RU2215783C2 - Mutant n-acetylglutamate synthase (variants), dna fragment, strain of microorganism escherichia coli as producer of arginine and method for preparing l-arginine - Google Patents

Abstract

FIELD: biotechnology, biochemistry, molecular biology, microbiology, amino acids. SUBSTANCE: L-arginine is prepared by culturing microorganism of genus Escherichia transformed with DNA fragment encoding mutant N-acetylglutamate synthase. Mutant N-acetylglutamate synthase is the synthase wherein sequence of amino acids corresponding to positions from 15 to 19 in natural N-acetylglutamate synthase is replaced by any of sequence of amino acids at numbers 1-4 (SEQ ID NOS: 1-4) or the synthase showing indicated properties and containing additionally deletions, substitutions, insertions or additions of one or some amino acids by one multiple positions except for positions from 15 to 19. Strains of Escherichia coli 237/pMADS11, E. coli 237/pMADS12 or E. coli 237/pMADS13 can be used strain-producers of L-arginine. EFFECT: enhanced yield of arginine. 9 cl, 3 tbl, 1 dwg, 1 ex

Description

 The invention relates to the microbiological industry, to a method for producing L-arginine, and for the use of a new mutant feedback resistance enzyme in the arginine biosynthesis pathway in E. coli strains producing L-arginine.

Description of existing methods
The biosynthesis of arginine from glutamate in E. coli cells occurs through a series of reactions starting with acetylation of glutamate with N-acetylglutamate synthase (NAGS), encoded by the argA gene. This process is regulated by repressing the transcription of arg-regulon and inhibition of NAGS with arginine in the feedback manner (Cunin R. et al, Microbiol. Rev., v. 50, p. 314-352, 1986). L-Arginine inhibits the expression of the argA gene in a ratio of more than 250 times and inhibits the activity of NAGS (K _i = 0.02 mM) (Leisinger T., Haas D., J. Biol. Chem., V. 250, p. 1690- 1693, 1975). Enhanced arginine biosynthesis in E. coli cells requires NAGS enzymes that are resistant to feedback inhibition (also referred to as “fbr”).

 Feedback inhibition resistant enzyme mutants can be obtained by spontaneous, chemical, or site-directed mutagenesis.

 Some of the fbr argA mutants have been isolated and studied. Serratia marcescens cells containing fbr argA mutations on the chromosome were unstable and gave rise to argA mutants with reduced activity or altered glutamate affinity (Takagi T. et al, J. Biochem., V.99, p. 357-364, 1986) .

 The fbr argA genes from five E. coli strains containing fbr NAGS were cloned, and in each of these strains various single base substitutions were found in the argA genes. It was found that these substitutions cause the substitution of His-15 for Tur, Tur-19 for Cys, Ser-54 for Asn, Arg-58 for His, Gly-287 for Ser, Gln-432 for Arg (Rajagopal BS et al, Appl . Environ. Microbiol., 1998, v. 64, No. 5, p. 1805-1811).

 Typically, an fbr phenotype of an enzyme results from the replacement of one amino acid residue with another at one or more places in a protein sequence, and these substitutions result in a decrease in enzyme activity. For example, replacing natural Met-256 with the remaining 19 amino acid residues in serine acetyltransferase (SAT) (cysE gene) from E. coli in most cases leads to the appearance of fbr phenotype, but the level of activity of mutant SAT proteins does not reach the level of activity of natural SAT (Nakamori S. et al, AEM, 64 (5): 1607-11, 1998).

 Thus, a drawback of mutant enzymes obtained by these methods is a decrease in the activity of mutant enzymes in comparison with natural enzymes.

Summary of the invention
An object of the present invention is to provide a mutant feedback inhibition resistant mutant and a highly active enzyme that plays a key role in the biosynthesis of arginine by E. coli bacteria.

 The present invention provides a new method for the synthesis of a large set of mutant argA genes using complete randomization of a fragment of the argA gene. The simultaneous replacement of several amino acid residues in a fragment of a protein sequence in which fbr mutations can be localized can lead to the formation of mutant proteins with restored activity, the level of which is close to natural, since the three-dimensional structure of the enzyme becomes more regular. Thus, the invention described below has been completed.

Here is what the present invention provides:
(1) A mutant N-acetylglutamate synthase in which the amino acid sequence corresponding to positions 15 to 19 in the natural N-acetylglutamate synthase is replaced by any of the amino acid sequences numbered 1-4 (SEQ ID NOS: 1-4) and the inhibition of L- feedback arginine is lost;
(2) The mutant N-acetylglutamate synthase according to claim 1, which is a naturally occurring mutant N-acetylglutamate synthase from Escherichia coli;
(3) The mutant N-acetylglutamate synthase according to claim 1, which contains deletions, substitutions, insertions or additions of one or more amino acids in one or many positions, except positions 15 to 19, and in which feedback inhibition by L-arginine is lost ;
(4) DNA encoding the mutant N-acetylglutamate synthase according to any one of claims 1-3;
(5) A bacterium belonging to the genus Escherichia, transformed with DNA according to paragraph 4 and having activity on the production of L-arginine; and
(6) A method for producing L-arginine, comprising the steps of growing the bacterium according to paragraph 5 in a culture medium for producing and storing L-arginine in a culture medium and collecting L-arginine from the culture fluid.

 A NAGS protein containing any of the fbr mutations described above may be referred to as a "mutant NAGS", DNA encoding a mutant NAGS may be referred to as a "mutant argA gene", a NAGS protein without mutations may be referred to as "natural NAGS".

 Further, the present invention will be explained in more detail.

 <1> Mutant NAGS proteins and mutant argA genes.

Mutant NAGS proteins and mutant argA genes encoding these proteins were obtained by mutagenesis by randomizing a fragment of a nucleotide sequence of a gene. To obtain multiple mutations in the argA gene, a fragment of 15 nucleotides was completely randomized, encoding the region in the protein sequence from the 15th to the 19th amino acid residue, inclusive. Complete randomization of a fragment of 15 nucleotides gives 4 ¹⁵ or about 10 ⁹ different DNA sequences encoding 20 ⁵ different amino acid residues in a 5-dimensional peptide. The probability of non-occurrence of stop codons in the reading frame in this sequence is 0.95 ⁵ or about 78%. Thus, complete randomization of the fragment of the argA gene encoding a peptide from the 15th to the 19th amino acid residue should produce approximately 2.5 million different protein sequences that differ in this peptide fragment in the NAGS structure. Sequential selection and selection of recombinant clones carrying mutant argA genes cloned into expression vectors allows the selection of fbr variants of mutant NAGS with different levels of their biological activity up to the level of activity of natural NAGS. In breeding, the inventors decided that strains carrying the mutant argA gene can be obtained using strains argD ^- and proB ^- or proA ^- because such strains cannot produce L-proline due to inhibition of NAGS and therefore cannot grow if there is an excess of L-arginine in the culture medium, but the strains carrying fbr NAGS can grow on minimal medium because glutamate semi-aldehyde, a precursor of L-proline, can be obtained using acetylornithine deacetylase (argE gene product) from N- acetylglutamate semial dehydrate, the precursor of L-arginine (Eckhardt, T., Leisinger, T., Mol. Gen. Genet., v. 138, p. 225-232, 1975). However, the inventors have found that using the method described above, as described in the following examples, it is difficult to obtain a high activity fbr NAGS, and that such a high activity fbr NAGS can be obtained by introducing a mutant argA gene into a natural strain and selection of strains showing growth retardation.

 In the framework of the present invention, amino acid sequences of mutant NAGS corresponding to the fbr phenotype of NAGS were determined. As a result, mutant NAGS can be obtained from these sequences by introducing mutations into the natural argA gene using conventional methods. As an example of the natural argA gene, E. coli argA gene (GenBank number Y00492) may be mentioned.

 The amino acid sequence at position 15-19 in the mutant NAGS according to the present invention corresponds to any of the sequences 1-4 (SEQ ID NOS: 1-4). The corresponding sequence of the known mutant NAGS, in which tyrosine at position 19 is replaced by cysteine, as well as the natural NAGS from E. coli are shown under numbers 5 and 6 (SEQ ID NOS: 5 and 6). Examples of nucleotide sequences encoding these amino acid sequences are given under the numbers 7-12 (SEQ ID NOS: 7-12). These sequences are presented in table 1 (see the end of the description).

 Mutant NAGS may contain deletions, substitutions, insertions or additions of one or more amino acids in one or many positions, except positions 15 to 19, provided that the activity of NAGS, which is the activity of catalysis of the acetylation reaction of L-glutamic acid to obtain N- acetylglutamate, not worsened.

 The value of "several" in relation to the number of amino acids differs depending on the position or type of amino acid residues in the three-dimensional structure of the protein for the following reason. Some amino acids have a high homology with respect to other amino acids, and the difference in their replacement does not significantly affect the three-dimensional structure of the protein. For this reason, the mutant NAGS according to the present invention may be such NAGS, the homology of which is not less than 30-50%, preferably 50-70%, in comparison with the full amino acid sequence established for NAGS, and which has fbr NAGS activity.

 In the present invention, “amino acid sequence corresponding to the sequence at position 15 to 19” means an amino acid sequence corresponding to the sequence of amino acids at position 15 to 19 in the naturally occurring NAGS sequence from E. coli. The position of amino acid residues may vary. For example, if an amino acid residue is introduced into the N-terminal portion, then the amino acid residue located naturally at position 15 moves to position 16. In this case, the amino acid residue corresponding to the initial position 15 is designated in the present invention as the amino acid residue in position 15.

 DNA encoding essentially the same protein as the mutant NAGS described above can be obtained, for example, by modifying the nucleotide sequence, for example, by site-directed mutagenesis, so that instead of one or more amino acid residues at the indicated location deletions, substitutions, insertions or additives. The modified DNA described above can be created using traditional processing methods to obtain mutations. Processing for mutations includes a method for processing DNA containing the argA gene in vitro, for example using hydroxylamine, and a method for treating a microorganism, for example a bacterium belonging to the genus Escherichia, carrying the argA gene, ultraviolet radiation, or an agent that causes mutations, such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and nitrous acid, commonly used in processing to produce mutations.

 Substitution, deletion, insertion or addition of a nucleotide, as described above, also includes mutations that occur naturally (mutant or variant), for example, based on individual differences or differences in the level of species or genus of the bacterium containing NAGS.

 DNA encoding essentially the same protein as the argA gene is detected by expression of the DNA containing the mutation described above in a suitable cell and by examining the activity of the expression product.

 Also, DNA encoding essentially the same protein as mutant NAGS can be obtained by isolating DNA that hybridizes with DNA having a known argA gene sequence or probe derived from it under stringent conditions and which encodes a protein having activity NAGS, from a cell carrying a mutant NAGS subjected to mutation treatment.

The term “stringent conditions” referred to here means the conditions under which a so-called specific hybrid (duplex) is formed, but a non-specific hybrid is not formed. It is rather difficult to clearly describe these conditions using numerical values. However, for example, stringent conditions are those under which DNA molecules with high homology, for example DNA with homology with each other at least 50%, hybridize, and DNA with less homology does not hybridize. As an alternative, stringent conditions are exemplified by the conditions under which DNA hybridizes to each other at a salt concentration corresponding to the usual concentration during washing during Southern hybridization, i.e. 60 ^° C, 1xSSC, 0.1% SDS, preferably 0.1xSSC, 0.1% SDS.

 Genes that hybridize under the conditions described above include genes that contain a stop codon inside the coding region of the gene, as well as those that encode an inactive protein due to mutations in the active center. However, such difficulties can be easily resolved by ligating the gene into a commercially available vector for expression and study of NAGS activity.

 <2> A bacterium belonging to the genus Escherichia according to the present invention.

 A bacterium belonging to the genus Escherichia according to the present invention is a bacterium belonging to the genus Escherichia, into which the mutant argA gene described above has been introduced. An example of a bacterium belonging to the genus Escherichia. is E. coli. A mutant argA gene can be introduced, for example, by transforming a bacterium belonging to the genus Escherichia, a recombinant plasmid containing a vector functioning in a bacterium belonging to the genus Escherichia, and a mutant gene argA. The mutant argA gene can also be introduced by replacing the argA gene with the mutant argA gene on the chromosome.

 Examples of vectors that can be used to introduce the mutant argA gene are plasmid vectors such as pBR322, pMW118, pUC19 or the like, phage vectors such as 11059, 1BF101, M13mp9 or the like, and transposons such as Mu, Tn10, Tn5 or like that.

 The introduction of DNA into a bacterium belonging to the genus Escherichia can be carried out, for example, by the method of D.A. Morrison (Methods in Enzymology, 68, 326 (1979)) or a method in which a recipient bacterial cell is treated with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol. 53, 159 (1970)) or a similar method.

 If the mutant argA gene is introduced into a bacterium belonging to the genus Escherichia, the producer of L-arginine, the amount of L-arginine produced can be increased. In addition, the ability to produce L-arginine can be given to bacteria into which the mutant argA gene has already been introduced.

 An example of a bacterium belonging to the genus Escherichia with activity in the production of L-arginine is E. coli 237 strain (VKPM B-7925). Strain 237 was deposited in the All-Russian collection of industrial microorganisms (VKPM).

 <3> A method for producing L-arginine.

 L-arginine can be obtained with high efficiency when growing bacteria, into which the mutant argA gene is introduced, and which is capable of producing L-arginine, in the culture medium, production and accumulation of L-arginine in the culture medium and collecting L-arginine from the culture liquids.

In the method according to the present invention, the cultivation of bacteria belonging to the genus Escherichia, the accumulation and collection of L-arginine from the culture fluid can be carried out in a manner similar to the method traditionally used for the production of L-arginine by fermentation using bacteria. The growth medium can be either synthetic or natural, provided that it contains sources of carbon and nitrogen, mineral compounds and, if necessary, nutritional supplements in the amount necessary for the growth of bacteria. Carbon sources include various carbohydrates, such as glucose and sucrose, and various organic acids, depending on the ability of the bacteria to absorb them. Alcohols such as ethanol and glycerin may be used. Ammonia, various ammonium salts such as ammonium sulfate, other nitrogen compounds such as amines, natural nitrogen sources such as peptone, soybean hydrolyzate and microbial fermentation are used as nitrogen sources. As mineral compounds, monosubstituted potassium phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium carbonate are used. Cultivation is preferably carried out under aerobic conditions such as agitation and agitation with stirring. The temperature of the culture is usually maintained from 20 ^{° C.} to 40 ^{° C.} , preferably from 30 ^{° C.} to 38 ^° C. The pH is in the range of 5 to 9, preferably in the range of 6.5 to 7.2. The pH of the medium can be adjusted by ammonia, calcium carbonate, various acids, bases and buffers. Typically, growing for 1 to 3 days leads to the accumulation of L-arginine in the culture fluid.

 The collection of L-arginine can be carried out by removing insoluble components such as cells from the culture medium after cultivation by centrifugation or filtration on a membrane, followed by collection and purification of L-arginine by ion exchange methods, concentration and fractional crystallization or the like.

 A brief description of the drawings.

 The drawing shows a diagram of the construction of a pool of mutant argA genes.

 The best way of carrying out the invention.

 The present invention will be explained in more detail with reference to the following examples.

 Example 1

 <1> Mutagenesis by randomizing a fragment of a nucleotide sequence of a gene.

The BamHI-SalI fragment of chromosomal DNA (2.02 kb) containing the argA gene was cloned into plasmid pUC19 (plasmid pUC19-argA). For amplification by PCR was used DNA polymerase Pyrobest ^TM of Takara Shuzo Co. (Japan), it was used in the conditions recommended by the manufacturer.

 To construct a pool of mutant argA genes in the first stage, a fragment of the argA gene was amplified, coding the sequence from the 20th amino acid residue to the end of NAGS (drawing). The pUC19-argA plasmid was used as a matrix, P1: 5'-CGAGGGATTCCGCNNNNNNNNNNNNNNNATCAATACCCACCGGG-3 '(SEQ ID NO: 13) seed primer was constructed based on the argA nucleotide sequence, and the reverse seed sequence M was used as P2 antisense13. A fixed sequence of 16 nucleotides at the 3'-end of the P1 seed is homologous to the argA gene sequence after the Tur-19 codon, and a fixed sequence of 13 nucleotides at the 5'-end of the seed is before the His-15 codon. The homology to the argA gene sequence at the 3'-end of the P1 seed was used to synthesize a DNA fragment (1.75 kb) during twenty PCR cycles.

As a matrix, 100 ng of the plasmid pUC19-argA was added to a PCR solution (50 μl) containing both seeds (40 pmol each). Twenty PCR cycles were performed (94 ^° C for 0.6 min, 55 ^° C for 0.5 min, 72 ^° C for 2 min) in a 2400 DNA thermal cycler (Perkin-Elmer Co., Foster City, CA )

In the second stage, eight amplification cycles (94 ^° C for 1 min, 37 ^° C for 1 min, 72 ^° C for 0.5 min) were carried out, during which the (-) chain of this fragment acted as a seed for it extensions to obtain the complete gene sequence.

In a third step, an aliquot of 10 μl of the reaction mixture was added to a fresh reaction mixture (40 μl) containing 100 pmol of seed P3: 5′-TGCCATGGTAAAGGAACGTAAAACC-3 ′ (SEQ ID NO: 14) homologous to the 5 ′ end of the argA gene sequence, and antisense seed P2, then ten cycles of amplification were carried out (94 ^o C for 0.5 min, 52 ^o C for 0.5 min, 72 ^o C for 2 min),
The DNA fragment (1.78 kb), encoding a pool of mutant variants of the full-sized argA gene, was purified by agarose gel electrophoresis, digested with NcoI enzymes (the recognition site includes the initial ATG codon of the argA gene) and HmdIII, and then ligated into the pKK233 vector -2 (Pharmacia, Sweden) digested with NcoI and HindIII enzymes.

 About 150 ng of donated DNA was used in subsequent experiments to transform E. coli recipient cells in order to produce about 2000 recombinant clones in each case.

 <2> Isolation of new argA mutants and the effect of amino acid substitutions on the catalytic properties of NAGS.

The plasmid vector pKK233-2 (Pharmacia, Sweden) was used to clone and express argA gene variants. TG1 (supE hsdΔ5 thi Δ (lacropooV) F '[traD36 proAB ⁺ lacI ^q lacZΔM15]) was used as the E. coli recipient strain (Sambrook, J. et al. Molecular Cloning. 1989). The selection of TG1 cells containing a set of recombinant plasmids pKK-argA-random (with mutant argA genes)? was carried out on plates with LB agar. A slowdown in the cell growth of certain mutant clones was observed and it was suggested that this effect correlates with the production of the active mutant fbr enzyme NAGS. Plasmids were isolated from some clones and the DNA sequence of the 5'-terminal fragment of the argA gene was determined by sequencing (a chain termination method using dideoxynucleotides) (table 2).

To determine the activity of NAGS, the E. coli strain B3083 (argA ^- . MetB ^- ) - auxotroph was transformed by arginine with the obtained plasmids. Enzymes in soluble fractions obtained from recombinant cells destroyed by ultrasound were partially purified by precipitation with ammonium sulfate and analyzed as described below. NAGS activity from strains containing plasmids pKK-argA-r11 (3390 nmol / min x mg), pKK-argA-r12 (1220 nmol / min x mg) pKK-argA-r13 (3120 nmol / min x mg), significantly higher NAGS activity from a strain containing plasmid pKK-argA-r4 (300 nmol / min x mg). The latter plasmid contains a mutant argA gene with the same substitution (Y19C) as the most active single-substitution argA gene described by Rajagopal BS et al (Rajagopal BS et al. Appl. Environ. Microbiol., 1998, v. 64, No. .5, p. 1805-1811).

 Also, the activity of NAGS in the strain containing the plasmid pKK-argA (wt) (natural argA) is lower than in the case of pKK-argA-r11, -r12 and -r13. The level of activity of mutant enzymes in the presence of 10 mM arginine is approximately the same, although the natural enzyme was significantly inhibited by arginine (less than one tenth of the initial activity). These results show that the peptide fragment from 15 to 19 amino acid residues is responsible for the feedback inhibition of NAGS with arginine and for the level of catalytic activity.

(Verification of enzyme activity)
Acetyl coenzyme A and all chemicals are purchased from Sigma Chemical Co., St. Louis, Mo. To determine the activity of NAGS, E. coli B3083 cells (argA ^- , metB ^- ) containing recombinant plasmids were grown in M9 medium (5 ml) until the late exponential phase, washed with 0.14 M NaCl solution and resuspended in 2 ml of 40 mM potassium phosphate buffer (pH 7.0) with 100 mM KCl. Cells were destroyed by ultrasound and centrifuged. Fractions containing NAGS were precipitated with 5 volumes of saturated (NH ₄ ) ₂ SO ₄ and the precipitates were dissolved in 2 ml of 40 mM potassium phosphate buffer (pH 7.0) with 100 mM KC1 and 30% (v / v) glycerol. A solution with NAGS was added to 0.1 ml of the reaction mixture (100 mM Tris-HCl (pH 8.5), 35 mM KCl, 20 mM L-glutamate, 1.2 mM acetyl coenzyme A) and the reaction mixture was incubated at 37 ^° C within 10 minutes The reaction was stopped by adding 0.3 ml of ethanol, followed by centrifugation of the reaction mixture. 0.95 ml of a 0.24 mM DTNB solution (5.5-dithio-bis-2-nitrobenzoate) was added to the supernatant and the mixture was incubated for 15 minutes. NAGS activity was determined by measuring absorbance at 412 nm.

<3> Isolation of new mutants of the argA gene by selection among B 16-4 cells (pro ^- argD ^- ).

The selection of mutant argA genes from E. coli strain B 16-4 (pro ^- argD ^- ) carrying the pKK-argA-random plasmid was carried out by the method described above. Recombinant clones from agar plates were suspended in M9 medium with L-arginine and grown to the stationary phase. An aliquot of the culture was suspended in fresh medium and the growing procedure was repeated four times. After this, an aliquot of the culture was transferred to agar medium M9 containing 5 mg / ml L-arginine and 100 μg of ampicillin. Plasmids were isolated from some clones, sequences of 5'-terminal fragments of mutant argA genes were determined, and the level of activity of mutant NAGS was verified as described above. 60% of the mutants contained the sequence -Gly-Trp-Pro-Cys-Val- (SEQ ID NO: 4) in the mutagenized fragment of the enzyme and had weak but fbr NAGS activity. Obviously, such a mutant protein has an optimal level of NAGS activity for the growth of B16-4 (pro ^- argD ^- ) cells under the conditions used for selection. Thus, the selection conditions determine the activity of the obtained mutant NAGS.

 <4> L-arginine production using mutant argA genes.

 Recombinant plasmids pKKArgA-r4, 11, 12, 13 and 32 were digested with restriction enzymes BamHI and SalI, and fragments containing mutant argA genes under the control of the trc promoter were re-cloned into the low copy plasmid pMW119 (Nippon Gene Co., Tokyo). The resulting plasmids were named pMADS4, pMADS11, pMADS12, pMADS13 and pMADS32, respectively. These plasmids were introduced into the strain E. coli 237 - producer of L-arginine (VKPM B-7925). The production of L-arginine (Arg) and citruline (Cit) transformants is shown in Table 3. Most producer strains with the new mutant NAGS genes had higher Arg and Cit productivity than the recipient strain or the strain with the known mutant Tyrl9Cys NAGS (pMADS4).

(Test conditions for fermentation in vitro)
The fermentation medium contained 60 g / L glucose, 25 g / L ammonium sulfate, 2 g / L KH ₂ PO ₄ , 1 g / L MgSO ₄ , 0.1 mg thiamine, 5 g / L Difco yeast extract, 25 g / l chalk per 1 liter of water (pH 7.2). Glucose and chalk were sterilized separately. 2 ml of culture medium was placed in a test tube, one loop of the test microorganism was seeded, cultivation was continued at 32 ^{° C.} for 3 days with shaking.

 A sequence listing is provided at the end of the description.

Claims (9)

 1. Mutant N-acetylglutamate synthase, in which the amino acid sequence corresponding to positions 15 to 19 in the natural N-acetylglutamate synthase is replaced by any of the amino acid sequences numbered 1-4 (SEQ ID NOS: 1-4).  2. The mutant N-acetylglutamate synthase according to claim 1, wherein the natural N-acetylglutamate synthase is N-acetylglutamate synthase from Escherichia coli.  3. A mutant N-acetylglutamate synthase in which the amino acid sequence corresponding to positions 15 to 19 in the natural N-acetylglutamate synthase is replaced by any of the amino acid sequences numbered 1-4 (SEQ ID NOS: 1-4), and such synthase contains deletions, substitutions, insertions, or additions of one or more amino acids in one or a plurality of positions, except positions 15 to 19.  4. The mutant N-acetylglutamate synthase according to claim 3, wherein the natural N-acetylglutamate synthase is N-acetylglutamate synthase from Escherichia coli.  5. A DNA fragment encoding a mutant N-acetylglutamate synthase according to any one of paragraphs. 1-4.  6. The bacterial strain Escherichia coli 237 / pMADS11, transformed with a DNA fragment encoding a mutant N-acetylglutamate synthase, in which the amino acid sequence corresponding to positions 15 to 19 in the natural N-acetylglutamate synthase is replaced by the amino acid sequence of SEQ ID NO: 1, - L-arginine producer.  7. The bacterial strain Escherichia coli 237 / pMADS12, transformed with a DNA fragment encoding a mutant N-acetylglutamate synthase, in which the amino acid sequence corresponding to positions 15 to 19 in the natural N-acetylglutamate synthase is replaced by the amino acid sequence SEQ ID NO: 2, - L-arginine producer.  8. The bacterial strain Escherichia coli 237 / pMADS13, transformed with a DNA fragment encoding a mutant N-acetylglutamate synthase, in which the amino acid sequence corresponding to positions 15 to 19 in the natural N-acetylglutamate synthase is replaced by the amino acid sequence SEQ ID NO: 3, - L-arginine producer.  9. A method for producing L-arginine, comprising the steps of growing a bacterium belonging to the genus Escherichia, a producer of L-arginine in a culture medium and isolating L-arginine from the culture fluid, characterized in that a strain of a bacterium belonging to the genus Escherichia, transformed with a DNA fragment according to claim 5.

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