine (AEC), S-methyllysine, α-chlorocaprolactam, and so forth. Mutants having resistance to these lysine analogues can be obtained by subjecting bacteria belonging to the genus Escherichia to a conventional artificial mutagenesis treatment. Specific examples of bacterial strains useful for producing L-lysine include Escherichia coli AJl 1442 (FERM BP-1543, NRRL B-12185; see U.S. Patent 4,346,170) and Escherichia coli VL611. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is desensitized.

The strain WC 196 may be used as an L-lysine producing bacterium of Escherichia coli. This bacterial strain was bred by conferring the AEC resistance to strain W3110, which was derived from Escherichia coli K- 12. The resulting strain was designated Escherichia coli AJ13069 and was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on December 6, 1994 and received an accession number of FERM P- 14690. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on September 29, 1995, and received an accession number of FERM BP-5252 (US Patent 5,827,698).

Examples of parent strains for deriving L-lysine-producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L- lysine biosynthetic enzyme are enhanced. Examples of such genes include, but are not limited to, genes encoding dihydrodipicolinate synthase (dapA), aspartokinase (fysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (IysA), diaminopimelate dehydrogenase (ddh) (U.S. Patent No. 6,040,160), phosphoenolpyrvate carboxylase (ppc), aspartate semialdehyde dehydrogenase (asd), and aspartase (aspA) (EP 1253195 A). In addition, the parent strains may have increased expression of the gene involved in energy efficiency (cyo) (EP 1170376 A), the gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (U.S. Patent No. 5,830,716), the ybjE gene (WO2005/073390), or combinations thereof. Examples of parent strains for deriving L-lysine-producing bacteria of the present invention also include strains having decreased or eliminated activity of an enzyme that catalyzes a reaction for generating a compound other than L-lysine by branching off from the biosyntlietic pathway of L-lysine. Examples include homoserine dehydrogenase, lysine decarboxylase (U.S. Patent No. 5,827,698), and the malic enzyme (WO2005/010175).

L-cysteine-producing bacteria

Examples of parent strains for deriving the L-cysteine-producing bacteria of the present invention include, but are not limited to, L-cysteine-producing bacteria belonging to the genus Escherichia, such as E. colt JMl 5, which is transformed with different cysE alleles encoding feedback-resistant serine acetyltransferases (US Patent 6,218,168, Russian patent application 2003121601); E. coli W3110 having over-expressed genes which encode proteins suitable for secreting substances toxic for cells (US Patent 5,972,663); E. coli strains having lowered cysteine desulfohydrase activity (JP 11-15557 IA); E. coli W3110 with increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene (WOO 127307 'A 1 ), and the like.

L-leucine-producing bacteria

Examples of parent strains for deriving the L-leucine-producing bacteria of the present invention include, but are not limited to, L-leucine-producing bacteria belonging to the genus Escherichia, such as E. coli strains resistant to leucine analogs, including β-2- thienylalanine, 3-hydroxyleucine, 4-azaleucine, and 5,5,5-trifluoroleucine (JP 62-34397B and JP 08-70879 A); E. coli strains obtained by the gene-engineering method described in WO96/06926; E. coli H-9068 (JP 08-70879A), and the like.

The bacterium of the present invention may be improved by enhancing the expression of one or more genes involved in L-leucine biosynthesis. Examples include genes of the leuABCD operon, which are preferably represented by the mutant leuA gene encoding isopropylmalate synthase freed from feedback inhibition by L-leucine (US Patent 6,403,342). In addition, the bacterium of the present invention may be improved by enhancing the expression of one or more genes encoding proteins which secrete L-amino acid from the bacterial cell. Examples of such genes include the b2682 and b2683 genes (ygaZH genes, EP1239041 A2). L-histidine-producing bacteria

Examples of parent strains for deriving the L-histidine-producing bacteria of the present invention include, but are not limited to, L-histidine-producing bacteria belonging to the genus Escherichia, such as E. coli strain 24 (VKPM B-5945, RU2003677); E. coli strain 80 (VKPM B-7270, RU2119536); E. coli NRRL B-12116 - B12121 (US Patent 4,388,405); E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (US Patent 6,344,347); E. coli H-9341 (FERM BP-6674) (EP1085087); E. coli AI80/pFM201 (US Patent 6,258,554), and the like.

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

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

Specific examples of strains having an L-histidine-producing ability include E. coli FERM P-5038 and 5048 which have been introduced with a vector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP 56-005099 A), E. coli strains introduced with rht, a gene for an amino acid-export (EPl 01671 OA), E. coli 80 strain imparted with sulfaguanidine, DL-l,2,4-triazole-3-alanine, and streptomycin-resistance (VKPM B-7270, Russian Patent No. 2119536), and so forth.

L-glutamic acid-producing bacteria

Examples of parent strains for deriving the L-glutamic acid-producing bacteria of the present invention include, but are not limited to, L-glutamic acid-producing bacteria belonging to the genus Escherichia, such as E. coli VL334thrC⁺ (EP 1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotrophic strain having mutations in the thrC and HvA genes (US Patent 4,278,765). A wild-type allele of the thrC gene was transferred by the method of general transduction, using bacteriophage Pl grown on wild-type E. coli Kl 2 (VKPM B-7) cells. As a result, an L-isoleucine auxotrophic strain VL334thrC⁺ (VKPM B-8961) was obtained. This strain is able to produce L-glutamic acid.

Examples of parent strains for deriving the L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains in which expression of one or more genes encoding an L-glutamic acid biosynthetic enzyme are enhanced. Examples of such genes include genes encoding glutamate dehydrogenase (gdh), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, ipdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase {end), phosphoglyceromutase (pgmA, pgml), phosphoglycerate kinase (pgk), glyceraldehyde-3-phophate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pflcA, pflcB), and glucose phosphate isomerase (pgi).

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

Examples of parent strains for deriving the L-glutamic acid-producing bacteria of the present invention also include strains having decreased or eliminated activity of an enzyme that catalyzes synthesis of a compound other than L-glutamic acid, and branching off from an L-glutamic acid biosynthesis pathway. Examples of such enzymes include isocitrate lyase (aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (ptd), acetate kinase (act), acetohydroxy acid synthase (UvG), acetolactate synthase (JIvI), formate acetyltransferase (pfl), lactate dehydrogenase (Idh), and glutamate decarboxylase (gadAB).

Bacteria belonging to the genus Escherichia deficient in α-ketoglutarate dehydrogenase activity or having a reduced α-ketoglutarate dehydrogenase activity and methods for obtaining them are described in US Patents 5,378,616 and 5,573,945. Specifically, these strains include the following: E. coli W3110sucA::Kmr E. coli AJ12624 (FERM BP-3853) E. coli AJ12628 (FERM BP-3854) K coli AJ12949 (FERM BP-4881)

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

Other examples of L-glutamic acid-producing bacterium include those which belong to the genus Escherichia and have resistance to an aspartic acid antimetabolite. These strains can also be deficient in the α-ketoglutarate dehydrogenase activity and include, for example, E. coli AJ13199 (FERM BP-5807) (U.S. Patent No. 5.908,768), FFRM P-12379, which additionally has a low L-glutamic acid decomposing ability (U.S. Patent No. 5,393,671); AJ13138 (FERM BP-5565) (U.S. Patent No. 6,110,714), and the like.

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

L-phenylalanine-producing bacteria

Examples of parent strains for deriving the L-phenylalanine-producing bacteria of the present invention include, but are not limited to, L-phenylalanine-producing bacteria belonging to the genus Escherichia, such as E. coli AJ12739 (tyrA::TnlO, tyrR) (VKPM B-8197); E. coli HW1089 (ATCC 55371) harboring the pheA34 gene (U.S. Patent 5,354,672); K coli MWEC101-b (KR8903681); E. colϊNRBL B-12141, NRRL B-12145, NRRL B-12146, and NRRL B-12147 (U.S. Patent 4,407,952). Also, as a parent strain, E. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662), and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named 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 HheyedA gene or the yddG gene may also be used (US patent applications 2003/0148473 Al and 2003/0157667 Al).

L-tryptophan-producing bacteria

Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention include, but are not limited to, L-tryptophan-producing bacteria belonging to the genus Escherichia, such as E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) deficient in tryptophanyl-tRNA synthetase encoded by a mutant trpS gene (US Patent 5,756,345); E. coli SVl 64 (pGH5) having the serA allele free from feedback inhibition by serine (US Patent 6,180,373); E. coli AGXl 7 (pGX44) (NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient in the enzyme tryptophanase (US Patent 4,371,614); E. coli AGX17/pGX50,pACKG4-ρps in which a phosphoenolpyruvate-producing ability is enhanced (WO9708333, US Patent 6,319,696), and the like may be used.

Previously, it was identified that the yddG gene encodes a membrane protein which is not involved in a biosynthetic pathway of any L-amino acid, and imparts to a microorganism resistance to L-phenylalanine and several amino acid analogues when the wild-type allele of the gene is amplified on a multi-copy vector in the microorganism. Besides, HhβyddG gene can enhance production of L-phenylalanine or L-tryptophan when additional copies are introduced into the cells of the respective producing strain (WO03044192). So it is desirable that the L-tryptophan-producing bacterium be further modified to have enhanced expression of the yddG open reading frame.

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 SV 164 which harbors desensitized anthranilate synthase and a transformant strain obtained by introducing into the E. coli SVl 64 the plasmid pGH5 (WO 94/08031), which contains a mutant serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase.

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

L-proline-producing bacteria

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

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

L-arginine-producing bacteria

Examples of parent strains for deriving the L-arginine-producing bacteria of the present invention include, but are not limited to, L-arginine-producing bacteria, such as E. coli strain 237 (VKPM B-7925) (US Patent Application US2002058315) and its derivative strains harboring mutant N-acetylglutamate synthase (Russian Patent Application No. 2001112869), E. coli strain 382 (VKPM B-7926) (EP1170358A1), an arginine-producing strain which has the argA gene encoding N-acetylglutamate synthetase introduced therein (JP 57-5693A), and the like.

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

L-valine-producing bacteria

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

Examples of parent strains for deriving L-valine-producing bacteria of the present invention include also include mutants having a mutation of amino-acyl t-RNA synthetase (U.S. Patent No. 5,658,766). For example, E. coli VL1970, which has a mutation in the HeS gene encoding isoleucine tRNA synthetase, can be used. E. coli VL 1970 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 113545 Moscow, 1 Dorozhny Proezd, 1) on June 24, 1988 under accession number VKPM B-4411. Furthermore, mutants requiring lipoic acid for growth and/or lacking H⁺-ATPase can also be used as parent strains (WO96/06926).

L-isoleucine-producing bacteria

Examples of parent strains for deriving L-isoleucine producing bacteria of the present invention include, but are not limited to, mutants having resistance to 6- dimethylaminopurine (JP 5-304969 A), mutants having resistance to an isoleucine analogue such as thiaisoleucine and isoleucine hydroxamate, and mutants additionally having resistance to DL-ethionine and/or arginine hydroxamate (JP 5-130882 A). In addition, recombinant strains transformed with genes encoding proteins involved in L- isoleucine biosynthesis, such as threonine deaminase and acetohydroxate synthase, can also be used as parent strains (JP 2-458 A, FR 0356739, and U.S. Patent No. 5,998,178).

2. Method of the present invention

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

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

A medium used for culture may be either a synthetic or natural medium, so long as the medium includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth. The carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the mode of assimilation of the used microorganism, alcohol, including ethanol and glycerol, may be used. As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism can be used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used. As vitamins, thiamine, yeast extract, and the like, can be used. The cultivation is preferably performed under aerobic conditions, such as a shaking culture, and a stirring culture with aeration, at a temperature of 20 to 40 ⁰C, preferably 30 to 38 ⁰C. The pH of the culture is usually between 5 and 9, preferably between 6.5 and 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5-day cultivation leads to accumulation of the target L-amino acid in the liquid medium.

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

Examples

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

Example 1. Preparation of the E. coli strain TGlΔptsGΔmanXYZ.

1. Construction of E. coli strain TGlΔptsG.

E. coli strain TGlΔptsG was constructed by inactivation of the native ptsG gene in E. coli strain TGl by introducing the cat gene.

To inactivate the native ptsG gene, a DNA fragment carrying the chloramphenicol resistance marker (Cm^R) encoded by the cat gene was integrated into the chromosome of the E. coli strain TGl in place of the native gene by the method described by Datsenko K.A. and Wanner BX. (Proc.Natl. Acad. ScLUSA, 2000, 97, 6640-6645) which is also called "Red-mediated integration" and/or "Red-driven integration". The recombinant plasmid pKD46 (Datsenko, K.A., Wanner, B.L., Proc.Natl.Acad.Sci.USA, 2000, 97, 6640- 6645) with the thermosensitive replicon was used as the donor of the phage λ-derived genes responsible for the Red-mediated recombination system. Escherichia coli strain BW25113 containing the recombinant plasmid pKD46 can be obtained from the E. coli Genetic Stock Center, Yale University, New Haven, USA, the accession number of which is CGSC7630.

A DNA fragment containing a Cm^R marker encoded by cat gene was obtained by PCR using the commercially available plasmid pACYC184 (GenBank/EMBL accession number X06403, "Fermentas", Lithuania) as the template, and primers Pl (SEQ ID NO: 3) and P2 (SEQ ID NO: 4). Primer Pl contains 38 nucleotides homologous to the 5'-region of the ptsG gene introduced into the primer for further integration into the bacterial chromosome. Primer P2 contains 41 nucleotides complementary to the 3 '-region of the ptsG gene introduced into the primer for further integration into the bacterial chromosome.

PCR was provided using the "Gene Amp PCR System 2700" amplificatory (Applied Biosystems). The reaction mixture (total volume - 50 μl) consisted of 5 μl of 10x PCR- buffer with 25 mM MgCl₂ ("Fermentas", Lithuania), 200 μM each of dNTP, 25 pmol each of the exploited primers and 1 U of Taq-polymerase ("Fermentas", Lithuania). Approximately 5 ng of the plasmid DNA was added to the reaction mixture as a template DNA for the PCR amplification. The temperature profile was the following: initial DNA denaturation for 5 min at 95 °C, followed by 25 cycles of denaturation at 95 °C for 30 sec, annealing at 55 ⁰C for 30 sec, elongation at 72 °C for 40 sec; and the final elongation for 5 min at +72 °C. Then, the amplified DNA fragment was purified by agarose gel- electrophoresis, extracted using "GenElute Spin Columns" ("Sigma", USA) and precipitated by ethanol.

The obtained DNA fragment was used for electroporation and Red-mediated integration into the bacterial chromosome of the E. coli strain TGl/pKD46.

TGl/pKD46 cells were grown overnight at 30 ⁰C in the liquid LB-medium with addition of ampicillin (100 μg/ml), then diluted 1 : 100 by the SOB-medium (Yeast extract, 5 g/1; NaCl, 0.5 g/1; Tryptone, 20 g/1; KCl, 2.5 mM; MgC12, 10 mM) with addition of ampicillin (100 μg/ml) and L-arabinose (10 mM) (arabinose is used for inducing the plasmid encoding genes of Red system) and grown at 30 °C to reach the optical density of the bacterial culture OD₆o₀=0.4-0.7. The grown cells from 10 ml of the bacterial culture were washed 3 times by the ice-cold de-ionized water, followed by suspending in 100 μl of the water. 10 μl of DNA fragment (100 ng) dissolved in the de-ionized water was added to the cell suspension. The electroporation was performed by "Bio-Rad" electroporator (USA) (No. 165-2098, version 2-89) according to the manufacturer's instructions. Shocked cells were added to 1-ml of SOC medium (Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989)), incubated 2 hours at 37 ⁰C₅ and then were spread onto L-agar containing 25 μg/ml of chloramphenicol. Colonies which had grown within 24 h were tested for the presence of Cm^R marker instead of native ptsG gene by PCR using primers P3 (SEQ ID NO: 5) and P4 (SEQ ID NO: 6). For this purpose, a freshly isolated colony was suspended in 20μl water and then lμl of obtained suspension was used for PCR. The temperature profile follows: initial DNA denaturation for 5 min at 95 ⁰C; then 30 cycles of denaturation at 95 ⁰C for 30 sec, annealing at 55 °C for 30 sec and elongation at 72 ⁰C for 1 min; the final elongation for 5 min at 72 ⁰C. A few Cm^R colonies tested contained the desired 1200 bp DNA fragment, confirming the presence of Cm^R marker DNA instead of 1840 bp fragment of native ptsG gene. One of the obtained strains was cured from the thermosensitive plasmid pKD46 by culturing at 37 ⁰C and the resulting strain was named as E. coli strain TGlΔptsG.

2. Construction of E. coli strain TGlΔptsGΔman.

E. coli strain TGlΔptsGΔman was constructed by inactivation of the native manXYZ operon in E. coli strain TGlΔpts by introducing the kan gene. Nucleotide sequences of manX, manY and manZ genes are shown in the SEQ ID NOS: 15, 16 and 17, respectively.

To inactivate the native manXYZ operon, the DNA fragment carrying the kanamicin resistance marker (Km^R) encoded by the kan gene was integrated into the chromosome of the E. coli strain TGlΔpts in place of the native operon by the method described by Datsenko K.A. and Wanner B.L. (Proc.Natl.Acad.Sci.USA, 2000, 97, 6640-6645) which is also called as a "Red-mediated integration" and/or "Red-driven integration". The recombinant plasmid pKD46 (Datsenko, K.A., Wanner, B.L., Proc.Natl.Acad.Sci.USA, 2000, 97, 6640-6645) with the thermosensitive replicon was used as the donor of the phage λ-derived genes responsible for the Red-mediated recombination system. Escherichia coli strain BW25113 containing the recombinant plasmid pKD46 can be obtained from the E. coli Genetic Stock Center, Yale University, New Haven, USA, the accession number of which is CGSC7630.

A DNA fragment containing a Km^R marker encoded by the kan gene was obtained by PCR using the commercially available plasmid pACYC177 (GenBank/EMBL accession number X06402, "Fermentas", Lithuania) as the template, and primers P5 (SEQ ID NO: 7) and P6 (SEQ ID NO: 8). Primer P5 contains 40 nucleotides homologous to the 5'-region of the manX gene introduced into the primer for further integration into the bacterial chromosome. Primer P6 contains 41 nucleotides complementary to the 3 '-region of the manZgfas, introduced into the primer for further integration into the bacterial chromosome. PCR was provided using the "Gene Amp PCR System 2700" amplificatory (Applied Biosystems). The reaction mixture (total volume - 50 μl) consisted of 5 μl of 1Ox PCR- buffer with 25 mM MgCl₂ ("Fermentas", Lithuania), 200 μM each of dNTP, 25 pmol each of the exploited primers and 1 U of Taq-polymerase ("Fermentas", Lithuania). Approximately 5 ng of the plasmid DNA was added in the reaction mixture as a template DNA for the PCR amplification. The temperature profile was the following: initial DNA denaturation for 5 min at 95 °C, followed by 25 cycles of denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec, elongation at 72 ⁰C for 50 sec; and the final elongation for 5 min at +72 °C. Then, the amplified DNA fragment was purified by agarose gel- electrophoresis, extracted using "GenElute Spin Columns" ("Sigma", USA) and precipitated by ethanol.

The obtained DNA fragment was used for electroporation and Red-mediated integration into the bacterial chromosome of the E. coli strain TGlΔptsG/pKD46.

Substitution of the native manXYZ operon in E. coli TGlΔptsG strain by kan gene was carried out as described above.

Shocked cells were added to 1-ml of SOC medium (Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989)), incubated 2 hours at 37 ⁰C, and then were spread onto L-agar containing 20 μg/ml of kanamicin. Colonies grown within 24 h were tested for the presence of Km marker instead of native manXYZ operone by PCR using primers P7 (SEQ ID NO: 9) and P8 (SEQ ID NO: 10). For this purpose, a freshly isolated colony was suspended in 20μl water and then lμl of obtained suspension was used for PCR. The temperature profile follows: initial DNA denaturation for 5 min at 95 ⁰C; then 30 cycles of denaturation at 95 ⁰C for 30 sec, annealing at 55 °C for 30 sec and elongation at 72 ⁰C for 45 sec; the final elongation for 5 min at 72 ⁰C. A few Km^R colonies tested contained the desired ~1065 bp DNA fragment, confirming the presence of Cm^R marker DNA instead of -2780 bp fragment of manXYZ operone. One of the obtained strains was cured from the thermosensitive plasmid pKD46 by culturing at 37 ⁰C and the resulting strain was named E. coli TGlΔptsGΔmanXYZ.

Example 2. Preparing mutant ystG gene. The wild type ptsG (ptsG-wt) gene and the mutant ptsG (ptsG-mut) gene were obtained by PCR with using primers P9 (SEQ ID NO: 11) and PlO (SEQ ID NO: 12). The mutant type ptsG gene was obtained by PCR under error-prone conditions.

Primer Pl contains a BamHl recognition site at the 5 '-end thereof, and primer P2 contains an EcoRl recognition site at the 3 '-end thereof which are necessary for further cloning of ptsG genes.

PCR was provided using the "Gene Amp PCR System 2700" amplificatory (Applied Biosystems). The reaction mixture (total volume - 50 μl) consisted of 5 μl of 1Ox PCR- buffer, 2.5 mM MgCl₂ (for ptsG-wt) or 2.0 mM MnCl₂ (for ptsG-mut), 5 μM of dNTP mixture (2.5 mM each), 25 pmol each of the exploited primers and 1 U of AccuTaq - polymerase ("Sigma", USA). Approximately 20 ng of the E. coli MGl 655 genomic DNA was added to the reaction mixtures as a template DNA for the PCR amplification. The temperature profile was the following: initial DNA denaturation for 5 min at 95 °C, followed by 35 cycles of denaturation at 95 ⁰C for 30 sec, annealing at 55 ⁰C for 30 sec, elongation at 72 ⁰C for 1 min; and the final elongation for 5 min at +72 °C. Then, the amplified DNA fragments were purified by agarose gel-electrophoresis, extracted using "GenElute Spin Columns" ("Sigma", USA) and precipitated by ethanol.

Both of the obtained fragments were digested by BamHl and EcoRl and cloned into pMWl 18/ BamHl- EcoRl vector, resulting in plasmid pMWl 18-ptsG-wt and set of plasmids pMWl 18-ptsG-mut accordingly.

Then the strain E. coli TGlΔptsGΔman was transformed with these plasmids.

The strains TGl ΔptsGΔman / pMWl 18-ptsG-mut, containing plasmids with mutant ptsG gene were selected on the M9-plates (ampicillin (100 μg/ml), glucose 0.4%, casaminoacids 0.2%). Then clones grown on plates with 0.4% glucose were picked on M9- plates (ampicillin, mannose 0.4 %, casaminoacids 0.2%). Strains with good growth on plates with both sugars were tested on M9-medium with 0.4% glucose and on the M9 medium with 0.4% mannose (table 2). The strain TGl ΔptsGΔman / pMWl 18-ρtsG-43 was chosen, since its doubling time on glucose and on mannose was less than for the strain carryings the plasmid pt$G-vrt. Table 2.

The ptsG-wt gene was sequenced and native sequence of gene was confirmed. The ptsG-mut-43 gene was sequenced and mutations were found: Ala263Val and Ile359Leu in SEQ ID NO:2 .

Example 3. The effect of introduction of the mutant ptsG gene on L-threonine production.

To evaluate the effect of introduction of the mutant ptsG gene on L-threonine production, strain B-3996 was transformed with plasmid pMWl 18-ptsG-mut or pMWl 18- ptsG-wt.

The resulting strains, B-3996/ pMWl 18-ptsG-mut-43 and strain B-3996/ pMWl 18- ptsG-wt, were each cultivated at 37⁰C for 18 hours in a nutrient broth and 0.3 ml of each of the obtained cultures was inoculated into 3 ml of fermentation medium having the following composition in a 20x200 mm test tube and cultivated at 37⁰C for 48 hours with a rotary shaker.

After cultivation, the amount of L-threonine, which had accumulated in the medium, was determined by paper chromatography using the following mobile phase: butanol - acetic acid - water = 4 : 1 : 1 (v/v). A solution of nmhydrin (2%) in acetone was used as a visualizing reagent. A spot containing L-threonine was cut out, L-threonine was eluted with 0.5 % water solution of CdCl₂, and the amount of L-threonine was estimated spectrophotometrically at 540 nm. The results often independent test tube fermentations are shown in Table 3.

Fermentation medium composition (g/1):

Glucose 40

(NILO₂SO₄ 16

K₂HPO₄ 0.7

MgSO₄-7H₂O 1.0 MnSO₄-5H₂O 0.01

FeSO₄-7H₂O 0.01

Thiamine hydrochloride 0.002

Yeast extract 2.0

L-isoleucine 0.05

CaCO₃ 33

MgSO₄-TH₂O and CaCO₃ were each sterilized separately.

Table 3

It can be seen from the Table 3, strain B3996/ pMWl 18-ptsG-mut-43 was able to cause accumulation of a higher amount of L-threonine as compared with B3996/ pMW118-ptsG-wt.

Example 4. The effect of introduction of the mutant ptsG gene on L-lysine production.

To evaluate the effect of introduction of the mutant ptsG gene on L-lysine production, strain AJl 1442 can be transformed with plasmid pMWl 18-ptsG-mut or pMWl 18-ptsG-wt. The strain AJl 1442 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, the independent administrative corporation, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Tsukuba Central 6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postal code: 305-8566) on May 1, 1981, and received an accession number of FERM P-5084. Then, it was converted from the above original deposit to an international deposit under the provisions of the Budapest Treaty on October 29, 1987 and received an accession number of FERM BP- 1543.

Both E. coli strains, AJl 1442/pMWl 18-ptsG-mut and AJl 1442/pMWl 18-ptsG-wt, can be cultured in L-medium at 37⁰C, and 0.3 ml of the obtained culture can be inoculated into 20 ml of the fermentation medium in a 500-ml flask. The cultivation can be carried out at 37⁰C for 16 h by using a reciprocal shaker at the agitation speed of 115 rpm. After the cultivation, the amounts of L-lysine and residual glucose in the medium can be measured by a known method (Biotech-analyzer AS210 manufactured by Sakura Seiki Co.). Then, the yield of L-lysine can be calculated relative to consumed glucose for each of the strains.

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

Glucose 40

(NH₄)₂SO₄ 24

K₂HPO₄ 1.0

MgSO₄-7H₂O 1.0

FeSO₄-7H₂O 0.01

MnSO₄-5H₂O 0.01

Yeast extract 2.0

The pH is adjusted to 7.0 by KOH and the medium is autoclaved at 115⁰C for 10 min. Glucose and MgSO₄-7H₂O are sterilized separately. CaCO₃ is dry-heat sterilized at 180⁰C for 2 hours and added to the medium for a final concentration of 30 g/1.

Example 5. The effect of introduction of the mutant ptsG gene on L-cvsteine production.

To evaluate the effect of introduction of the mutant ptsG gene on L-cysteine production, strain JM15(ydeD) can be transformed with plasmid pMWl 18-ptsG-mut or pMW118-ptsG-wt.

E. coli JM15(ydeD) is a derivative of E. coli JM15 (US Patent No. 6,218,168), which can be transformed with DNA having the ydeD gene encoding a membrane protein, and is not involved in a biosynthetic pathway of any L-amino acid (US Patent No. 5,972,663). The strain JMl 5 (CGSC # 5042) can be obtained from The Coli Genetic Stock Collection at the E.coli Genetic Resource Center, MCD Biology Department, Yale University (http://cgsc.biology.yale.edu/).

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

Example 6. The effect of introduction of the mutant ptsG gene on L-leucine production To evaluate the effect of introduction of the mutant ptsG gene on L-leucine production, the E. coli L-leucine-producing strain 57 can be transformed with plasmid pMWl 18-ptsG-mut or pMWl 18-ptsG-wt. The strain 57 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1^st Dόrozhny proezd, 1) on May 19, 1997 under accession number VKPM B-7386.

Both E. coli strains, 57/pMWl 18-ptsG-mut and 57/pMWl 18-ptsG-wt, can be cultured for 18-24 hours at 37°C on L-agar plates. To obtain a seed culture, the strains can be grown on a rotary shaker (250 rpm) at 32°C for 18 hours in 20x200-mm test tubes containing 2 ml of L-broth supplemented with 4% sucrose. Then, the fermentation medium can be inoculated with 0.21 ml of seed material (10%). The fermentation can be performed in 2 ml of a minimal fermentation medium in 20x200-mm test tubes. Cells can be grown for 48-72 hours at 32°C with shaking at 250 rpm. The amount of L-leucine can be measured by paper chromatography (liquid phase composition: butanol - acetic acid - water = 4:1:1).

The composition of the fermentation medium (g/1) (pH 7.2) is as follows: Glucose 60.0

(NHU)₂SO₄ 25.0

K₂HPO₄ 2.0

MgSO₄-7H₂O 1.0

Thiamine 0.01

CaCO₃ 25.0

Glucose and CaCO₃ are sterilized separately.

Example 7. The effect of introduction of the mutant ptsG gene on L-histidine production

To evaluate the effect of introduction of the mutant ptsG gene on L-histidine production, the E. coli L-histidine-producing strain 80 can be transformed with plasmid pMWl 18-ptsG-mut or pMWl 18-ptsG-wt. The strain 80 has been described in Russian patent 2119536 and deposited in the Russian National Collection of Industrial Microorganisms (Russia, 117545 Moscow, 1st Dorozhny proezd, 1) on October 15, 1999 under accession number VKPM B-7270 and then converted to a deposit under the Budapest Treaty on My 12, 2004. Both E. coli strains, 80/pMWl 18-ptsG-mut and 80/pMWl 18-ρtsG-wt, can each be cultured in L-broth for 6 h at 29°C. Then, 0.1 ml of obtained culture can be inoculated into 2 ml of fermentation medium in a 20x200-mm test tube and cultivated for 65 hours at 29⁰C with shaking on a rotary shaker (350 rpm). After cultivation, the amount of histidine which accumulates in the medium can be determined by paper chromatography. The paper can be developed with a mobile phase consisting of n-butanol : acetic acid : water = 4 : 1 : 1 (v/v). A solution of ninhydrin (0.5%) in acetone can be used as a visualizing reagent.

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

Glucose 100.0

Mameno (soybean hydrolysate) 0.2 of as total nitrogen

L-proline 1.0

(NHU)₂SO₄ 25.0

KH₂PO₄ 2.0

MgSO₄-7H₂0 1.0

FeSO₄-7H₂0 0.01

MnSO₄ 0.01

Thiamine 0.001

Betaine 2.0

CaCO₃ 60.0

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

Example 8. The effect of introduction of the mutant ptsG gene on L-glutamate production.

To evaluate the effect of introduction of the mutant ptsG gene on L-glutamate production, the E. coli L-glutamate-producing strain VL334thrC⁺ can be transformed with plasmid pMWl 18-ρtsG-mut or pMWl 18-ρtsG-wt. The strain VL334thrC⁺ has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1^st Dorozhny proezd, 1) on December 6, 2004 under the accession number VKPM B-8961 and then converted to a deposit under the Budapest Treaty on December 8, 2004. Both strains, VL334thrC7pMWl 18-ptsG-mut and VL334thrC⁺/pMWl 18-ptsG-wt, can be grown for 18-24 hours at 37⁰C on L-agar plates. Then, one loop of the cells can be transferred into test tubes containing 2ml of fermentation medium. The fermentation medium contains glucose (60g/l), ammonium sulfate (25 g/l)₅ KH₂PO₄ (2g/l), MgSO₄ (1 g/lj, thiamine (0.1 mg/ml), L-isoleucine (70 μg/ml), and CaCO₃ (25 g/1). The pH is adjusted to 7.2. Glucose and CaCO₃ are sterilized separately. Cultivation can be carried out at 3O⁰C for 3 days with shaking. After the cultivation, the amount of L-glutamic acid which is produced can be determined by paper chromatography (liquid phase composition of butanol-acetic acid-water=4:l:l) with subsequent staining by ninhydrin (1% solution in acetone) and further elution of the compounds in 50% ethanol with 0.5% CdCl₂.

Example 9. The effect of introduction of the mutant ptsG gene on L- phenylalanine production.

To evaluate the effect of introduction of the mutant ptsG gene on L-phenylalanine production, the E. coli L-phenylalanine-producing strain AJ12739 can be transformed with plasmid pMWl 18-ptsG-mut or pMWl 18-ptsG-wt. The strain AJ12739 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1^st Dorozhny proezd, 1) on November 6, 2001 under accession no. VKPM B- 8197 and then converted to a deposit under the Budapest Treaty on August 23, 2002.

Both strains, AJ12739/ρMWl 18-ptsG-mut and AJ 12739/pMWl 18-ptsG-wt, can be cultivated at 37⁰C for 18 hours in a nutrient broth, and 0.3 ml of the obtained culture can each be inoculated into 3 ml of a fermentation medium in a 20x200-mm test tube and cultivated at 37°C for 48 hours with shaking on a rotary shaker. After cultivation, the amount of phenylalanine which accumulates in the medium can be determined by TLC. The 10xl5-cm TLC plates coated with 0.11 -mm layers of Sorbfil silica gel containing no fluorescent indicator (Stock Company Sorbpolymer, Krasnodar, Russia) can be used. The Sorbfil plates can be developed with a mobile phase consisting of propan-2-ol : ethylacetate : 25% aqueous ammonia : water = 40 : 40 : 7 : 16 (v/v). A solution of ninhydrin (2%) in acetone can be used as a visualizing reagent.

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

Glucose 40.0

(NHU)₂SO₄ 16.0 K₂HPO₄ 0.1

MgSO₄-7H₂O 1.0

FeSO₄-7H₂O 0.01

_. MnSO₄-5H₂O 0.01

Thiamine HCl 0.0002

Yeast extract 2.0

Tyrosine 0.125

CaCO₃ 20.0

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

Example 10. The effect of introduction of the mutant ptsG gene on L-tryptophan production.

To evaluate the effect of introduction of the mutant ptsG gene on L-tryptophan production, the E. coli L-tryptophan-producing strain SV 164 can be transformed with plasmid pMWl 18-ptsG-mut or pMWl 18-ptsG-wt. The strain SV164 has the trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan. The plasmid pGH5 harbors a mutant serA gene encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine. The strain SVl 64 (pGH5) was described in detail in US patent No. 6,180,373 or European patent 0662143.

Both strains, SV164(pGH5)/pMW118-ptsG-mut and SV164(pGH5)/pMW118-ptsG- wt can be cultivated with shaking at 32°C for 18 hours in 3 ml of nutrient broth supplemented with tetracycline (10 mg/ml, marker of pGH5 plasmid). The obtained cultures (0.3 ml each) can be inoculated into 3 ml of a fermentation medium containing tetracycline (10 mg/ml) in 20 x 200-mm test tubes, and cultivated at 32⁰C for 72 hours with a rotary shaker at 250 rpm. After cultivation, the amount of tryptophan which accumulates in the medium can be determined by TLC as described in Example 9.

The fermentation medium components are listed in Table 4, but should be sterilized in separate groups (A, B, C, D, E, F, and G)₃ as shown, to avoid adverse interactions during sterilization. Table 4

Solution A had pH 7.1 adjusted by NH₄OH.

Example 11 The effect of introduction of the mutant ptsG gene on L-proline production.

To evaluate the effect of introduction of the mutant ptsG gene on L-proline production, the E. coli L-proline-producing strain 702ilvA can be transformed with plasmid pMWl 18-ρtsG-mut or pMWl 18-ρtsG-wt. The strain 702ilvA has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1^st Dorozhny proezd, 1) on July 18, 2000 under accession number VKPM B- 8012 and then converted to a deposit under the Budapest Treaty on May 18, 2001.

Both E. coli strains, 702ilvA/pMWl 18-ptsG-mut and 702ilvA/pMWl 18-ptsG-wt, can be grown for 18-24 hours at 37°C on L-agar plates. Then, these strains can be cultivated under the same conditions as in Example 8.

Example 12. The effect of introduction of the mutant ytsG gene on L-arginine production. To evaluate the effect of introduction of the mutant ptsG gene on L-arginine production, the E. coli L-arginine-producing strain 382 can be transformed with plasmid pMWl 18-ρtsG-mut or pMWl 18-ρtsG-wt. The strain 382 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1^st Dorozhny proezd, 1) on April 10, 2000 under accession number VKPM B- 7926 and then converted to a deposit under the Budapest Treaty on May 18, 2001.

Both strains, 382/ρMWl 18-ρtsG-mut and 382/pMWl 18-ptsG-wt, can be cultivated with shaking at 37°C for 18 hours in 3 ml of nutrient broth, and 0.3 ml of the obtained cultures were inoculated into 2 ml of a fermentation medium in 20 x 200-mm test tubes and cultivated at 32⁰C for 48 hours on a rotary shaker.

After the cultivation, the amount of L-arginine which had accumulated in the medium can be determined by paper chromatography using the following mobile phase: butanol : acetic acid : water = 4 : 1 : 1 (v/v). A solution of ninhydrin (2%) in acetone can be used as a visualizing reagent. A spot containing L-arginine can be cut out, L-arginine can be eluted with 0.5% water solution of CdCl₂, and the amount of L-arginine can be estimated spectrophotometrically at 540 nm.

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

Glucose 48.0

(NHM)₂SO₄ 35.0

KH₂PO₄ 2.0

MgSO₄-7H₂O 1.0

Thiamine HCl 0.0002

Yeast extract 1.0

L-isoleucine 0.1

CaCO3 5.0

Glucose and magnesium sulfate were sterilized separately. CaCO₃ was dry-heat sterilized at 18O⁰C for 2 hours. The pH was adjusted to 7.0.

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. Industrial Applicability

The present invention provides a novel mutant II^GI°, L-amino acid-producing bactiera whose amino acid production is enhanced, and a method for producing L-amino acids using these bacteria. The present invention is useful for amino acid production.

Claims

1. A mutant IIB/IIC subunit of glucose-specific PTS permease selected from the group consisting of:

(A) a protein comprising the amino acid sequence of SEQ ID NO: 2, except that the L-amino acids at positions 263 and/or 359 are replaced with other L-amino acids; and

(B) a variant of protein (A) which has glucose-specific PTS permease activity when combined with the HA (Crr) subunit.

2. The mutant IIB/IIC subunit of glucose-specific PTS permease according to claim 1, wherein the alanine at position 263 is replaced with a valine, and/or the isoleucine at. position 359 is replaced with valine or leucine.

3. A DNA coding for the mutant IIB/IIC subunit of glucose-specific PTS permease according to any of claims 1 to 2.

4. A mutant glucose-specific PTS permease comprising the IIB/IIC subunit according to any of claims 1 to 2.

5. An L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein said bacterium has been modified to contain the mutant glucose-specific PTS permease according to claim 4.

6. The bacterium according to claim 5, wherein the bacterium has been transformed with the DNA according to claim 3.

7. The bacterium according to claim 5, wherein expression of the gene encoding the mutant IIB/IIC subunit of glucose-specific PTS permease according to claim 1 is enhanced by increasing the copy number of said gene or by modifying an expression regulating sequence of said gene.

8. The bacterium according to claim 5, wherein said bacterium is selected from the group consisting of Escherichia, Enter obacter, Erwinia, Klebsiella, Pantoea, Providencia, Salmonella, Serratia, Shigella, and Morganella.

9. The bacterium according to claim5, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

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

11. The bacterium according to claim 5, wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L- leucine, L-isoleucine, L-valine₅ L-histidine, glycine, L-serine, L-alanine, L-asparagine, L- aspartate, L-glutamine, L-glutamic acid, L-proline, and L-arginine.

12. The bacterium according to claim 11, wherein said bacterium has been further modified to enhance expression a gene selected from the group consisting of

- the mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I and is resistant to feedback inhibition by threonine, the thrB gene which codes for homoserine kinase, the thrC gene which codes for threonine synthase, the rhtA gene which codes for a putative transmembrane protein,

- the asd gene which codes for aspartate-β-semialdehyde dehydrogenase;

- the aspC gene which codes for aspartate aminotransferase (aspartate transaminase), and

- combinations thereof.

13. The bacterium according to claim 12, wherein said bacterium has been modified to increase expression of said mutant thrA gene, said thrB gene, said thrC gene, and said rhtA gene.

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

- cultivating the bacterium according to claim 5 in a medium, and

- collecting said L-amino acid from the medium.

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

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

17. The method according to claim 15, wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L- leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L- aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine.