WO2007013638A1

WO2007013638A1 - A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE ENTEROBACTERIACEAE FAMILY WITH ATTENUATED EXPRESSION OF THE pnp GENE

Info

Publication number: WO2007013638A1
Application number: PCT/JP2006/315079
Authority: WO
Inventors: Konstantin Vyacheslavovich Rybak; Aleksandra Yurievna Skorokhodova; Elvira Borisovna Voroshilova
Original assignee: Ajinomoto Co., Inc.
Priority date: 2005-07-25
Filing date: 2006-07-24
Publication date: 2007-02-01

Abstract

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

Description

DESCRIPTION

A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE ENTEROBACTERIACEAE FAMILY WITH ATTENUATED EXPRESSION OF THE /røj? 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 Enterohacteriaceaβ family which has been modified to attenuate expression of the pnp gene.

Background Art

Polynucleotide phosphorylase (PNPase), a 3' to 5' exonuclease encoded by ihepnp gene, plays a key role in Escherichia coli RNA decay. The enzyme, made of three identical 71 l-amino acid subunits, may also be assembled in the RNA degradosome, a heteromultimeric complex involved in RNA degradation.

PNPase autogenously regulates its expression by promoting the decay of pnp mRNA, by binding at the 5'-untranslated leader region of an RNase Ill-processed form of this transcript. The KH and Sl RNA-binding domains at the C terminus of the protein (amino acids 552-711) are involved in pnp mRNA recognition (Regonesi, M.E. et al. A mutation in polynucleotide phosphorylase from Escherichia coli impairing RNA binding and degradosome stability. (Nucleic Acids Res., 2004, 32(3): 1006-1017).

It has been shown that simultaneous inactivation of PNPase and RNase II (both are 3' to 5' exonucleases) in Escherichia coli leads to the loss of cell viability and the accumulation of partially degraded mRNA species. In exponentially growing cells, inactivation of PNPase leads to an increase in the steady-state level of more expressed mRNAs (17.3%) than inactivation of RNase II (7.3%) (Mohanly, B.K. and Kushner, S.R. Genomic analysis in Escherichia coli demonstrates differential roles for polynucleotide phosphorylase and RNase II in mRNA abundance and decay. MoI. Microbiol., 2003, 50(2):645-658). It has also been shown that transiently increasing intracellular poly(A) levels stabilize the pnp transcripts, leading to increased PNPase level. The half-live of the pnp transcripts is dependent on the intracellular level of polyadenylated transcripts in Escherichia coli (Mohanly, B. K. and Kushner, S.R. Polyadenylation of Escherichia coli transcripts plays an integral role in regulating intracellular levels of polynucleotide phosphorylase and RNase E. MoL Microbiol, 2002, 45(5): 1315-1324).

The PNPase synthesis is autocontrolled at a post-transcriptional level in an RNase Ill-dependent mechanism. RNase III cleaves a long stem-loop in the pnp leader, which triggers pnp mRNA instability, resulting in a decrease in the synthesis of polynucleotide phosphorylase (Jarrige, A.C., Mathy, N., and Portier, C. PNPase autocontrols its expression by degrading a double-stranded structure in the pnp mRNA leader. EMBO J., 2001, 20(23):6845-6855).

Expression of the pnp gene is increased at low temperatures. InE. coli cells grown at 18^aC, the amount of PNPase is twice of that found in cells grown at 30^sC (Mathy, N., Jarrige, A.C., Robert-Le Meur, M., and Portier, C. Increased expression of Escherichia coli polynucleotide phosphorylase at low temperatures is linked to a decrease in the efficiency of autocontrol. J. Bacterid., 2001, 183(13):3848-3854). It has been shown that PNPase plays a critical role in cold shock adaptation by repressing cold shock protein production at the end of the acclimation phase (Yamanaka, K. and Inouye, M. Selective mRNA degradation by polynucleotide phosphorylase in cold shock adaptation in Escherichia coli. J. BacterioL, 2001, 183(9):2808-2816).

But currently, there have been no reports of inactivating the pnp gene for the purpose of producing L-amino acids.

Disclosure of the Invention

Objects of the 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 pnp gene can enhance 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 ihtpnp gene.

It is a further object of the present invention to provide the bacterium as described above, wherein the expression of th&pnp gene is attenuated by inactivation of ύxzpnp 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-aspartic acid, 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-aspartic acid, L-glutamine, L- glutamic acid, L-proline, and L-arginine.

The present invention is described in detail below.

Detailed Description of the Preferred Embodiments 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 pnp gene.

In the present invention, "L-amino acid-producing bacterium" means a bacterium which has an ability to produce and excrete an L-amino acid into a medium, when the bacterium is cultured in the medium.

The term "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 by 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 acid" 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-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. 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, Enterobactβr, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella , Morganella Yersinia, etc. Specifically, those classified into the Enterobacteriaceae 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 into 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, e.g., 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 into 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. Bacterid., 43, 162-173 (1993)).

The phrase "bacterium has been modified to attenuate expression of the pnp gene" means that the bacterium has been modified in such a way that the modified bacterium contains a reduced amount of the PNPase protein, as compared with an unmodified bacterium, or the modified bacterium is unable to synthesize the PNPase protein. The phrase "bacterium has been modified to attenuate expression of the pnp gene" also means that the target gene is modified in such a way that the modified gene encodes a mutant PNPase protein which has a decreased activity.

The phrase "inactivation of the pnp 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 pnp gene encodes the PNPase protein, polynucleotide phosphorylase (synonyms - B3164, BfI). Thepnp gene (nucleotide positions 3,309,259 to 3,307,055; GenBank accession no. NC_000913.2; gi:49175990; SEQ ID NO: 1) is located between the nlpl and rpsO genes on the chromosome of E. coli K-12. The nucleotide sequence of the pnp gene and the amino acid sequence of PNPase encoded by the pnp gene are shown in SΕQ ID NO: 1 and SΕQ ID NO: 2, respectively.

Since there may be some differences in DNA sequences between the genera or strains of the Enterobacteriaceae family, the pnp gene to be inactivated on the chromosome is not limited to the gene shown in SΕQ ID No: 1, but may include genes homologous to SΕQ ID No: 1 encoding a variant protein of the PNPase 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 PNPase 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 SΕQ ID NO: 2. These changes in the variants 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. These changes in the variant protein can occur in regions of the protein which are not critical for the function of the protein. Therefore, the protein variant encoded by the pnp 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 SΕQ ID NO. 2, as long as the ability of PNPase to degrade RNA prior to inactivation is maintained.

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.

Moreover, the pnp gene may be a variant which hybridizes under stringent conditions with the nucleotide sequence shown in SΕQ ID NO: 1, or a probe which can be prepared from the nucleotide sequence, provided that it encodes a functional PNPase protein prior to inactivation. "Stringent conditions" include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, preferably not less than 70%, more preferably not less than 80%, 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 or more, preferably two or three times, at a salt concentration of 1 X SSC, 0.1% SDS, preferably 0.1 X SSC, 0.1% SDS at 60^sC. Duration of washing depends on the type of membrane used for blotting and, as a rule, may 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 usually varies from 100 bp to 1 kbp.

Expression of the pnp gene can be attenuated by introducing a mutation into the gene on the chromosome so that intracellular activity of the protein encoded by the gene is decreased as compared with an unmodified strain. Such a mutation on the gene can be replacement of one base or more to cause amino acid substitution in the protein encoded by the gene (missense mutation), introduction of a stop codon (nonsense mutation), deletion of one or two bases to cause a frame shift, insertion of a drug-resistance gene, or deletion of a part 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)). Expression of the pnp gene can also be attenuated by modifying an expression regulating sequence such as the promoter, the Shine-Dalgarno (SD) sequence, etc. (WO95/34672, Carrier, T.A. and Keasling, J.D., Biotechnol Prog 15, 58-64 (1999)).

For example, the following methods may be employed to introduce a mutation by gene recombination. A mutant gene encoding a mutant protein having a decreased activity is prepared, and a bacterium to be modified is transformed with a DNA fragment containing the mutant gene. Then the native gene on the chromosome is replaced with the mutant gene by homologous recombination, and the resulting strain is selected. Such gene replacement using homologous recombination can be conducted by the method 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)), or by methods employing a plasmid containing a temperature-sensitive replication (U.S. Patent No. 6,303,383 or JP 05-007491A). 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. In a case where a marker gene such as antibiotic resistant gene is used for preparing the mutant gene or detecting recombination between the mutant gene and the native gene on the chromosome, the marker gene can be eliminated from the chromosome by, for example, a method described in Examples section.

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 of activity of the PNPase protein can be detected by complementation of mutation pnp^' by the method described, for example, in Fontanella, L. et al. Photometric assay for polynucleotide phosphorylase. Anal. Biochem., 1999, 269(2):353-358. Thus, the reduced or absent activity of the PNPase protein in the bacterium according to the present invention can be determined when compared to the parent unmodified bacterium.

The presence or absence of the pnp 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 of mRNA transcribed from the gene using various well-known methods, including Northern blotting, quantitative RT-PCR, and the like. Amount or molecular weight of the protein encoded by the gene can be measured by well-known methods, including SDS-PAGE followed by immunoblotting assay (Western blotting 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 pnp gene, bacteria which are able to produce either an aromatic or a non-aromatic L- amino acid may be used. The bacterium of the present invention can be obtained by attenuating expression of thepnp gene in a bacterium which inherently has the ability to produce an L-amino acid. 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 pnp 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 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) (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-threoninerproducing 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 yaaX open 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 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 Ε. 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 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 17th 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 al., 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 WC196 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 AJ13069 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 (lysA), diaminopimelate dehydrogenase (ddh) (U.S. Patent No. 6,040,160), phosphoenolpyrvate carboxylase (ppc), aspartate semialdehyde dehydrogenease (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), theybjE 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 JM15 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 (JP11155571A2); 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-hydroxyleucine, 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); E. coli NRRL B-12116 - B12121 (U.S. Patent No. 4,388,405); E. coli H- 9342 (FΕRM BP-6675) and H-9343 (FΕRM BP-6676) (U.S. Patent No. 6,344,347); E. coli H-9341 (FΕRM BP-6674) (ΕP1085087); E. coli AI80/pFM201 (U.S. 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 (JiisIE), phosphoribosylformimino-5- aminoimidazole carboxamide ribotide isomerase (hisA), amidotransferase (hisH), histidinol phosphate aminotransferase QiisC), histidinol phosphatase QiisB), histidinol dehydrogenase QiisD), and so forth.

It is known that L-histidine biosynthetic enzymes 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 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), which is able to produce L-glutamic acid, was obtained.

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, acriB), citrate synthase (gltA), phosphoenolpyruvate carboxylase (ppc), pyruvate dehydrogenase (aceEF,aceEF, ipdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsAppsA), enolase {end), phosphoglyceromutase (pgmA, pgml), phosphoglycerate kinase (pgk), glyceraldehyde-3-phophate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), 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 acidby branching off from an L-glutamic acid biosynthesis pathway. Examples of such genes include genes encoding isocitrate lyase (aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (HvG), acetolactate synthase (HvI), 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 W3Ϊ10sucA::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,^' £. 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 AJ13356 is deficient in the α-ketoglutarate dehydrogenase activity as a result of disruption of the αKGDH-El subunit gene (sucA). The above strain was identified as Enterobacter agglomerans when it was isolated and deposited as the Enterobacter agglomerans AJ13356. 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 AJ12739 (tyrA::Tnl0, tyrR) (VKPM B-8197); E. coli HW1089 (ATCC 55371) harboring the muiantpheA34 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 (FΕRM 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) which is deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Patent No. 5,756,345); E. coli SV164 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine and a trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan (U.S. Patent No. 6,180,373); E. coli AGXIl (pGX44) (NRRL B- 12263) and AGX6(pGX50)aroP (NRRL B-12264) which is deficient in the enzyme tryptophanase (U.S. Patent No. 4,371,614); E. coli AGX17/pGX50,pACKG4-pps in which a phosphoenolpyruvate-producing ability is enhanced (WO9708333, U.S. Patent No. 6,319,696), and the like may be used. L- tryptophan-producing bacteria belonging to the genus Escherichia with an enhanced activity of the 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).

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 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 (trpBA). The tryptophan synthase consists of α and β subunits which are encoded by the trpA and trpB genes, respectively. In addition, L-tryptophan-producing ability may be improved by enhancing expression of the isocitrate lyase-malate synthase operon (WO2005/103275).

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, p.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) (EP1170358A1), an arginine-producing strain into which argA gene encoding N-acetylglutamate synthetase is introduced therein (EP1170361A1), 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 (carAB).

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 HvGMEDA 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 HvA 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 ileS gene encoding isoleucine tRNA synthetase, can be used. E. coli VL1970 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. Brief Description of Drawings

Figure 1 shows the construction of the pMW118-attL-Cm-attR plasmid used as a template for PCR.

Figure 2 shows the relative positions of primers P17 and Pl 8 on plasmid pMW118- attl_^Cm-attR used for PCR amplification of the cat gene.

Figure 3 shows the construction of the chromosomal DNA fragment comprising the inactivated pnp gene.

Examples

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

Example 1. Preparation of the PCR template and helper plasmids

The PCR template plasmid pMW118-attL-Cm-attR and the helper plasmid pMW- intxis-ts were prepared as follows: (1) pMW118-attL-Cm-attR

The pMW118-attL-Cm-attR plasmid was constructed on the basis of pMW118- attL-Tc-attR that was obtained by ligation of the following four DNA fragments:

1) the Bglϊl-EcoRl fragment (114 bp) carrying αttL (SEQ ID NO: 3) which was obtained by PCR amplification of the corresponding region of the E. coli W3350 (contained λ prophage) chromosome using oligonucleotides Pl and P2 (SΕQ ID NOS: 4 and 5) as primers (these primers contained the subsidiary recognition sites for BgIU and EcoRl endonucleases);

2) the Pstl-HindlU fragment (182 bp) carrying attR (SΕQ ID NO: 6) which was obtained by PCR amplification of the corresponding region of the E. coli W3350 (contained λ prophage) chromosome using the oligonucleotides P3 and P4 (SΕQ ID NOS: 7 and 8) as primers (these primers contained the subsidiary recognition sites for Pstl and Hindϊll endonucleases);

3) the large Bglll-Hindlll fragment (3916 bp) of pMW118-ter_mtB. The plasmid pMW118-ter_rm5 was obtained by ligation of the following three DNA fragments: • the large DNA fragment (2359 bp) carrying theylαtII-Ecøi?I fragment of pMWllδ that was obtained by the following way: pMW118 was digested with EcoRl restriction endonuclease, treated with Klenow fragment of DNA polymerase I, and then digested with^latll restriction endonuclease;

• the small Aatϊl-Bglϊϊ fragment (1194 bp) of pUC19 carrying the bla gene for ampicillin resistance (Ap^R) was obtained by PCR amplification of the corresponding region of the pUC19 plasmid using oligonucleotides P5 and P6 (SΕQ ID NOS: 9 and 10) as primers (these primers contained the subsidiary recognition sites fory4αtll and5g/II endonucleases);

• the small Bglll-Pstl fragment (363 bp) of the transcription terminator ter jrrnB was obtained by PCR amplification of the corresponding region of the E. coli MGl 655 chromosome using oligonucleotides P7 and P8 (SΕQ ID NOS: 11 and 12) as primers (these primers contained the subsidiary recognition sites for

B gill and Pstl endonucleases);

4) the small Ecoi?I-PstI fragment (1388 bp) (SΕQ ID NO: 13) of pML-Tc-ter_t/*rL bearing the tetracycline resistance gene and the ttτJhrL transcription terminator; the pML-Tc-ter_t/zrL plasmid was obtained in two steps:

• the pML-ter_t&rL plasmid was obtained by digesting the pML-MCS plasmid (Mashko, S.V. et al, Biotekhnologiya (in Russian), 2001, no. 5, 3-20) with the Xbal and BamHl restriction endonucleases, followed by ligation of the large fragment (3342 bp) with the Xbal-BamHl fragment (68 bp) carrying terminator t&τ_thrL obtained by PCR amplification of the corresponding region of the E. coli MG1655 chromosome using oligonucleotides P9 and PlO (SΕQ ID NOS: 14 and 15) as primers (these primers contained the subsidiary recognition sites for theZbαl andifømHl endonucleases);

• the pML-Tc-ter_t/irL plasmid was obtained by digesting the pML-ter_t/jrL plasmid with the Kpnl andZfoαl restriction endonucleases followed by treatment with Klenow fragment of DNA polymerase I and ligation with the small EcoRl-Van91l fragment (1317 bp) of pBR322 bearing the tetracycline resistance gene (pBR322 was digested with EcoRl and Van91l restriction endonucleases and then treated with Klenow fragment of DNA polymerase I); The above strain E. coli W3350 is a derivative of wild type strain E. coli K-12. The strain E. coli MG1655 (ATCC 700926) is a wild type strain and can be obtained from American Type Culture Collection (P.O. Box 1549 Manassas, VA 20108, United States of America). The plasmid pMW118 and pUC19 are commercially available. The BgHl- EcoRI fragment carrying attL and the Bglϊϊ-Pstϊ fragment of the transcription terminator ter jrrnB can be obtained from the other strain of E. coli in the same manner as describe above.

The pMW118-attL-Cm-attR plasmid was constructed by ligation of the large BamHl-Xbal fragment (4413 bp) of pMW118-attL-Tc-attR and the artificial DNA Bglϊl- Xbal fragment (1162 bp) containing the P_A2 promoter (the early promoter of the phage T7), the cat gene for chloramphenicol resistance (Cm^R), the teτ_thrL transcription terminator, and attR. The artificial DNA fragment (SΕQ ID NO: 16) was obtained as follows:

1. The pML-MCS plasmid was digested with the Kpήl and Xbal restriction endonucleases and ligated with the small Kpnϊ-Xbal fragment (120 bp), which included the PA₂ promoter (the early promoter of phage T7) obtained by PCR amplification of the corresponding DNA region of phage T7 using oligonucleotides PIl and P12 (SΕQ ID NOS: 17 and 18, respectively) as primers (these primers contained the subsidiary recognition sites toxKpήl and Xbal endonucleases). As a result, the PML-P_A2-MCS plasmid was obtained. Complete nucleotide sequence of phage T7 has been reported (J. MoI. Biol, 166: 477-535 (1983).

2. The Xbal site was deleted from PML-P_A2-MCS. AS a result, the PML-P_A2- MCS(ZbflI^") plasmid was obtained.

3. The small Bglll-Hindϊll fragment (928 bp) of pML-P_A2-MCS(X&αr) containing the P_A2 promoter (the early promoter of the phage T7) and the cat gene for chloramphenicol resistance (Cm^R) was ligated with the small HindlH-Hindlϊl fragment (234 bp) of pMW118-attL-Tc-attR containing the teτJhrL transcription terminator and attR.

4. The required artificial DNA fragment (1156 bp) was obtained by PCR amplification, of the ligation reaction mixture using oligonucleotides P9 and P4 (SΕQ ID NOS: 14 and 8) as primers (these primers contained the subsidiary recognition sites for HmdIII and Xbal endonucleases). .

(2) pMW-intxis-ts Recombinant plasmid pMW-intxis-ts containing the cl repressor gene and the int- xis genes of phage λ under control of promoter P_R was constructed on the basis of vector pMWP_laclacI-ts. To construct the pMWP_laclacI-ts variant, the AatU-EcoRV fragment of the pMWP_laclacI plasmid (Skorokhodova, A. Yu. et al., Biotekhnologiya (in Russian), 2004, no. 5, 3-21) was substituted with th&Aatll-EcoRV fragment of the pMAN997 plasmid (Tanaka, K. et al., J. Bacterid., 2001, 183(22): 6538-6542, WO99/03988) bearing the par and ori loci and the repA^ts gene (a temperature sensitive-replication origin) of the pSClOl replicon. The plasmid pMAN997 was constructed by exchanging the Vspl-Hindlll fragments of pMAMBl (J. Bacteriol., 162, 1196 (1985)) and pUC19.

Two DNA fragments were amplified using phage λ DNA ("Fermentas") as a template. The first one contained the DNA sequence from 37168 to 38046, the cl repressor gene, promoters P_RM and P_R, and the leader sequence of the cro gene. This fragment was PCR-amplified using oligonucleotides P13 and P14 (SEQ ID NOS: 19 and 20) as primers. The second DNA fragment containing the xis-int genes of phage λ and the DNA sequence from 27801 to 29100 was PCR-amplified using oligonucleotides P15 and P16 (SEQ ID NOS: 21 and 22) as primers. All primers contained the corresponding restriction sites.

The first PCR-amplified fragment carrying the cl repressor was digested with restriction endonuclease CIaI, treated with Klenow fragment of DNA polymerase I, and then digested with restriction endonuclease Eco RI. The second PCR-amplified fragment was digested with restriction endonucleases Eco RI and Pstl. The pMWP_laclacI-ts plasmid was digested with the BgIlI endonuclease, treated with Klenow fragment of DNA polymerase I, and digested with the Pstl restriction endonuclease. The vector fragment of pMWPlaclacI-ts was eluted from agarose gel and ligated with the above-mentioned digested PCR-amplified fragments to obtain the pMW-intxis-ts recombinant plasmid.

Example 2. Construction of a strain with the inactivated pnp gene .1. Deletion of the pnp gene

A strain having deletion of the pnp 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". The DNA fragment containing the Cm^R marker encoded by the cat gene was obtained by PCR, using primers P17 (SEQ ID NO: 23) and P18 (SEQ ID NO: 24) and plasmid pMW118-attL-Cm-attR as a template (for construction see Example 1). Primer P17 contains both a region complementary to the 36- nt region located at the 5' end of the pnp gene and a region complementary to the attL region. Primer P18 contains both a region complementary to the 35-nt region located at the 3' end of the pnp gene and a region complementary to the attR region. Conditions for PCR were as follows: denaturation step: 3 min at 95⁰C; profile for two first cycles: 1 min at 95°C, 30 sec at 50⁰C, 40 sec at 72°C; profile for the last 25 cycles: 30 sec at 95°C, 30 sec at 54°C, 40 sec at 72°C; final step: 5 min at 72°C.

A 1699-bp PCR product (Fig. 2) was obtained and purified in agarose gel and was used for electroporation of E. coli MGl 655 (ATCC 700926), which contains the pKD46 plasmid having temperature-sensitive replication. The pKD46 plasmid (Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645) includes a 2,154- bp DNA fragment of phage λ (nucleotide positions 31088 to 33241, 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_araB promoter. The plasmid pKD46 is necessary for integration of the PCR product into the chromosome of strain MG1655. The strain MG1655 can be obtained from American Type Culture Collection. (P.O. Box 1549 Manassas, VA 20108, U.S.A.).

Εlectrocompetent cells were prepared as follows: E. coli MG1655 was grown at 30⁰C in LB medium containing ampicillin (100 mg/1), and the culture was 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 cells were grown with aeration at 30⁰C to an OD_60O of «0.6 and then were made electrocompetent by 100-fold concentrating and washing three times with ice-cold deionized H₂O. Electroporation was performed using 70 μl of cells and «100 ng of the 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 37⁰C for 2.5 hours and then were plated onto L-agar containing chloramphenicol (30 μg/ml) and were grown at 37°C to select Cm^R recombinants. Then, to eliminate the pKD46 plasmid, two passages on L-agar with Cm at 42°C were performed and the obtained colonies were tested for sensitivity to ampicillin.

2. Verification of the pnp gene deletion by PCR The mutants having the pnp gene deleted and marked with the Cm resistance gene were verified by PCR. Locus-specific primers P19 (SEQ ID NO: 25) and P20 (SEQ ID NO: 26) 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 72⁰C. The PCR product obtained in the reaction with the parental pnp⁺ MG1655 strain as a template was 2301 bp in length. The PCR product obtained in the reaction with the mutant strain as the template was 1796 bp in length (Fig.3). The mutant strain was named MG1655 Δpnp::cat.

Example 3. Production of L-threonine by E. coli B-3996-Δpnp

To test the effect of inactivation of the pnp gene on threonine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 Δpnp::cat were transferred to the threonine-producing E. coli strain VKPM B-3996 by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain B-3996-Δpnp.

Both E. coli strains, B-3996 and B-3996-Δpnp, were grown for 18-24 hours at 37°C on L-agar plates. To obtain a seed culture, the strains were grown on a rotary shaker (250 rpm) at 32⁰C for 18 hours in 20x200-mm test tubes containing 2 ml of L-broth supplemented with 4% glucose. 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 65 hours at 32°C 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 of ninhydrin (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 of ten independent test tube fermentations are shown in Table 1.

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

Glucose 80.0

(NH₄)₂SO₄ 22.0 NaCl 0.8 KH₂PO₄ 2.0 MgSO₄-7H₂O 0.8 FeSO₄ TH₂O 0.02 MnSO₄-5H₂O 0.02 Thiamine HCl 0.0002 Yeast extract 1.0 CaCO₃ 30.0

Glucose and magnesium sulfate were sterilized separately. CaCO₃ was sterilized by dry-heat at 180⁰C for 2 hours. The pH was adjusted to 7.0. The antibiotic was introduced into the medium after sterilization.

Table 1

As follows from Table 1, B-3996-Δpnp caused accumulation of a higher amount of L-threonine, as compared with B-3996.

Example 4. Production of L-lysine by E. coli WC196(pCABD2)-Δpnp

To test the effect of inactivation of thepnp gene on lysine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 Δpnp::cat can be transferred to the lysine-producing E. coli strain WC196 (pCABD2) by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain WC196(pCABD2)-Δpnp. The pCABD2 plasmid includes the dapA gene encoding dihydrodipicolinate synthase having a mutation which desensitizes feedback inhibition by L-lysine, the lysC gene encoding aspartokinase III having a mutation which desensitizes feedback inhibition by L-lysine, the dapB gene encoding dihydrodipicolinate reductase, and the ddh gene encoding diaminopimelate dehydrogenase (U.S. Patent No. 6,040,160). Both E. coli strains, WC196(pCABD2) and WC196(pCABD2)-Δpnp can be cultured in L-medium containing streptomycin (20 mg/1) at 37°C, 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°C 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 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 SH₂O 0.01

Yeast extract 2.0

The 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. 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. Production of L-cysteine by E. coli JM15(ydeD)-Δpnp

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

E. coli JM15(ydeD) is a derivative of E. coli JM15 (U.S. 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 (U.S. Patent No. 5,972,663). The strain JM15 (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 U.S. Patent No. 6,218,168.

Example 6. Production of L-leucine by E. coli 57-Δpnp

To test the effect of inactivation of the pnp gene on L-leucine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 Δpnp::cat can be transferred to the L-leucine-producing E. coli strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121) by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain 57- Δpnp. 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 accession number VKPM B-7386.

Both E. coli strains, 57 and 57-Δpnp, can be cultured for 18-24 hours at 37⁰C 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

(NH₄)₂SO₄ 25.0

K₂HPO₄ 2.0

MgSO₄-7H₂O 1.0

Thiamine 0.01

CaCO₃ 25.0

Glucose and CaCO₃ are sterilized separately.

Example 7. Production of L-histidine by E. coli 80-Δpnp

To test the effect of inactivation of the pnp gene on L-histidine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 Δpnp::cat can be transferred to the histidine-producing E. coli strain 80 by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain 80-Δpnp. 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 July 12, 2004.

Both E. coli strains, 80 and 80-Δpnp, can be cultured in L-broth for 6 hours at 29°C. Then, 0.1 ml of obtained cultures can each be inoculated into 2 ml of fermentation medium in a 20x200-mm test tube and cultivated for 65 hours at 29° C 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) (pH 6.0) is as follows:

Glucose 100.0

Mameno (soybean hydrolysate) 0.2 as total nitrogen

L-proline 1.0

(NH₄)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. Production of L-glutamate by E. coli VL334thrC⁺-Δpnp

To test the effect of inactivation of the pnp gene on L-glutamate production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 Δpnp::cat can be transferred to the L-glutamate-producing E. coli strain VL334thrC⁺ (EP 1172433) by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain VL334thrC⁺-Δpnp. 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 E. coli strains, VL334thrC⁺ and VL334thrC⁺-Δpnp, can be grown for 18-24 hours at 37°C 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/1), KH₂PO₄ (2g/l), MgSO₄ (1. g/1), 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 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 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. Production of L- phenylalanine by E. coli AJ12739-Δpnp

To test the effect of inactivation of the pnp gene on L-phenylalanine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 Δpnp::cat can be transferred to the phenylalanine-producing E. coli strain AJ12739 by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain AJ12739-Δpnp. The strain AJ12739 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 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 E. coli strains, AJ12739-Δpnp and AJ12739, can be cultivated at 37⁰C for 18 hours in a nutrient broth, and 0.3 ml of the obtained cultures can each be inoculated into 3 ml of a fermentation medium in a 20x200-mm test tube and cultivated at 37⁰C 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

(NH₄)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 180⁰C for 2 hours. The pH is adjusted to 7.0.

Example 10. Production of L- tryptophan by E. coli SV164 (pGH5)-Δpnp

To test the effect of inactivation of the^np gene on L- tryptophan production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 Δpnp::cat can be transferred to the tryptophan-producing E. coli strain SVl 64 (pGH5) by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain SV164(pGH5)-Δpnp. 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 SV164 (pGH5) is described in detail in U.S. Patent No. 6,180,373.

Both E. coli strains, SV164(pGH5)-Δpnp and SV164(pGH5), can be cultivated with shaking at 37°C for 18 hours in 3 ml of nutrient broth supplemented with tetracycline (20 mg/1, marker of pGH5 plasmid). The obtained cultures (0.3 ml each) 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 37⁰C 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 listed in Table 2, and are sterilized in separate groups (A, B, C, D, E, F, and H), as shown, to avoid adverse interactions during sterilization.

Table 2

Group A has pH 7.1 adjusted by NH₄OH. Each of groups A, B, C, D, E, F and H is sterilized separately, chilled, and mixed together, and then CaCO₃ sterilized by dry heat is added to the complete fermentation medium.

Example 11. Production of L-proline by E. coli 702ilvA-Δpnp

To test the effect of inactivation of the pnp gene on L-proline production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 Δpnp::cat can be transferred to the proline-producing E. coli strain 702ilvA by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain 702ilvA-Δpnp. The strain 702ilvA has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 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-Δpnp, 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. Production of L-arginine by E. coli 382-Δpnp

To test the effect of inactivation of the pnp gene on L-arginine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 Δpnp::cat can be transferred to the arginine-producing E. coli strain 382 by Pl transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain 382-Δpnp. 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 E. coli strains, 382-Δpnp and 382, can be cultivated with shaking at 37°C for 18 hours in 3 ml of nutrient broth. The obtained cultures (0.3 ml each) can each be inoculated into 3 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 accumulates 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-argiήine can be estimated . spectrophotometrically at 540 nm.

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 are sterilized separately. CaCO₃ is dry-heat sterilized at 180 ⁰C for 2 hours. The pH is adjusted to 7.0.

Example 13. Elimination of the Cm resistance gene (cat gene) from the chromosome of L- amino acid-producing E. coli strains.

The Cm resistance gene {cat gene) can be eliminated from the chromosome of the L- amino acid-producing strain using the int-xis system. For that purpose, an L-amino acid- producing strain having DNA fragments from the chromosome of the above-described E. coli strain MG1655 Δpnp::cat transferred by Pl transduction (see Examples 3-12), can be transformed with plasmid pMWts-Int/Xis. Transformant clones can be selected on the LB- medium containing 100 μg/ml of ampicillin. Plates can be incubated overnight at 30⁰C. Transformant clones can be cured from the cat gene by spreading the separate colonies at 37°C (at that temperature repressor Cits is partially inactivated and transcription of the int/xis genes is derepressed) followed by selection of Cm^sAp^R variants. Elimination of the cat gene from the chromosome of the strain can be verified by PCR. Locus-specific primers P21 (SEQ ID NO: 27) and P22 (SEQ ID NO: 28) can be used in PCR for the verification. Conditions for PCR verification can be as described above. The PCR product obtained in reaction with cells having the eliminated cat gene as a template, should be 0.2 kbp in length. Thus, the L-amino acid-producing strain with the inactivated pnp gene and eliminated cat gene can be obtained.

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 pnp gene.

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

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

Escherichia.

4. The bacterium according to claim 1, wherein said bacterium belongs to 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-aspartic acid, 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-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine.