RU2468083C1 - Method of obtaining l-amino acid with application of bacterium of enterobacteriaceae family, with weakened expression of genes, coding lysine/arginine/ornithine transporter - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: claimed is bacterium-producent of diaminomonocarboxylic L-amino acid, selected from L-lysine, L-arginine, L-ornithine and L-citrulline, belonging to Enterobacteriaceae family, modified in such a way that in said bacterium weakened is expression of one or several genes, coding lysine/arginine/ornithine transporter and one or several genes, coding histidine transporter, represented by cluster argT-hisJQMP. Claimed is method of obtaining diaminomonocarboxylic L-amino acid, which includes: growing of said bacterium in nutritional medium, inducing production and secretion of said L-amino acid into culture liquid and isolation of said L-amino acid from culture liquid.

EFFECT: invention makes it possible to obtain said diaminomonocarboxylic L-amino acid in larger volume.

9 cl, 2 tbl, 5 ex

Description

Technical field

The invention relates to the microbiological industry, in particular to a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, modified in such a way that the expression of genes encoding the lysine / arginine / ornithine transporter is reduced in said bacterium.

Description of the Related Art

Traditionally, L-amino acids on an industrial scale can be obtained by fermentation using strains of microorganisms obtained from natural sources, or their mutants. Typically, microorganisms are modified in order to increase the production of L-amino acids.

Many methods have been described to increase the production of L-amino acids, for example, by transforming a microorganism of recombinant DNA (see, for example, US patent 4,278,765). Other methods are based on increasing the activity of enzymes involved in the biosynthesis of amino acids, and / or reducing the sensitivity of the target enzyme to feedback inhibition produced by the L-amino acid (see, for example, US patents 4,346,170; 5,661,012 and 6,040,160).

Other methods for increasing the production of L-amino acids are to weaken the expression of one or more genes involved in the degradation of the target L-amino acid; genes whose expression leads to the distraction of the precursors of the target amino acid from the pathway of biosynthesis of L-amino acids; genes involved in the redistribution of carbon, nitrogen and phosphorus streams; genes encoding toxins, etc.

The Salmonella typhimurium hisJ and argT genes encode two periplasmic binding proteins, J and LAO, involved in histidine and arginine transport, respectively, and interacting with a common membrane-bound component, protein P. The complete nucleotide sequence of these two genes has been determined. Genes are highly homologous (70%) and are thought to have arisen as a result of tandem duplication of a single gene. Two proteins encoded by these genes perform different functions, but retain a degree of homology sufficient to interact with one site of the membrane-bound protein P (Higgins CF, Ames GF, Proc Natl Acad Sci USA .; 78 (10): 6038-42 (1981)) .

The superfamily of transport ATPases (ABC transporters) includes bacterial periplasmic transport systems (permeases) and various eukaryotic transporters. The histidine permease of S. typhimurium and Escherichia coli consists of a soluble receptor, including a membrane-bound complex containing four subunits, a substrate-binding protein, and receives energy from ATP. It was shown that histidine transport is completely dependent on ATP and HisJ ligand; it is affected by pH, temperature, and salt concentration. Transport is irreversible, and accumulation reaches a plateau when the transport stops. Permease inhibit ADP and high concentrations of histidine inside the cell. Histidine inhibition probably indicates that the membrane-bound HisQ / M / P complex includes a substrate-binding site. The activity of reconstructed permease corresponds to a level of 40-70% of the in vivo transport rate (Liu C.E., Ames G.F., J Biol Chem .; 272 (2): 859-66 (1997)).

The membrane-bound complex of Salmonella typhimurium periplasmic histidine permease consists of two integral membrane proteins, HisQ and HisM, and two copies of the ATP-binding subunit, HisP. The complex hydrolyzes ATP by inducing the activity of a soluble receptor and periplasmic histidine-binding protein, HisJ. It was shown that the nucleotide-binding component can be separated from the integral membrane components, HisQ and HisM. The complex can be reconstructed using HisP membranes with HisQ and HisM removed and pure soluble HisP. HisP has been shown to have high affinity for the complex with the deleted HisP, HisQM, and two HisP molecules are attached independently to each HisQM unit. The complex assembled in vitro has completely normal properties and reacts to HisJ and ATPase inhibitors in the same way as the original complex, as opposed to soluble HisP. These results show the absolute need of HisP for ATP hydrolysis, HisQM cannot hydrolyze ATP, HisP depends on HisQM in order to transmit a signal from HisJ soluble receptor, and HisQM regulates HisP ATPase activity. It was also shown that HisP conformation changes upon interaction with phospholipids (Liu P.Q., Ames G.F., Proc Natl Acad Sci USA; 95 (7): 3495-500 (1998)).

The E. coli ArgT protein may be in the form of a cytoplasmic precursor protein with a molecular weight of 28 kDa and in the form of a mature periplasmic protein with a molecular weight of 25.8 kDa. To study the role of proteolysis in the regulation of adaptation in the stationary phase of growth, the clpA, clpX and clpP mutants of Escherichia coli were studied using proteomic analysis during growth and carbon starvation. In the clpA, clpX, and clpP mutants, the periplasmic lysine-arginine-ornithine-binding protein ArgT did not show the induction typical of wild-type cells in the late stationary growth phase (Weichart D. et al., Journal of Bacteriology, vol. 185, No.1 p.115-125 (2003)).

There are currently no reports describing the use of attenuation of the expression of genes encoding the lysine / arginine / ornithine transporter to produce L-amino acids.

Description of the invention

The objectives of the present invention are to increase the productivity of strains producing L-amino acids and the provision of a method for producing L-amino acids using these strains.

It has been found that a decrease in the expression of the argT-hisJQMP cluster can lead to an increase in the production of diaminomonocarboxylic carotenes such as L-lysine, L-arginine, L-ornithine and L-citrulline.

The present invention provides a bacterium belonging to the Enterobacteriaceae family with increased ability to produce amino acids such as L-lysine, L-arginine, L-ornithine and L-citrulline.

An object of the present invention is to provide a bacterium belonging to the Enterobacteriaceae family, an L-amino acid producer, modified in such a way that the expression of one or more genes encoding the lysine / arginine / ornithine transporter is attenuated.

It is also an object of the present invention to provide the bacteria described above, characterized in that the argT-hisJQMP cluster includes genes encoding the lysine / arginine / ornithine transporter.

It is also an object of the present invention to provide a bacterium as described above, characterized in that said argT-hisJQMP cluster is inactivated.

It is also an object of the present invention to provide a bacterium as described above, characterized in that one or more genes of said argT-hisJQMP cluster is inactivated.

It is also an object of the present invention to provide a bacterium as described above, characterized in that said bacterium belongs to the genus Escherichia.

It is also an object of the present invention to provide a bacterium as described above, characterized in that said bacterium belongs to the genus Pantoea.

It is also an object of the present invention to provide a bacterium as described above, characterized in that said L-amino acid is diaminomonocarboxylic acid.

It is also an object of the present invention to provide a bacterium as described above, characterized in that said diaminomonocarboxylic acid is selected from the group consisting of L-lysine, L-arginine, ornithine and citrulline.

Another objective of the present invention is the provision of a method for producing L-amino acids, including:

- growing the bacteria described above in a nutrient medium, causing production and secretion of the specified L-amino acid into the culture fluid;

and

- the allocation of the specified L-amino acids from the culture fluid.

It is also an object of the present invention to provide the process described above, characterized in that said L-amino acid is diaminomonocarboxylic acid.

It is also an object of the present invention to provide the method described above, characterized in that said diaminomonocarboxylic acid is selected from the group consisting of L-lysine, L-arginine, ornithine and citrulline.

In more detail, the present invention is described below.

BEST MODE FOR CARRYING OUT THE INVENTION

1. The bacterium according to the present invention

A bacterium according to the present invention is an L-amino acid producing bacterium belonging to the Enterobacteriaceae family, capable of producing an L-amino acid, such as diaminomonocarboxylic acid, characterized in that the lysine / arginine / ornithine transporter in said bacterium is inactivated.

The phrase “L-amino acid producing bacterium” may mean a bacterium capable of producing and secreting an L-amino acid into a culture medium when the bacterium is grown in said culture medium.

The term “bacterium producing L-amino acids” can also mean a bacterium that is capable of producing and accumulating L-amino acids, such as diaminomonocarboxylic acid, in large amounts of fermentation medium compared to the natural or parent E. coli strain, such as strain E coli K-12, and may also mean that the bacterium is able to accumulate in the medium the target L-amino acid in an amount of not less than 0.5 g / l or not less than 1.0 g / l in another example.

The term “diaminomonocarboxylic acid” may include L-lysine, L-arginine, L-ornithine and L-citrulline. These amino acids have a positive charge due to the presence of an amino group.

The Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc. More specifically, bacteria classified as belonging to the Enterobacteriaceae family according to the taxonomy used in the NCBI database (National Center for Biotechnology Information) (http://www.ncbi.nlm.nih.gov/htbinpost/Taxonomy/ can be used. wgetorg? mode = Tree & id = 1236 & lvl = 3 & keep = l & srchmode = l & unlock). A bacterium belonging to the genus Escherichia or Pantoea is preferred.

The term "bacterium belonging to the genus Escherichia" means that the bacterium belongs to the genus Escherichia in accordance with the classification known to the person skilled in the field of microbiology. As an example of a microorganism belonging to the genus Escherichia used in the present invention, the bacterium Escherichia coli (E. coli) may be mentioned.

The range of bacteria belonging to the genus Escherichia that can be used in the present invention is not limited in any way, however, for example, the bacteria described in Neidhardt, F.C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1), can be included in the number of bacteria according to the present invention.

The term "bacterium belonging to the genus Pantoea" means that the bacterium belongs to the genus Pantoea in accordance with the classification known to the specialist in the field of microbiology. Recently, several Enterobacter agglomerans species have been classified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like based on analysis of the 16S rRNA nucleotide sequence, etc. (Int. J. Syst. Bacteriol., 43, 162-173 (1993)).

The term “bacterium is modified in such a way that expression of the argT-hisJQMP cluster is weakened” means that the bacterium is modified in such a way that, as a result of the modification, such a bacterium contains a reduced amount of ArgT, HisJ, HisQ, HisM and HisP proteins compared to an unmodified bacterium, or this may also mean that the modified bacterium is not able to synthesize ArgT, HisJ, HisQ, HisM and HisP.

The phrase "inactivation of the argT-hisJQMP cluster" means that the modified gene encodes a completely inactive protein. It is also possible that natural expression of the modified DNA region is not possible due to deletion of a portion of the gene, shift of the reading frame, insertion of the missense / nonsense mutation (s), or modification of regions adjacent to the gene that include sequences that control gene expression, such as promoters, enhancers , attenuators, etc.

The presence or absence of an argT-hisJQMP cluster on the chromosome can be determined by well-known methods, including PCR, Southern blotting, etc. In addition, the level of gene expression can be estimated by determining the amount of RNA transcribed from the gene using various known methods, including Northern blotting, quantitative RT-PCR, etc. The amount and molecular weight of the proteins encoded by the genes can be determined by known methods, including electrophoresis in SDS-PAGE with subsequent immunoblotting (Western blotting), etc.

The artT gene (synonyms: ECK2304, b2310) encodes the ArtT protein, a subunit of the ABC of the lysine / arginine / ornithine transporter (synonym for B2310). The artT gene (nucleotides complementary to the nucleotides 2,425,031 to 2,425,813 in sequence with accession number NC_000913.2 in the GenBank database; gi: 16130244) is located between the hisJ gene and the ubiX gene, oriented in the same direction as the artT gene, on the chromosome of E. coli strain K-12. The nucleotide sequence of the artT gene and the amino acid sequence of the ArtT protein encoded by the artT gene are shown in the Sequence Listing Numbers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2), respectively.

The hisJ gene (synonyms: ECK2303, b2209) encodes a HisJ protein, a subunit of the ABC histidine transporter (synonym B2309). HisJ gene (nucleotides complementary to 2,424,028 to 2,424,810 in sequence with accession number NC_000913.2 in the GenBank database; gi: 16130244) is located between the argT gene and hisQ gene, oriented in the same direction as hisJ gene, on the chromosome of E. coli strain K-12. The nucleotide sequence of the hisJ gene and the amino acid sequence of HisJ protein encoded by the hisJ gene are shown in the Sequence Listing under numbers 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4), respectively.

The hisQ gene (synonyms: ECK2302, b2208) encodes HisQ protein, a subunit of the ABC of the lysine / arginine / ornithine transporter (synonym B2308). HisQ gene (nucleotides complementary to nucucleotides 2,423,252 through 2,423,938 in sequence with accession number NC_000913.2 in the GenBank database; gi: 16130243) is located between the hisJ gene and hisM gene, oriented in the same direction as the hisQ gene, on the chromosome of E. coli strain K-12. The nucleotide sequence of the hisQ gene and the amino acid sequence of HisQ protein encoded by the hisQ gene are shown in the Sequence Listing Nos. 5 (SEQ ID NO: 5) and 6 (SEQ ID NO: 6), respectively.

The hisM gene (synonyms: HisM) encodes a HisM protein, the ABC subunit of the lysine / arginine / ornithine transporter (synonym B2307). HisM gene (nucleotides complementary to n-nucleotides 2,422,539 to 2,423,255 in sequence with accession number NC_000913.2 in the GenBank database; gi: 16130242) is located between the hisQ gene and hisP gene, oriented in the same direction as the hisM gene, on the chromosome of E. coli strain K-12. The nucleotide sequence of the hisM gene and the amino acid sequence of the HisM protein encoded by the hisM gene are listed in the Sequence Listing Numbers 7 (SEQ ID NO: 7) and 8 (SEQ ID NO: 8), respectively.

The hisP gene (synonyms: ECK2300, b2306) encodes HisP protein, a subunit of the ABC of the lysine / arginine / ornithine transporter (synonym B2308). HisP gene (nucleotides complementary to nucleotides 2,421,758 to 2,422,531 in sequence with accession number NC_000913.2 in the GenBank database; gi: 16130241) is located between the hisM gene and the open yfcI reading frame, oriented in the same direction as the hisP gene on the E chromosome coli K-12. The nucleotide sequence of the hisP gene and the amino acid sequence of the HisP protein encoded by the hisP gene are listed in the Sequence Listing Nos. 9 (SEQ ID NO: 9) and 10 (SEQ ID NO: 10), respectively.

Since representatives of various genera and strains of the Enterobacteriaceae family may experience some variations in the nucleotide sequences, the concept of the argT-hisJQMP cluster is not limited to the genes whose sequences are listed in the Sequence Listing Numbers SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 , SEQ ID NO: 7 and SEQ ID NO: 9, but may also include genes homologous to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9 encoding variants of ArgT, HisJ, HisQ, HisM, and HisP proteins. The term "protein variant" may mean a protein with changes in the sequence, whether deletion, insertion, addition or replacement of amino acids, in which the functional activity of the protein is maintained. The number of changes in a protein variant depends on the position or type of amino acid residue in the tertiary structure of the protein. It can be from 1 to 30, or in another example from 1 to 15, or in another example from 1 to 5 in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10. These variations in variants may occur in areas not critical to protein function. These changes are possible because some amino acids have a high homology to each other, therefore, such changes do not affect the tertiary structure or activity. Therefore, a variant of the protein encoded by the argT-hisJQMP cluster gene can be represented by proteins with a homology of at least 80%, or in another example, at least 90%, or in another example, at least 95%, relative to the complete amino acid sequence shown in The sequence listing under the numbers SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, while maintaining the functionality of the protein.

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

In addition, the argT-hisJQMP cluster genes can be variants if they hybridize under stringent conditions with nucleotide sequences complementary to the nucleotide sequences shown in the Sequence Listing under the number SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, or with a probe that can be synthesized based on the indicated nucleotide sequence, provided that it encodes a functional protein. "Stringent conditions" include those conditions under which specific hybrids, for example hybrids with a homology of at least 60%, or in another example, at least 70%, or in another example, at least 80%, or in another example, at least 90%, or in another example, at least 95% are formed, and non-specific hybrids, for example hybrids with less homology than indicated above, are not formed. A practical example of harsh conditions is a single wash, preferably two or three times, at a salt concentration of 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, at 60 ° C. The duration of washing depends on the type of membrane used for blotting and, as a rule, is as recommended by the manufacturer. For example, the recommended washing time for Hybond ™ N ⁺ nylon membrane (Amersham) under severe conditions is 15 minutes. Two to three times washing is preferred. The length of the probe can be selected depending on hybridization conditions and is usually about 100-1000 bp.

The expression of the argT-hisJQMP cluster can be attenuated by introducing a mutation into the gene such that the intracellular activity of the protein encoded by the gene is reduced compared to the unmodified strain. Mutations that result in diminished gene expression include the substitution of one or more bases for amino acid substitution in the encoded protein (missense mutation), the introduction of a stop codon (nonsense mutation), deletion of one or more bases for frame shift reading, inserting an antibiotic resistance gene or deleting a gene or part thereof (Qiu, Z. and Goodman, MF, J. Biol. Chem., 272, 8611-8617 (1997); Kwon, DH et al., J. Antimicrob. Chemother., 46, 793-796 (2000)). The expression of the artI gene can also be attenuated by modifying the expression of regulatory sequences, such as a promoter, a Shine-Dalgarno (SD) sequence, etc. (PCT application WO 95/34672; Carrier, T.A. and Keasling, J.D., Biotechnol Prog 15, 58-64 (1999)).

For example, the following methods can be used to introduce mutations by gene recombination. A mutant gene is constructed that encodes a mutant protein with reduced activity, and the bacterium for its modification is transformed with a DNA fragment containing the mutant gene. Then the native gene on the chromosome is replaced by homologous recombination with the mutant gene, and the resulting strain is selected. Substitution of a gene using homologous recombination can be carried out using a linear DNA method known as "Red-dependent integration" or "Red-system integration" (Datsenko, KA, Wanner, BL, Proc.Natl.Acad.Sci. USA, 97, 12, 6640-6645 (2000)), or by a method using a plasmid whose replication is temperature sensitive (US patent 6,303,383 or Japanese patent application JP 05-007491 A). In addition, the introduction of a site-specific mutation by replacing a gene using the aforementioned homologous recombination can also be carried out using a plasmid with reduced replication ability in the host cell.

Gene expression can also be attenuated by inserting a transposon or IS factor into the coding region of the gene (US Pat. No. 5,175,107) or by conventional methods such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine).

Gene inactivation can also be carried out by traditional methods such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine), site-specific mutagenesis, gene destruction using homologous recombination and / or mutagenesis due to insertion deletions (Yu, D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97: 12: 5978-83, and Datsenko, KA and Wanner, BL, Proc. Natl. Acad. Sci USA, 2000, 97: 12: 6640-45), also called "Red-Dependent Integration".

Methods for preparing plasmid DNA, DNA restriction and ligation, transformation, selection of nucleotides as a primer, etc. may be conventional methods known to a person skilled in the art. These methods are described, for example, 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 bacterium

In the method according to the present description of the invention, a bacterium modified in such a way that the gene expression (s) of the argT-hisJQMP cluster in the specified bacterium is weakened and capable of producing an L-amino acid such as diaminomonocarboxylic acid can be used.

Said bacterium can be obtained by attenuating the expression of the gene / s of the argT-hisJQMP cluster in a bacterium already possessing the ability to produce an L-amino acid, such as diaminomonocarboxylic acid. On the other hand, said bacterium can be obtained by imparting the ability to produce an L-amino acid, such as diaminomonocarboxylic acid, a bacterium in which the expression of the argT-hisJQMP cluster gene / s is already attenuated.

L-lysine producing bacterium

Examples of bacteria producing L-lysine belonging to the genus Escherichia include mutants that are resistant to the L-lysine analogue. The L-lysine analogue inhibits the growth of bacteria belonging to the genus Escherichia, but this inhibition is completely or partially removed when L-lysine is also present in the 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 on. Mutants that are resistant to these lysine analogues can be obtained by treating bacteria belonging to the genus Escherichia with traditional mutagens. Specific examples of bacterial strains used to produce L-lysine include Escherichia coli strain AJ11442 (FERM BP-1543, NRRL B-12185; see US patent 4346170) and Escherichia coli strain VL611. In these microorganisms, aspartokinase is resistant to feedback inhibition by L-lysine.

Strain WC196 can be used as a bacterium producer of L-lysine Escherichia coli. This bacterial strain was obtained by selection of the phenotype of resistance to AEC in strain W3110, derived from strain Escherichia coli K-12. The resulting strain was named Escherichia coli AJ13069 and was deposited at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry), currently called the National Institute of Advanced Industrial Science and Technology, International Depository of Organizations for Patenting Purposes, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan ( National in 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), December 6, 1994 and received accession number FERM P -14690. Then, the strain was internationally deposited in accordance with the terms of the Budapest Treaty on September 29, 1995, and the strain received accession number FERM BP-5252 (US patent 5827698).

Examples of parental strains for producing L-lysine producing bacteria of the present invention also include strains in which the expression of one or more genes encoding L-lysine biosynthesis enzymes is enhanced. Examples of enzymes involved in the biosynthesis of L-lysine include, but are not limited to, dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate carboxylase (lysA), diaminoproimene-patent-yl-6-azolephenol-6-enumero-carboxymene-6-carboxylic acid. ppc), aspartate semialdehyde dehydrogenase (asd), nicotinamide adenine dinucleotide transhydrogenase (pntAB) and aspartase (aspA) (European application EP 1 253 195 A). In addition, parental strains may have an increased level of expression of the gene involved in the respiration process (suo) (European application EP 1170376 A), the gene encoding nicotinamide nucleotranshydrogenase (pntAB) (US patent 5,830,716), the ybjE gene (PCT application WO 2005/073390) or combinations of these genes.

Examples of parent strains for the production of L-lysine producing bacteria according to the present invention also include strains in which enzyme activity is reduced or absent which catalyze the formation of compounds other than L-lysine branching off from the main L-lysine biosynthesis pathway. Examples of enzymes that catalyze the formation of non-L-lysine compounds branching off from the main L-lysine biosynthesis pathway include homoserine dehydrogenase, lysine decarboxylase (US Pat. No. 5,827,698) and malate dehydrogenase (PCT application WO 2005/010175).

L-arginine producing bacterium

Examples of parent strains used to produce the L-arginine producing bacterium 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) (US patent application 2002 / 058315 A1) and its derivatives containing mutant N-acetylglutamate synthase (RF patent application 2001112869), E. coli 382 strain (VKPM B-7926) (European patent application EP 1170358 A1), arginine producing strain into which the argA gene is introduced encoding N-acetylglutamate synthetase (European Patent Application EP 1170361 A1), and the like.

Examples of parental strains used to produce the L-arginine producing bacterium of the present invention also include strains in which the expression of one or more genes encoding L-arginine biosynthesis enzymes is enhanced. Examples of L-arginine biosynthesis enzymes include N-acetylglutamylphosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetyl glutamate kinase (argB), acetylornithine transaminase (argD), ornithinecarbamoyltransurase arginic acid, argN, argin, argin, argin, argin, carbamoylphosphate synthetase (carAB).

L-citrulline producing bacterium

Examples of parental strains used to produce the citrulline producer bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as N-acetylglutamate synthase mutant strains of E. coli 237 / pMADS11, 237 / pMADS12 and 237 / pMADS13 (RU 2215783, EP 1170361 B1, US 6790647 B2).

Also, the citrulline producing bacterium can be easily obtained from any arginine producing bacterium, for example, from E. coli 382 strain (VKPM B-7926), by inactivation of arginine succinate synthase encoded by the argG gene.

The phrase "inactivation of arginine succinate synthase" means that the bacterium is modified so that the modified bacterium contains inactive arginine succinate synthase, or it may also mean that the bacterium is not capable of synthesizing arginine succinate synthase. Inactivation of arginine succinate synthase can be accomplished by inactivation of the argG gene.

The phrase "inactivation of the argG gene" means that the modified gene encodes a fully non-functional protein. It is also possible that the modified DNA region is not capable of naturally expressing the gene due to deletion of part of the gene or the entire gene, shift of the reading frame of the gene, insertion of the missense / nonsense mutation (s), or modification of regions adjacent to the gene, including sequences that control gene expression, such as promoter, enhancer, attenuator, ribosome binding site, etc.

The presence or absence of the argG gene on the bacterial chromosome can be determined by well-known methods, including PCR, Southern blotting, and the like. In addition, the level of gene expression can be estimated by determining the amount of RNA transcribed from the gene using various known methods, including Northern blotting, quantitative RT-PCR, etc. The amount of protein encoded by the argG gene can be determined by known methods, including SDS-PAGE electrophoresis followed by immunoblotting (Western blotting), etc.

The expression of the argG gene can be attenuated by introducing a mutation into the gene on the chromosome such that the intracellular activity of the protein encoded by the gene is reduced compared to that in the unmodified strain. Such a gene mutation may be the replacement of one or more bases for amino acid substitution in the encoded protein (the Missense mutation), the introduction of a stop codon (the "nonsense" mutation), the deletion of one or more bases for reading frame shift, insertion of the resistance gene an antibiotic or deletion of a gene or part thereof (Qiu, Z. and Goodman, MF, J. Biol. Chem., 272, 8611-8617 (1997); Kwon, DH et al., J. Antimicrob. Chemother., 46, 793-796 (2000)). The expression of the argG gene can also be attenuated by modifying the expression of regulatory sequences, such as a promoter, a Shine-Dalgamo (SD) sequence, etc. (PCT application WO 95/34672; Carrier, T.A. and Keasling, J.D., Biotechnol Prog 15, 58-64 (1999)).

For example, the following methods can be used to introduce mutations by gene recombination. A mutant gene is constructed that encodes a mutant protein with reduced activity, and the bacterium for its modification is transformed with a DNA fragment containing the mutant gene. Then the native gene on the chromosome is replaced by homologous recombination with the mutant gene, and the resulting strain is selected. Such gene substitution using homologous recombination can be carried out using a linear DNA method known as “Red-dependent integration” or “Red-system integration” (Datsenko, KA, Wanner, BL, Proc.Natl.Acad.Sci.USA , 97, 12, 6640-6645 (2000), PCT application WO 2005/010175), or by a method using a plasmid whose replication is temperature sensitive (US Pat. No. 6,303,383 or Japanese Patent Application JP 05-007491 A). Further, the introduction of a site-specific mutation by replacing a gene using the aforementioned homologous recombination can also be carried out using a plasmid with reduced replication ability in the host cell.

Gene expression can also be attenuated by inserting a transposon or IS factor into the coding region of the gene (US Pat. No. 5,175,107) or by traditional methods such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine).

L-ornithine producing bacterium

An ornithine producing bacterium can be easily obtained from any arginine producing bacterium, for example E. coli 382 strain (VKPM B-7926), by inactivating the ornithine carbamoyltransferase encoded by the argF and argI genes. Methods for inactivating ornithine carbamoyltransferase are described above.

2. The method according to the present invention

The method according to the present invention is a method for producing an L-amino acid, comprising the steps of growing a bacterium according to the present invention in a culture medium in order to produce and accumulate the L-amino acid in the culture medium and isolate the L-amino acid from the culture fluid.

According to the present invention, the cultivation, isolation and purification of the L-amino acid from a culture or similar liquid can be carried out in a manner similar to traditional fermentation methods in which the amino acid is produced using bacteria.

The nutrient medium used for growing can be either synthetic or natural, provided that the medium contains sources of carbon, nitrogen, mineral additives and, if necessary, the appropriate amount of nutrient additives necessary for the growth of microorganisms. Ethanol is used as a carbon source. Various inorganic ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, natural nitrogen sources, such as peptone, soybean hydrolyzate, microorganism fermentolizate, can be used as a nitrogen source. As mineral additives, potassium phosphate, magnesium sulfate, sodium chloride, iron 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 carried out under aerobic conditions, such as mixing the culture fluid on a rocking chair, shaking with aeration, at a temperature in the range from 20 to 40 ° C, preferably in the range from 30 to 38 ° C. The pH of the medium is maintained in the range from 5 to 9, preferably from 6.5 to 7.2. The pH of the medium can be adjusted by ammonia, calcium carbonate, various acids, bases and buffer solutions. Typically, growing for 1 to 5 days leads to the accumulation of the target L-amino acid in the culture fluid.

After growth, solid residues, such as cells, can be removed from the culture fluid by centrifugation or filtration through a membrane, and then the L-amino acid can be isolated and purified by ion exchange chromatography, concentration and / or crystallization.

Examples

The present invention will be described in more detail below with reference to the following non-limiting examples of the invention.

Example 1. Construction of a strain with an inactivated cluster argT-hisJQMP

1. Cluster divisions argT-hisJQMP

The strain containing the argT-hisJQMP cluster deletion was constructed using a technique developed by Datsenko, K.A. and Wanner, BL (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645), known as "Red-Dependent Integration." A DNA fragment containing the Cm ^R marker encoded by the cat gene was obtained by PCR using primers PI (SEQ ID NO: 11) and P2 (SEQ ID NO: 12) and plasmid pMW118-attL-Cm-attR (WO 05/010175 ) as a matrix. Primer P1 contains a region complementary to the region localized at the 5 ′ end of the argT gene and a region complementary to the attR region. Primer P2 contains a region complementary to the region localized at the 3 ′ end of the hisP gene and a region complementary to the attL region. The following temperature profile for PCR was used: denaturation at 95 ° C for 3 min; the first two cycles: 1 min at 95 ° C, 30 sec at 50 ° C, 40 sec at 72 ° C; subsequent 25 cycles: 30 sec at 95 ° C, 30 sec at 54 ° C, 40 sec at 72 ° C; and final polymerization: 5 min at 72 ° C.

The resulting PCR product with a length of 1699 bp purified on an agarose gel and used for electroporation into E. coli strain MG1655 (ATCC 700926) containing the plasmid pKD46 with a heat-sensitive replicon. Plasmid pKD46 (Datsenko, K.A. and Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 6640-6645) contains a 2154 bp DNA fragment of phage λ. (positions 31088 through 33241 of the nucleotide sequence with accession number J02459 in the GenBank database), and also contains the genes of the λ Red-homologous recombination system (γ, β, exo genes) under the control of the _araB -induced P _araB promoter. Plasmid pKD46 is required for integration of the PCR product into the chromosome of strain MG1655.

Electrocompetent cells were obtained as follows: an overnight culture of E. coli strain MG1655 was grown at 30 ° C in LB medium supplemented with ampicillin (100 mg / L), diluted 100 times by adding 5 ml of SOB medium (Sambrook et al., "Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989) containing ampicillin and L-arabinose (1 mM). The resulting culture was grown with stirring at 30 ° С until OD ₆₀₀ ≈0.6 was reached, after which the cells were made electrocompetent by 100-fold concentration and three times washing with ice-cold deionized Н ₂ O. Electroporation was performed using 70 μl of cells and ≈100 ng of PCR product. After electroporation, the cells were incubated in 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 hours, after which they were plated on plates with L-agar containing 30 μg / ml chloramphenicol and grown at 37 ° C to select Cm ^R recombinants. Then, to remove plasmid pKD46, 2 passages were performed on L-agar with Cm at 42 ° C and the obtained colonies were tested for sensitivity to ampicillin.

2. Verification of the argT-hisJQMP cluster deletion by PCR

Mutants with the chaC delegated gene containing the Cm resistance gene were verified by PCR. Locus-specific primers P3 (SEQ ID NO: 13) and P4 (SEQ ID NO: 14) were used to verify deletion by PCR. The following temperature profile was used for PCR testing: denaturation at 94 ° C for 3 min; profile for 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 length of the PCR product obtained by the reaction using a mutant strain as a matrix of cells is 1650 bp The mutant strain was named MG1655 ΔargT-hisJQMP :: cat.

Example 2. The production of L-arginine strain E. coli 382ΔargT-hisP

To assess the effect of inactivation of the argT-hisJQMP cluster on L-arginine production, DNA fragments of the chromosome of the above E. coli strain MG1655 ΔargT-hisP :: cat were transferred to the E. coli 382 L-arginine producing strain using P1 transduction (Miller , JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain 382-ΔargT-hisP. Strain 382 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, ^1st Dorozhniy proezd, 1) on April 10, 2000 with accession number VKPM B-7926, then on May 18, 2001, this strain was internationally deposited in accordance with the conditions Budapest Treaty.

Both strains, 382 and 382-ΔargT-hisP, were grown with stirring at 37 ° C for 18 hours in 3 ml of nutrient broth, 0.3 ml of the resulting cultures were introduced into 3 ml of fermentation medium in 20 × 200 mm tubes and cultures were grown at 32 ° C for 48 hours on a rotary shaker.

After growing, the amount of L-arginine accumulated in the medium was determined using paper chromatography, and the following composition of the mobile phase was used: butanol: acetic acid: water = 4: 1: 1 (v / v). A solution of ninhydrin (2%) in acetone was used for visualization. A spot containing L-arginine was excised; L-arginine was eluted with a 0.5% aqueous solution of CdCl ₂ , after which the amount of L-arginine was determined spectrophotometrically at a wavelength of 540 nm. The results of ten independent in vitro fermentations are presented in table 1. As follows from table 1, strain 382ΔargT-hisP accumulated a greater amount of L-arginine than strain 382.

The fermentation medium of the following composition was used (g / l):

Glucose 48.0 (NH ₄ ) ₂ SO ₄ 35.0 KH ₂ PO ₄ 2.0 MgSO ₄ 7H ₂ O 1.0 Thiamine 0.0002 Yeast extract 1.0 L-isoleucine 0.1 CaCO ₃ 5.0

Glucose and magnesium sulfate were sterilized separately. CaCO _{3 was} dry heat sterilized at 180 ° C for 2 hours. The pH was adjusted to 7.0.

Example 3. Production of L-lysine by E. coli strain AJ11442-ΔargT-hisJQMP

To assess the effect of inactivation of the argT-hisJQMP cluster on lysine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔargT-hisP :: cat described above can be transferred to the E. coli L-lysine producer strain AJ1442 using P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain AJ 11442-382-ΔargT-hisP. The plasmid pCABD2 contains the dapA gene, which encodes a dihydrodipicolinate synthase with a mutation that removes feedback inhibition of L-lysine, the lysC gene that encodes aspartokinase III with a mutation that removes feedback by L-lysine inhibition, the dapB gene, which encodes dihydrodipzucine and dihydrodipzicoline ddh encoding diaminopimelate dehydrogenase (US Patent 6,040,160).

Both strains of E. coli, AJ1442 and AJ11442-382-ΔargT-hisP, can be grown in L-medium containing 20 mg / l streptomycin at 37 ° C; and 0.3 ml of the obtained cultures can be introduced into 20 ml of a fermentation medium containing the necessary antibiotics into 500 ml flasks. Cultivation can be carried out at 37 ° C for 16 hours using a reciprocating rocking chair with a stirring speed of 115 rpm. After growing, the amount of L-lysine and residual glucose in the medium can be measured in a known manner (Biotech-analyzer AS210, manufacturer Sakura Seiki Co.). Then, for each of the strains, the yield of L-lysine can be calculated in terms of glucose consumed.

Can be used in a fermentation medium of the following composition (g / l):

Glucose 40 (NH ₄ ) ₂ SO ₄ 24 K ₂ HPO ₄ 1.0 MgSO ₄ · 7H ₂ O 1.0 FeSO ₄ · 7H ₂ O 0.01 MnSO ₄ · 5H ₂ O 0.01 Yeast extract 2.0

The pH was adjusted to 7.0 with KOH and the medium was autoclaved at 115 ° C for 10 minutes. Glucose and MgSO ₄ 7H ₂ O are sterilized separately. CaCO _{3 is} also added to a concentration of 30 g / l, previously sterilized by dry heat at 180 ° C for 2 hours.

Example 4. The production of citrulline strain E. coli 382 ilvA ⁺ ΔargG ΔargT-hisP

To evaluate the effect of inactivation of the argT-hisJQMP cluster on citrulline production, a producer strain of citrulline 382 ilvA ⁺ ΔargG was constructed. For this, strain 382 ilvA ⁺ was obtained from arginine producer strain 382 (VKPM B-7926) by the method of P1 transduction of the ilvA gene from wild E. coli K12 strain. 382 ilvA ⁺ clones were selected that grow well on plates with minimal agar. Then, a producer strain of citrulline 382 ilvA ⁺ ΔargG was obtained by removing the argG gene on the chromosome of strain 382 ilvA ⁺ using the method proposed by Datsenko, KA and Warmer, BL (Proc. Natl. Acad. Sci. USA, 2000, 97 (12) , 6640-6645), called "Red-Dependent Integration".

The DNA fragment containing the chloramphenicol resistance marker (Cm ^R ) encoded by the cat gene was obtained by PCR using primers P5 (SEQ ID NO: 15) and P6 (SEQ ID NO: 16) and plasmid pMW118-attL-Cm- attR (WO 05/010175) as a matrix. Primer P5 contains a region complementary to the region located at the 5 ′ end of the argG gene and a region complementary to the attR region. Primer P6 contains a region complementary to the region localized at the 3 ′ end of the argG gene and a region complementary to the attL region. Used the following temperature profile for PCR: denaturation for 3 min at 95 ° C; the first two cycles: 1 min at 95 ° C, 30 sec at 50 ° C, 40 sec at 72 ° C; subsequent 25 cycles: 30 sec at 95 ° C, 30 sec at 54 ° C, 40 sec at 72 ° C; and final polymerization: 5 min at 72 ° C.

The resulting PCR product is 1.85 kbp. purified on an agarose gel and used for electroporation into E. coli 382 ilvA ⁺ strain, which was previously transformed with the plasmid pKD46, necessary for integration of the PCR product into the bacterial chromosome. Electrocompetent cells were obtained as described in Example 1. Electroporation was performed using 70 μl of cells and ≈100 ng of PCR product. After electroporation, the cells were incubated and the plasmid pKD46 was removed as described in Example 1.

Mutants not containing the argG gene and labeled with the chloramphenicol resistance gene were tested by PCR. For testing, locus-specific primers P7 (SEQ ID NO: 17) and P8 (SEQ ID NO: 18) and cells of the mutant strain as a matrix were used. Used the following temperature profile for PCR: denaturation for 3 min at 94 ° C; 30 cycles: 30 sec at 94 ° C, 30 sec at 54 ° C, 1 min at 72 ° C; and final polymerization: 7 min at 72 ° C. A 1.35 kb PCR product was obtained. The resulting mutant strain was named 382 ilvA ⁺ ΔargG :: cat.

The chloramphenicol resistance gene (cat gene) was removed from the chromosome of E. coli 382 ilvA ⁺ ΔargG :: cat strain using the int-xis system. For this purpose, E. coli strain 382 ilvA ⁺ ΔargG :: cat was transformed with the plasmid pMWts-Int / Xis (WO 05/010175). Transformed clones were selected on LB medium containing ampicillin (100 μg / ml). The plates were incubated at 30 ° C. overnight. The cat gene and the plasmid pMWts-Int / Xis were removed from transformants by plating individual colonies at 37 ° C (at this temperature, the Cits repressor was partially inactivated and transcription of the int / xis genes was repressed) and selecting Cm ^S Ap ^R variants. Removal of the cat gene from the chromosome was verified by PCR. For testing, locus-specific primers P7 (SEQ ID NO: 17) and P8 (SEQ ID NO: 18) were used, and the cells without the cat gene were used as a matrix. The PCR conditions were the same as described above. A PCR product of ~ 0.44 kb was obtained. Thus, a citrulline producing strain 382 ilvA ⁺ ΔargG was obtained.

To assess the effect of argT-hisJQMP cluster inactivation on citrulline production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔargT-hisP :: cat described above were transferred to the E. coli 382 ilvA ⁺ ΔargG citrulline producing strain using P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain 382 ilvA ⁺ ΔargG ΔargT-hisP.

Both strains of E. coli, 382 ilvA ⁺ ΔargG and 382 ilvA ⁺ ΔargG ΔargT-hisP, were grown with stirring at 37 ° C for 18 hours in 3 ml of culture medium and 0.3 ml of the resulting cultures were transferred to 2 ml of fermentation medium in test tubes 20 × 200 mm and cultured at 32 ° C for 48 hours on a rotary shaker.

After growing, the amount of citrulline obtained was determined by paper chromatography using butanol: acetic acid: water = 4: 1: 1 (v / v) as the mobile phase. Subsequent staining was performed with ninhydrin (2% solution in acetone). The citrulline-containing spot was excised and citrulline was eluted using a 0.5% aqueous solution of CdCl ₂ . The amount of citrulline was determined spectrophotometrically at a wavelength of 540 nm.

The results of eight independent in vitro fermentations are shown in Table 2. As can be seen from table 2, strain 382 ilvA ⁺ ΔargG ΔargT-hisP produced L-citrulline in a larger amount than strain 382 ilvA ⁺ ΔargG.

The fermentation medium of the following composition was used (g / l):

Glucose 48.0 (NH ₄ ) ₂ SO ₄ 35.0 KH ₂ PO ₄ 2.0 MgSO ₄ · 7H ₂ O 1.0 Thiamine HCl 0.0002 Yeast extract 1.0 L-arginine 0.1 CaCO ₃ 5.0

Glucose and magnesium sulfate are sterilized separately. CaCO _{3 is} sterilized by dry heat at 180 ° C for 2 hours. The pH is adjusted to 7.0.

Example 5. Production of ornithine by E. coli strain 382 ΔargFΔargI ΔargT-hisP

To assess the effect of inactivation of the argT-hisJQMP cluster on ornithine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔargT-hisP :: cat described above can be transferred to the E. coli 382ΔargFΔargI ornithine producing strain using P1 transduction (Miller, JH ( 1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain 382 ΔargFΔargIΔargT-hisP. Strain 382ΔargFΔargI can be obtained by successive deletions of the argF and argI genes on the chromosome of strain 382 (VKPM B-7926) using the method proposed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645), called "Red-dependent Integration". In accordance with this procedure, two pairs of PCR primers can be constructed that are homologous to both the regions adjacent to the argF and argI genes and the gene responsible for antibiotic resistance. Plasmid pMW118-attL-Cm-attR (WO 05/010175) can be used as a template in PCR.

Both strains of E. coli, 382ΔargFΔargI and 382ΔargFΔargIΔargT-hisP, can be grown with stirring at 37 ° C for 18 hours in 3 ml of culture medium, and 0.3 ml of the resulting cultures can be transferred to 2 ml of fermentation medium in 20 × 200 mm tubes and can be cultured at 32 ° C for 48 hours on a rotary shaker.

After growing, the amount of ornithine obtained can be determined by paper chromatography using a mobile phase of the following composition: butanol-acetic acid-water = 4: 1: 1. Subsequent staining can be performed with ninhydrin (2% solution in acetone). A stain containing citrulline can be excised, citrulline can be eluted using a 0.5% aqueous solution of CdCl ₂ , and the amount of citrulline can be determined spectrophotometrically at a wavelength of 540 nm.

Possible composition of the fermentation medium (g / l):

Glucose 48.0 (NH ₄ ) OSO ₄ 35.0 KH ₂ PO ₄ 2.0 MgSO ₄ · 7H ₂ O 1.0 Thiamine Hcl 0.0002 Yeast extract 1.0 L-isoleucine 0.1 L-arginine 0.1 CaCO ₃ 5.0

Glucose and magnesium sulfate are sterilized separately. CaCO _{3 is} sterilized by dry heat at 180 ° C for 2 hours. The pH is adjusted to 7.0.

Although the invention has been described in detail with reference to the best mode of carrying out the invention, it will be apparent to those skilled in the art that various changes may be made and equivalent replacements may be made, and such changes and replacements are not outside the scope of the present invention.

Each of the above documents has a link, and all cited documents are part of the description of the present invention.

Table 1 Strain OD ₅₄₀ L-arginine, g / l 382 11.3 ± 1.9 10.7 ± 1.0 382ΔargT-hisP 13.2 ± 1.0 11.6 ± 0.7

table 2 Strain OD ₅₄₀ L-citrulline, g / l 382 ilvA ⁺ ΔargG
382 ilvA ⁺ ΔargG ΔargT-hisP 12.1 ± 0.3
10.4 ± 0.7 4.0 ± 0.3
4.3 ± 0.2

Claims (9)

1. A bacterium-producer of diaminomonocarboxylic L-amino acids belonging to the Enterobacteriaceae family, modified in such a way that the expression of one or more genes encoding the lysine / arginine / ornithine transporter and one or more genes encoding the histidine transporter is weakened in said bacterium. 2. The bacterium according to claim 1, characterized in that the argT-hisJQMP cluster includes genes encoding a lysine / arginine / ornithine transporter and a histidine transporter. 3. The bacterium according to claim 2, characterized in that said argT-hisJQMP cluster is inactivated. 4. The bacterium according to claim 2, characterized in that the argT gene and one or more genes of the hisJQMP operon are inactivated. 5. The bacterium according to claim 1, characterized in that said bacterium belongs to the genus Escherichia. 6. The bacterium according to claim 1, characterized in that said bacterium belongs to the genus Pantoea. 7. A bacterium-producer of diaminomonocarboxylic L-amino acids according to any one of claims 1 to 6, characterized in that said diaminomonocarboxylic L-amino acid is selected from the group consisting of L-lysine, L-arginine, L-ornithine, L-citrulline. 8. A method of producing diaminomonocarboxylic L-amino acids, including:
- growing bacteria according to any one of claims 1 to 7 in a nutrient medium, causing production and secretion of the specified L-amino acid into the culture fluid; and
- the allocation of the specified L-amino acids from the culture fluid. 9. The method of claim 8, wherein said diaminomonocarboxylic acid is selected from the group consisting of L-lysine, L-arginine, L-ornithine and L-citrulline.

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