WO2007086544A1 - A method for producing an l-amino acid using a bacterium of enterobacteriaceae family with attenuated expression of the bisc gene - Google Patents

Abstract

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

Description

DESCRIPTION

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

Technical Field

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

Background Art

The BisC protein belongs to the family of anaerobic dehydrogenases, which typically contain selenocysteine. It was first identified as a biotin sulfoxide reductase that reduces a spontaneous oxidation product of biotin, biotin J-sulfoxide (BDS), back to biotin. Biotin sulfoxide reductase requires a molybdenum containing cofactor, plus several accessory proteins. One of the accessory proteins is a small, heat-stable, thioredoxin-like protein which functions as a source for reducing equivalents and an unidentified flavoprotein. The function of the reductase is unknown, but it may allow scavenging of BDS as a biotin source. It may also protect the cell from oxidation damage. BisC also exhibits methionine-iS-sulfoxide reductase activity, acting specifically on the S enantiomer in the free, but not the protein-bound form (Ezraty, B. et al, J. Bacteriol., 187(l):231-7 (2005)).

It has been reported that biotin sulfoxide reductase is also capable of reducing nicotinamide N-oxide, trimethylamine-N-oxide, and dimethyl sulfoxide, although with varying efficiencies, and can directly utilize NADPH as a reducing agent for the reduction of both biotin sulfoxide and ferricyanide. The enzyme contains the prosthetic group molybdopterin guanine dinucleotide, and does not require any accessory proteins for functionality (Pollock, V. V. and Barber M. J., J Biol Chem., 272(6):3355-62 (1997)). The bisC gene (b3551) encoding the BisC protein from Escherichia coli has been described (del Campillo-Campbell, A. and Campbell A., J. Bacteriol., 149, 2, 469-78 (1982)).

But currently, there have been no reports of attenuating expression of the bisC gene for the purpose of improving L-amino-acid productivity.

Disclosure of the Invention

Objects of present invention include enhancing the productivity of L-amino acid producing strains and providing a method for producing an L-amino acid using these strains.

The above objects were achieved by finding that attenuating expression of the bisC gene can enhance the production of L-amino acids, such as L-threonine, L-lysine, L- cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L- alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine, L-phenylalanine, L-tyrosine, and L-tryptophan.

The present invention provides a bacterium of the Enterobacteriaceae family having an increased ability to produce amino acids, such as L-threonine, L-lysine, L- cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L- alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine, L-phenylalanine, L-tyrosine, and L-tryptophan.

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

It is a further object of the present invention to provide the bacterium as described above, wherein the expression of the bisC gene is attenuated by inactivation of the bisC gene.

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

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

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

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

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

- cultivating the bacterium as described above in a medium to produce and excrete said L-amino acid into the medium, and

- collecting said L-amino acid from the medium.

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

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

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

The present invention is described in detail below.

1. Bacterium of the present invention

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

In the present invention, "L-amino acid producing bacterium" means a bacterium which has an ability to produce and excrete L-amino acid into a medium, when the bacterium is cultured in the medium. 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 an amount larger than a wild-type or parental strain of the bacterium, for example, E. coli, such as E. coli K- 12, and preferably means that the 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. The term "L-amino acids" includes L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L- proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

The term "aromatic L-amino acid" includes L-phenylalanine, L-tyrosine, and L- tryptophan. The term "non-aromatic L-amino acid" includes L-threonine, L-lysine, L- cysteine, L-methionine₅ 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. L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L-phenylalanine, L-tryptophan, L-proline and L-arginine are particularly preferred.

The Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella , Morganella, Yersinia, etc.. Specifically, those classified into the Enterobacteriaceae according to the taxonomy used in 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 phrase "a bacterium belonging to the genus Pantoea" means that the bacterium is classified as the genus Pantoea according to the classification known to a person skilled in the art of microbiology. Some species of Enterobacter agglomerans have been recently re-classified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like, based on nucleotide sequence analysis of 16S rRNA, etc.

The phrase "bacterium has been modified to attenuate expression of the bisC gene" means that the bacterium has been modified in such a way that the modified bacterium contains a reduced amount of the BisC protein as compared with an unmodified bacterium, or is unable to synthesize the BisC protein. The phrase "bacterium has been modified to attenuate expression of the bisC gene" also includes that the bacterium has been modified to reduce an activity of biotin sulfoxide reductase.

Activity of the BisC protein can be measured by the method described in, for example, (Ezraty, B. et al, J. Bacteriol., 187(1):231-7(2005)). So, the reduced or absent activity of the BisC protein in the bacterium according the present invention can be determined when compared to the parent unmodified bacterium.

The phrase "inactivation of the bisC gene" means that the modified gene encodes a completely non-functional protein. It is also possible that the modified DNA region is unable to naturally express the gene due to the deletion of a part of the gene, the shifting of the reading frame of the gene, the introduction of missense/nonsense mutation(s), or the modification of an adjacent region of the gene, including sequences controlling gene expression, such as a promoter, enhancer, attenuator, ribosome-binding site, etc.

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

The bisC (b3551) gene encodes the BisC protein -biotin sulfoxide reductase with methionine-iS-sulfoxide reductase activity. The bisC gene (nucleotides complementary to nucleotides 3712084 to 3714303 in the GenBank accession number NC_000913.2; gi:49175990; SEQ ID NO: 1) is located between the yiaC and the yiaD on the chromosome of E. coli strain K- 12. The nucleotide sequence of the bisC gene and the amino acid sequence of BisC encoded by the bisC gene are shown in SEQ ID NO: 1 and SEQ ID NO:2, respectively.

Since there may be some differences in DNA sequences between the genera or strains of the Enterobacteriaceae family, the bisC gene to be inactivated on the chromosome is not limited to the gene shown in SEQ ID No:l, but may include genes homologous to SEQ ID No:l encoding a variant protein of the BisC protein. The phrase "variant protein" as used in the present invention means a protein which has changes in the sequence, whether they are deletions, insertions, additions, or substitutions of amino acids, but still maintains the activity of the product as the BisC protein. The number of changes in the variant protein depends on the position or the type of amino acid residues in the three dimensional structure of the protein. It may be 1 to 30, preferably 1 to 15, and more preferably 1 to 5 in SEQ ID NO: 2. These changes in the variants are conservative mutations that preserve the function of the protein. In other words, these changes can occur in regions of the protein which are not critical for the function of the protein. A conservative mutation is a mutation wherein substitution takes place mutually among Phe, Trp, Tyr, if the substitution site is an aromatic amino acid; among Leu, He, VaI, if the substitution site is a hydrophobic amino acid; between GIn, Asn, if it is a polar amino acid; among Lys, Arg, His, if it is a basic amino acid; between Asp, GIu, if it is an acidic amino acid; and between Ser, Thr, if it is an amino acid having a hydroxyl group. Typical conservative mutations are conservative substitutions. Specific examples of substitutions that are considered to be conservative include: substitution of Ala with Ser or Thr; substitution of Arg with GIn, His, or Lys; substitution of Asn with GIu, GIn, Lys, His, or Asp; substitution of Asp with Asn, GIu, or GIn; substitution of Cys with Ser or Ala; substitution of GIn with Asn, GIu, Lys, His, Asp, or Arg; substitution of GIu with GIy, Asn, GIn, Lys, or Asp; substitution of GIy with Pro; substitution of His with Asn, Lys, GIn, Arg, or Tyr; substitution of He with Leu, Met, VaI, or Phe; substitution of Leu with He, Met, VaI, or Phe; substitution of Lys with Asn, GIu, GIn, His, or Arg; substitution of Met with He, Leu, VaI, or Phe; substitution of Phe with Trp, Tyr, Met, He, or Leu; substitution of Ser with Thr or Ala; substitution of Thr with Ser or Ala; substitution of Trp with Phe or Tyr; substitution of Tyr with His, Phe, or Trp; and substitution of VaI with Met, He, or Leu. Substitutions, deletions, insertions, additions, or inversions and the like of the amino acids described above include naturally occurred mutations (mutant or variant) depending on differences in species, or individual differences of microorganisms that retain the bisC gene. Such a gene can be obtained by modifying the nucleotide sequences shown in SEQ ID NO: 1 using, for example, site-directed mutagenesis, so that the site-specific amino acid residue in the protein encoded includes substitutions, deletions, insertions, or additions. Moreover, the protein variant encoded by the bisC gene may have a homology of not less than 80 %, preferably not less than 90%, and most preferably not less than 95 %, with respect to the entire amino acid sequence shown in SEQ ID NO. 2, as long as the activity of the BisC protein is maintained.

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

Moreover, the bisC gene may be a variant which hybridizes with the nucleotide sequence shown in SEQ ID NO: 1, or a probe which can be prepared from the nucleotide sequence under stringent conditions, provided that it encodes a functional BisC protein prior to inactivation. "Stringent conditions" include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, more preferably not less than70%, further preferably not less than 80%, and still more preferably not less than 90%, and most preferably not less than 95% is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above is not formed. For example, stringent conditions are exemplified by washing one time, preferably two or three times at a salt concentration corresponding to 1 X SSC, 0.1% SDS, preferably 0.1 X SSC, 0.1% SDS at 6O⁰C. Duration of washing depends on the type of membrane used for blotting and, as a rule, should be what is recommended by the manufacturer. For example, the recommended duration of washing for the Hybond™ N+ nylon membrane (Amersham) under stringent conditions is 15 minutes. Preferably, washing may be performed 2 to 3 times. The length of the probe may be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp.

Examples of methods of attenuating expression of the bisC gene include mutating or deleting the bisC gene so that intracellular activity of the protein encoded by the bisC gene is reduced or eliminated as compared to a non-mutated strain or wild-type strain. For example, this can be achieved by using recombination to inactivate the bisC 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)).

Enzymatic activity can also be decreased or eliminated by constructing a gene encoding a mutant enzyme which lacks a coding region, using homologus recombination to replace the normal gene on the chromosome with this gene, and introducing a transposon or IS factor into the gene.

For example, the following methods may be employed to introduce a mutation causing a decrease of, or eliminating, the above enzyme activity by gene recombination. A portion of the sequence of the targeted gene is modified, a mutant gene that does not produce a normally functioning enzyme is prepared, DNA containing this gene is used to transform a microbe from the Enterobacteriaceae family, and the mutant gene is made to recombine with the gene on the chromosome, which results in replacing the target gene on the chromosome with the mutant gene.

Such gene substitution using homologous recombination can be conducted by methods employing linear DNA, such as the method known as "Red-driven integration" (Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)), or by methods employing a plasmid containing a temperature-sensitive replication (U.S. Patent No. 6,303,383 or JP 05-007491 A). Furthermore, the incorporation of a site- specific mutation by gene substitution using homologous recombination such as set forth above can also be conducted with a plasmid lacking the ability to replicate in the host.

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

The presence or absence of the bisC gene on the chromosome of a bacterium can be detected by well-known methods, including PCR, Southern blotting and the like. In addition, the level of gene expression can be estimated by measuring the amount or molecular weight of mRNA transcribed from the gene using various well-known methods, including Northern blotting, quantitative RT-PCR, and the like. The amount of the protein encoded by the gene can be measured by well-known methods, including SDS-PAGE followed by an immunoblotting assay (Western blot analysis), and the like. Methods for preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer, and the like may be ordinary methods well known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).

L- amino acid producing bacteria

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

The bacterium of the present invention can be obtained by attenuating expression of the bisC gene in a bacterium which inherently has the ability to produce L-amino acids. Alternatively, the bacterium of present invention can be obtained by imparting the ability to produce an L-amino acid to a bacterium already having attenuated expression of the bisC gene.

L-threonine-producing bacteria

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

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

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

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

- the mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine;

- the thrB gene which codes for homoserine kinase;

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

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

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

The thrA gene which encodes aspartokinase homoserine dehydrogenase I of . Escherichia coli has been elucidated (nucleotide positions 337 to 2799, GenBank accession 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 NC_000913.2, gi: 49175990). The thrB gene is located between the thrA and thrC genes on the chromosome of E. coli K-12. The thrC gene which encodes threonine synthase of Escherichia coli has been elucidated (nucleotide positions 3734 to 5020, GenBank accession NC_000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaXopen reading frame on the chromosome of E. coli K-12. All three genes functions as a single threonine operon. To enhance expression of the threonine operon, the attenuator region which affects the transcription is desirably removed from the operon (WO2005/049808, WO2003/097839).

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

The rhtA gene exists at 18 min on the E. coli chromosome close to the glnHPQ operon, which encodes components of the glutamine transport system. The rhtA gene is identical to ORFl (ybiF gene, nucleotide positions 764 to 1651, GenBank accession number AAA218541, gi:440181) and is 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 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 NC_000913.1, gi:16131307), and can be obtained by PCR (polymerase chain reaction; refer to White, TJ. et ah, Trends Genet, 5, 185 (1989)) utilizing primers prepared based on the nucleotide sequence of the gene. The asd genes of other microorganisms can be obtained in a similar manner.

Also, .the aspC gene of E.coli has already been elucidated (nucleotide positions 983742 to 984932, GenBank accession NC_000913.1, gi:16128895), and can be obtained by PCR. The aspC genes of other microorganisms can be obtained in a similar manner.

L-lysine-producing bacteria

Examples of L-lysine-producing bacteria belonging to the genus Escherichia include mutants having resistance to an L-lysine analogue. The L-lysine analogue inhibits growth of bacteria belonging to the genus Escherichia, but this inhibition is fully or partially desensitized when L-lysine coexists in a medium. Examples of the L-lysine analogue include, but are not limited to, oxalysine, lysine hydroxamate, S-(2-aminoethyl)- L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam and so forth. Mutants having resistance to these lysine analogues can be obtained by subjecting bacteria belonging to the genus Escherichia to a conventional artificial mutagenesis treatment. Specific examples of bacterial strains useful for producing L-lysine include Escherichia coli AJl 1442 (FERM BP-1543, NRRL B-12185; see U.S. Patent No. 4,346,170) and Escherichia coli VL611. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is desensitized.

The strain WC 196 may be used as an L-lysine producing bacterium of Escherichia coli. This bacterial strain was bred by conferring AEC resistance to the strain W3110, which was derived from Escherichia coli K- 12. The resulting strain was designated Escherichia coli AJ 13069 strain and was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on December 6, 1994 and received an accession number of FERM P- 14690. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on September 29, 1995, and received an accession number of FERM BP-5252 (U.S. Patent No. 5,827,698).

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

L-cysteine-producing bacteria

Examples of parent strains for deriving L-cysteine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JM 15 which is transformed with different cysE alleles coding for feedback- resistant serine acetyltransferases (U.S. Patent No. 6,218,168, Russian patent application 2003121601); E. coli W3110 having over-expressed genes which encode proteins suitable for secreting substances toxic for cells (U.S. Patent No. 5,972,663); E. coli strains having lowered cysteine desulfohydrase activity (JPl 1155571 A2); E. coli W3110 with increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene (WO0127307A1), and the like.

L-leucine-producing bacteria

Examples of parent strains for deriving L-leucine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strains resistant to leucine (for example, the strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121)) or leucine analogs including β-2-thienylalanine, 3 -hydroxy leucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A); E. coli strains obtained by the gene engineering method described in WO96/06926; E. coli H-9068 (JP 8- 70879 A), and the like.

The bacterium of the present invention may be improved by enhancing the expression of one or more genes involved in L-leucine biosynthesis. Examples include genes of the leuABCD operon, which are preferably represented by a mutant leuA gene coding for isopropylmalate synthase freed from feedback inhibition by L-leucine (U.S. Patent No. 6,403,342). In addition, the bacterium of the present invention may be improved by enhancing the expression of one or more genes coding for proteins which excrete L- amino acid from the bacterial cell. Examples of such genes include the b2682 and b2683 genes (ygaZH genes) (EP 1239041 A2).

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

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

It is known that the L-histidine biosynthetic enzyme encoded by hisG 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 (EP1016710A), E. coli 80 strain imparted with sulfaguanidine, DL- l,2,4-triazole-3 -alanine, and streptomycin-resistance (VKPM B-7270, Russian Patent No. 2119536), and so forth.

L-glutamic acid-producing bacteria

Examples of parent strains for deriving L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli VL334thrC⁺ (EP 1172433). E. coli VL334 (VKPM B-1641) is an L- isoleucine and L-threonine auxotrophic strain having mutations in thrC and UvA genes (U.S. Patent No. 4,278,765). A wild-type allele of the thrC gene was transferred by the method of general transduction using a bacteriophage Pl grown on the wild-type E. coli strain K12 (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, 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 (flip), phosphofructokinase (pfkA, pβB), and glucose phosphate isomerase (pgϊ).

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 (ptά), acetate kinase (ack), acetohydroxy acid synthase (UvG), acetolactate synthase (UvI), formate acetyltransferase (pfl), lactate dehydrogenase (Idh), and glutamate decarboxylase (gadAB). Bacteria belonging to the genus Escherichia deficient in the α-ketoglutarate dehydrogenase activity or having a reduced α-ketoglutarate dehydrogenase activity and methods for obtaining them are described in U.S. Patent Nos. 5,378,616 and 5,573,945. Specifically, these strains include the following:

E. coli W311 OsucA: :Kmr

E. coli AJ12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ12949 (FERM BP-4881) E. coli W3110sucA::Kmr is a strain obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter referred to as "sucA gene") of E. coli W3110. This strain is completely deficient in the α-ketoglutarate dehydrogenase.

Other examples of L-glutamic acid-producing bacterium include those which belong to the genus Escherichia and have resistance to an aspartic acid antimetabolite. These strains can also be deficient in the α-ketoglutarate dehydrogenase activity and include, for example, E. coli AJ13199 (FΕRM 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 (FΕRM BP-5565) (U.S. Patent No. 6,110,714), and the like.

Examples of L-glutamic acid-producing bacteria, include mutant strains belonging to the genus Pantoea which are deficient in the α-ketoglutarate dehydrogenase activity or have a decreased α-ketoglutarate dehydrogenase activity, and can be obtained as described above. Such strains include Pantoea ananatis AJ13356. (U.S. Patent No. 6,331,419). Pantoea ananatis AJ13356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on February 19, 1998 under an accession number of FERM P- 16645. It was then converted to an international deposit under the provisions of Budapest Treaty on January 11, 1999 and received an accession number of FERM BP-6615. Pantoea ananatis AJl 3356 is deficient in the 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 the 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 L-phenylalanine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli AJl 2739 (tyrA::TnlO, tyrR) (VKPM B-8197); E.coli HWl 089 (ATCC 55371) harboring the pheA34 gene (U.S. Patent No. 5,354,672); E.coli MWEC101-b (KR8903681); E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B- 12147 (U.S. Patent No. 4,407,952). Also, as a parent strain, E. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP- 12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used (EP 488424 Bl). Furthermore, L-phenylalanine producing bacteria belonging to the genus Escherichia with an enhanced activity of the protein encoded by the yedA gene or the yddG gene may also be used (U.S. 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, strains belonging to the genus Escherichia, such as E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Patent No. 5,756,345); E. coli SVl 64 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase which is free from feedback inhibition by serine and a trpE allele encoding anthranilate synthase which is free from feedback inhibition by tryptophan (U.S. Patent No. 6,180,373); E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient in the enzyme tryptophanase (U.S. Patent No. 4,371,614); E. coli _, AGX17/pGX50,pACKG4-pps in which a phosphoenolpyruvate-producing ability is enhanced (WO9708333, U.S. Patent No. 6,319,696), and the like may be used.

Previously, it was identified that the yddG gene encodes a membrane protein which is not involved in the biosynthetic pathway of any L-amino acid, and also imparts to a microorganism resistance to L-phenylalanine and several amino acid analogues when the wild-type allele of the gene was amplified on a multi-copy vector in the microorganism. Besides, the yddG 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 following enzymes anthranilate synthase, phosphoglycerate dehydrogenase, and tryptophan synthase are enhanced. The anthranilate synthase and phosphoglycerate dehydrogenase are both subject to feedback inhibition by L-tryptophan and L-serine, so that a mutation desensitizing the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such a mutation include a E. coli SVl 64 which harbors desensitized anthranilate synthase and a transformant strain obtained by introducing into the E. coli SV164 the plasmid pGH5 (WO 94/08031), which contains a mutant serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase.

Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention also include strains into which the tryptophan operon which contains a gene encoding desensitized anthranilate synthase has been introduced (JP 57-71397 A, JP 62-244382 A, U.S. Patent No. 4,371,614). Moreover, L-tryptophan-producing ability may be imparted by enhancing expression of a gene which encodes tryptophan synthase, among tryptophan operons (tiψBA). 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 L-proline-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli 702ilvA (VKPM B-8012) which is deficient in the UvA gene and is able to produce L-proline (EP 1172433). The bacterium of the present invention may be improved by enhancing the expression of one or more genes involved in L-proline biosynthesis. Examples of such genes for L-proline producing bacteria which are preferred include the proB gene coding for glutamate kinase of which feedback inhibition by L-proline is desensitized (DE Patent 3127361). In addition, the bacterium of the present invention may be improved by enhancing the expression of one or more genes coding for proteins excreting L-amino acid from bacterial cell. Such genes are exemplified by b2682 and b2683 genes (ygaZH genes) (EP1239041 A2). Examples of bacteria belonging to the genus Escherichia, which have an activity to produce L-proline include the following E. coli strains: NRRL B-12403 and NRRL B- 12404 (GB Patent 2075056), VKPM B-8012 (Russian patent application 2000124295), plasmid mutants described in DE Patent 3127361, plasmid mutants described by Bloom F.R. et al (The 15^th Miami winter symposium, 1983, ρ.34), and the like.

L-arginine-producing bacteria

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

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

L-valine-producing bacteria

Example of parent strains for deriving L-valine-producing bacteria of the present invention include, but are not limited to, strains which have been modified to overexpress the UvGMEDA operon (U.S. Patent No. 5,998,178). It is desirable to remove the region of the UvGMEDA operon which is required for attenuation so that expression of the operon is not attenuated by the 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 UeS 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 °C, preferably 30 to 38 °C. 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.

Brief Description of Drawings

Figure 1 shows the relative positions of primers bisCL and bisCR on plasmid pACYC184, which is used for amplification of the cat gene.

Figure 2 shows the construction of the chromosomal DNA fragment which includes the inactivated bisC gene.

Examples

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

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

1. Deletion of the bisC gene.

A strain having deletion of the bisC gene was constructed by the method initially developed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) called "Red-driven integration". According to this procedure, the PCR primers bisC L (SEQ ID NO: 3) and bisC R (SEQ ID NO: 4), which are complementary to both the region adjacent to the bisC gene and the gene conferring antibiotic resistance in the template plasmid, were constructed. The plasmid pACYC184 (NBL Gene Sciences Ltd., UK) (GenBank/EMBL accession number X06403) is used as a template in the PCR reaction. Conditions for PCR were as follows: denaturation step: 3 min at 95 ⁰C; profile for two first cycles: 1 min at 95 °C, 30 sec at 50 °C, 40 sec at 72 ⁰C; profile for the last 25 cycles: 30 sec at 95 °C, 30 sec at 54 °C, 40 sec at 72 ⁰C; final step: 5 min at 72 °C.

A 1152 bp PCR product (Fig. 1) was obtained and purified in agarose gel and was used for electroporation of the E. coli strain MGl 655 (ATCC 700926), which contains the plasmid pKD46 having a temperature-sensitive replication. The plasmid pKD46 (Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 2000, 97:12:6640-45) includes a 2,154 nucleotide (31088-33241) DNA fragment of phage λ (GenBank accession No. J02459), and contains genes of the λ Red homologous recombination system (γ, β, exo genes) under the control of the arabinose-inducible P^_B promoter. The plasmid pKD46 is necessary for integration of the PCR product into the chromosome of strain MG1655. The strain MGl 655 can be obtained from American Type Culture Collection. (P.O. Box 1549 Manassas, VA 20108, U.S.A.).

Electrocompetent cells were prepared as follows: a night culture of E. coli MGl 655 is grown at 30 °C in LB medium, supplemented with ampicillin (100 mg/1), diluted 100 times with 5 ml of SOB medium (Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989)) with ampicillin and L-arabinose (1 mM). The obtained cells were grown with aeration at 30 °C to an OD₆₀₀ of «0.6 and then were made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H₂O. Electroporation was performed using 70 μl of cells and «100 ng of PCR product. Cells after electroporation were incubated with 1 ml of SOC medium (Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989)) at 37 °C for 2.5 h and after that plated onto L-agar containing chloramphenicol (30 μg/ml) and were grown at 37 ⁰C to select Cm^R recombinants. Then, to eliminate the pKD46 plasmid, 2 passages on L-agar with Cm at 42 ⁰C were performed and the obtained colonies were tested for sensitivity to ampicillin.

2. Verification of the bisC gene deletion by PCR.

The mutants, which have the bisC gene deleted, and marked with Cm resistance gene, were verified by PCR. Locus-specific primers bisCl (SEQ ID NO: 5) and bisC2 (SEQ ID NO: 6) were used in PCR for the verification. Conditions for PCR verification were as follows: denaturation step: 3 min at 94 °C; profile for the 30 cycles: 30 sec at 94 °C, 30 sec at 54 °C, 1 min at 72 °C; final step: 7 min at 72 °C. The PCR product obtained in the reaction with the cells of parental bisC ⁺ strain MGl 655 as a template, was 2472 bp in length. The PCR product obtained in the reaction with the cells of mutant MGl 655 Δ bisC::cat strain as the template was 1332 nucleotides in length (Fig.2).

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

To test the effect of inactivation of the bisC gene on threonine production, DNA fragments from the chromosome of the above-described E. coli MGl 655 AbisC::cat were transferred to the threonine-producing E. coli strain VKPM B-3996 by Pl transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain B-3996-ΔbisC. The strain B-3996 was deposited on November 19, 1987 in the Ail-Union Scientific Center of Antibiotics (Nagatinskaya Street 3 -A, 117105 Moscow, Russian Federation) under the accession number RIA 1867. The strain was also deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Dorozhny proezd. 1, Moscow 117545, Russian Federation) under the accession number B-3996.

Both E. coli strains B-3996 and B-3996-ΔbisC were grown for 18-24 hours at 37 ⁰C on L-agar plates. To obtain a seed culture, the strains were grown on a rotary shaker (250 rpm) at 32 ⁰C for 18 hours in 20x200 mm test tubes containing 2 ml of L-broth with 4% sucrose. Then, the fermentation medium was inoculated with 0.21 ml (10%) of seed material. The fermentation was performed in 2 ml of minimal medium for fermentation in 20x200 mm test tubes. Cells were grown for 72 hours at 32⁰C with shaking at 250 rpm.

After cultivation, the amount of L-threonine which had accumulated in the medium was determined by paper chromatography using the following mobile phase: butanol : acetic acid : water = 4 : 1 : 1 (v/v). A solution (2%) of ninhydrin in acetone was used as a visualizing reagent. A spot containing L-threonine was cut off, L-threonine was eluted in 0.5 % water solution Of CdCl₂, and the amount of L-threonine was estimated spectrophotometrically at 540 nm. The results of 8 independent test tube fermentations are shown in Table 1.

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

Glucose 80.0

(NRO₂SO₄ 22.0

NaCl 0.8 KH₂PO₄ 2.0

MgSO₄7H₂O 0.8

FeSO₄7H₂O 0.02

MnSO₄ 5H₂O 0.02

Thiamine HCl 0.0002

Yeast extract 1.0

CaCO₃ 30.0

Glucose and magnesium sulfate are sterilized sepan at 180°C for 2 h. The pH is adjusted to 7.0. Antibiotic is introduced into the medium after sterilization.

Table 1

It can be seen from Table 1 that B-3996-ΔbisC caused a higher amount of accumulation of L-threonine as compared with B-3996.

Example 3. Production of L-lvsine by E. coli strain AJl 1442-ΔbisC.

To test the effect of inactivation of the bisC gene on lysine production, DNA fragments from the chromosome of the above-described E. coli MG1655 AbisC::cat can be transferred to the Iy sine-producing E. coli strain WC 196 (pCABD2) by Pl transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain WC196(pCABD2) ΔbisC::cat. pCABD2 is a plasmid which includes a dapA gene coding for a dihydrodipicolinate synthase having a mutation which desensitizes feedback inhibition by L-lysine, a lysC gene coding for aspartokinase III having a mutation which desensitizes feedback inhibition by L-lysine, a dapB gene coding for a dihydrodipicolinate reductase gene, and a ddh gene coding for diaminopimelate dehydrogenase (U.S. Patent No. 6,040,160).

Both E. coli strains WC196(pCABD2) and WC196(ρCABD2) ΔbisC::cat canbe cultured in the L-medium containing 20 mg/1 of streptomycin at 37 ⁰C, and 0.3 ml of the obtained culture can be inoculated into 20 ml of the fermentation medium containing the required drugs in a 500 ml-flask. The cultivation can be carried out at 37 ⁰C for 16 hours 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 relative to consumed glucose can be calculated for each of the strains.

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

Glucose 40

(NBU)₂SO₄ 24

K₂HPO₄ 1.0

MgSO₄-7H₂O 1.0

FeSO₄-7H₂O 0.01

MnSO-5H₂O 0.01

Yeast extract 2.0 pH is adjusted to 7.0 by KOH and the medium is autoclaved at 115°C for 10 min. Glucose and MgSO₄-7H₂O are sterilized separately. 30 g/1 OfCaCO₃, which has been dry- heat sterilized at 180°C for 2 hours, is added.

Example 4. Production of L-cvsteine by E. coli strain JM15(ydeDV A bisC.

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

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

Fermentation conditions for evaluation of L-cysteine production are described in detail in Example 6 of U.S. Patent No. 6,218,168. Example 5. Production of L-leucine by E. coli strain 57-ΔbisC.

To test the effect of inactivation of the bisC gene on L-leucine production, DNA fragments from the chromosome of the above-described E. coli MG1655 ΔbisC::cat can be transferred to the E. coli L-leucine producing strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121) by Pl transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain 57-pMW-ΔbisC. The strain 57 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow 1, Dorozhny proezd 1) on May 19, 1997 under the accession number VKPM B-7386.

Both E. coli strains 57 and 57-ΔbisC 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 ⁰C for 18 hours in 20x200 mm test tubes containing 2 ml of L-broth with 4% sucrose. Then, the fermentation medium can be inoculated with 0.21 ml (10%) seed material. The fermentation can be performed in 2 ml of minimal medium for fermentation 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) is as follows (pH 7.2):

Glucose 60.0

(NHU)₂SO₄ 25.0

K₂HPO₄ 2.0

MgSO₄-7H₂O 1.0

Thiamine 0.01

CaCO₃ 25.0

Glucose and chalk should be sterilized separately.

Example 6. Production of L-histidine by E. coli strain 80-ΔbisC.

To test the effect of inactivation of the bisC gene on L-histidine production, DNA fragments from the chromosome of the above-described E. coli MGl 655 ΔbisC::cat can be transferred to the histidine-producing E. coli strain 80 by Pl transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain 80-ΔbisC. The strain 80 has been described in Russian patent 2119536 and deposited in the Russian National Collection of Industrial Microorganisms (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on October 15, 1999 under accession number VKPM B-7270 and then converted to a deposit under the Budapest Treaty on July 12, 2004.

Both E. coli strains 80 and 80-ΔbisC can be cultivated in L-broth for 6 hours at 29 °C. Then, 0.1 ml of obtained culture can be inoculated into 2 ml of fermentation medium in 20x200mm test tube and cultivated for 65 hours at 29 °C with 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: 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 (pH 6.0) (g/1):

Glucose 100.0

Mameno 0.2 as total nitrogen

L-proline 1.0

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. pH is adjusted to 6.0 before sterilization.

Example 7. Production of L-glutamate by E. coli strain VL334thrC⁺-Δ bisC.

To test the effect of inactivation of the bisC gene on L-glutamate production, DNA fragments from the chromosome of the above-described E. coli MG1655 ΔbisCr.cat can be transferred to the E. coli L-glutamate producing strain VL334thrC⁺ (EP 1172433) by Pl transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain VL334thrC⁺-ΔbisC. The strain VL334thrC⁺ has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on December 6, 2004 under the accession number VKPM B-8961 and then converted to a deposit under the Budapest Treaty on December 8, 2004.

Both strains, VL334thrC⁺ and VL334thrC⁺-ΔbisC, can be grown for 18-24 hours at 37 ⁰C on L-agar plates. Then, one loop of the cells can be transferred into test tubes containing 2ml of fermentation medium. The fermentation medium should contain 60g/l glucose, 25 g/1 ammonium sulfate, 2g/l KH₂PO₄, 1 g/1 MgSO₄, 0.1 mg/ml thiamine, 70 μg/ml L-isoleucine and 25 g/1 CaCO₃ (pH 7.2). Glucose and CaCO₃ are sterilized separately. Cultivation can be carried out at 30 °C for 3 days with shaking. After the cultivation, the amount of L-glutamic acid produced can be determined by paper chromatography (liquid phase composition: butanol-acetic acid-water=4:l:l) with subsequent staining by ninhydrin (1% solution in acetone) and further elution of the compounds in 50% ethanol with 0.5% CdCl₂.

Example 8. Production of L- phenylalanine by E. coli strain AJ12739-ΔbisC.

To test the effect of inactivation of the bisC gene on L-phenylalanine production, DNA fragments from the chromosome of the above-described E. coli MGl 655 ΔbisCr.cat can be transferred to the phenylalanine-producing E. coli strain AJ 12739 by Pl transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY). 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 number VKPM B-8197 and then converted to a deposit under the Budapest Treaty on August 23, 2002.

Both strains, AJ12739-ΔbisC and AJ12739, can be cultivated at 37 ⁰C for 18 hours in a nutrient broth, and 0.3 ml of the obtained culture can be inoculated into 3 ml of a fermentation medium in a 20 x 200 mm test tube and cultivated at 37 ⁰C for 48 hours with a rotary shaker. After cultivation, the amount of phenylalanine which accumulates in the medium can be determined by TLC. 10 x 15 cm TLC plates coated with 0.11 mm layers of Sorbfil silica gel without fluorescent indicator (Stock Company Sorbpolymer, Krasnodar, Russia) can be used. Sorbfil plates can be developed with a mobile phase: propan-2-ol : ethylacetate : 25% aqueous ammonia : water = 40 : 40 : 7 : 16 (v/v). A solution (2%) of ninhydrin in acetone can be used as a visualizing reagent.

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

Glucose 40.0

(NKO₂SO₄ 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 dry-heat sterilized at 180° for 2 h. pH is adjusted to 7.0.

Example 9. Production of L- tryptophan by E. coli strain SVl 64 (pGH5VΔbisC.

To test the effect of inactivation of the bisC gene on L-tryptophan production, DNA fragments from the chromosome of the above-described E. coli MGl 655 ΔbisC::cat can be transferred to tryptophan-producing E. coli strain SVl 64 (pGH5) by Pl transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain SV164(ρGH5)-ΔbisC. The strain SV164 has the trpE allele encoding anthranilate synthase which is 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) is described in detail in U.S. patent No. 6,180,373 or European patent 0662143.

Both strains, SV164(ρGH5)-ΔbisC and SV164(pGH5), can be cultivated with shaking at 37 °C for 18 hours in a 3 ml of nutrient broth supplemented with 20 mg/ml of tetracycline (marker of pGH5 plasmid). 0.3 ml of the obtained cultures can each be inoculated into 3 ml of a fermentation medium containing tetracycline (20 mg/1) in 20 x 200 mm test tubes, and cultivated at 37 ⁰C for 48 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 8. The fermentation medium components are set forth in Table 2, but should be sterilized in separate groups A, B, C, D, E, F, and H, as shown, to avoid adverse interactions during sterilization.

Table 2

Solution A had pH 7.1 adjusted by NH₄OH.

Example 10. Production of L-proline by E. coli strain 702ilvA-ΔbisC.

To test the effect of inactivation of the bisC gene on L-proline production, DNA fragments from the chromosome of the above-described E. coli MG1655 ΔbisCr.cat can be transferred to the proline-producing E. coli strain 702ilvA by Pl transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain 702ilvA-ΔbisC. 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 and 702ilvA-ΔbisC 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 7.

Example 11. Production of L-arginine by E. coli strain 382-ΔbisC.

To test the effect of inactivation of the bisC gene on L-arginine production, DNA fragments from the chromosome of the above-described E. coli MGl 655 ΔbisCr.cat were transferred to the arginine-producing E. coli strain 382 by Pl transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain 382-ΔbisC. The strain 382 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on April 10, 2000 under accession number VKPM B-7926 and then converted to a deposit under the Budapest Treaty on May 18, 2001.

Both strains, 382-ΔbisC and 382, were separately cultivated with shaking at 37 ⁰C for 18 hours in a 3 ml of nutrient broth. 0.3 ml of the obtained cultures can each be inoculated into 3 ml of a fermentation medium in 20 x 200 mm test tubes, and cultivated at 32 °C for 48 hours on a rotary shaker.

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

The results of 9 independent test tube fermentations are shown in Table 3.

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

Glucose 48.0

(NH4)₂SO₄ 35.0

KH₂PO₄ 2.0

MgSO₄-7H₂O 1.0

Thiamine HCl 0.0002

Yeast extract 1.0

L-isoleucine 0.1 CaCO₃ 5.0

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

Table 3

It can be seen from Table 3 that strain 382-ΔbisC caused a slightly higher amount of accumulation of L-arginine as compared with 382.

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

Industrial Applicability

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

Claims

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

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

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

Escherichia.

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

Pantoea.

5. The L-amino acid producing bacterium according to any of claims 1 to 4, wherein said

L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

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

7. The L-amino acid producing bacterium according to claim 5, wherein said non-aromatic

L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L- alanine, L-asparagine, L-aspartate, L-glutamine, L-glutamic acid, L-proline, and L- arginine.

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

- cultivating the bacterium according to any of claims 1 to 7 in a medium to produce and excrete said L-amino acid into the medium, and

- collecting said L-amino acid from the medium.

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

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

11. The method according to claim 9, wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L- leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartate, L-glutamine, L-glutamic acid, L-proline, and L-arginine.