RU2550269C2 - METHOD OF PRODUCING L-ARGININE USING Enterobacteriaceae BACTERIA, CONTAINING DISRUPTED-ACTIVITY N-ACETYLORNITHINE DEACETYLASE - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and provides a method of producing an L-amino acid, such as L-arginine, via fermentation using Escherichia bacteria, modified to contain an argJ gene which codes an enzyme having at least ornithine acetyltransferase activity, wherein the bacteria are modified to contain disrupted-activity N-acetylornithine deacetylase.

EFFECT: invention enables to obtain L-arginine with high efficiency.

10 cl, 2 dwg, 4 tbl, 7 ex

Description

FIELD OF THE INVENTION

The present invention relates to the microbiological industry, in particular to a method for producing L-amino acids, such as L-arginine, by fermentation of a bacterium of the Enterobacteriaceae family, which is modified so that it contains the argJ gene encoding an enzyme having at least ornithine acetyltransferase activity, and a disrupted argE gene encoding N-acetylornithine deacetylase.

State of the art

Traditionally, L-amino acids in industry are produced by the fermentation method using strains of microorganisms obtained from natural sources, or their mutants. Typically, microorganisms are modified 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 recombinant DNA microorganisms (see, for example, US Pat. No. 4,278,765) and changing regulatory regions, such as a promoter, leader sequence and / or attenuators, or others known to the person skilled in the art. area (see, for example, US Patent application No. 20060216796 A1 and WO9615246 A1). Other yield enhancing methods include increasing the activity of enzymes involved in amino acid biosynthesis and / or removing feedback inhibition of key enzymes produced by the L-amino acid (see, for example, Japanese Patent Publication No. 56-18596 (1981), WO 95 / 16042 or US Pat. Nos. 5,661,012 and 6,040,160).

Another way to increase the production of L-amino acids is to weaken the expression of one or more genes involved in the degradation of the target L-amino acid, genes involved in the removal of the precursors of the target L-amino acid from the biosynthetic pathway of the L-amino acid, genes involved in the redistribution of carbon and nitrogen flows and phosphorus, and genes encoding toxins, etc.

It is generally accepted that, for example, in bacteria, the biosynthesis of L-arginine from L-glutamate can take place in two ways, linear or cyclic, depending on the particular microorganism (Cunin R. et al., Microbiol. Rev., 1986, 50: 314 -352). In bacteria, such as a bacterium belonging to the Enterobacteriaceae family, in particular Escherichia coli (E. coli), there is a linear pathway that includes 8 steps until complete formation of L-arginine from L-glutamate (Vogel HJ and MacLellan WL, Methods Enzymol. 1970, 17A, 265-269). Biosynthesis is initiated by the acetylation of L-glutamate with the amino acid N-acetyltransferase (EC 2.3.1.1, also called N-acetyl-L-glutamate synthetase) encoded by the argA gene. Subsequent biosynthetic reactions are catalyzed by enzymes commonly referred to as N-acetylglutamate kinase, N-acetyl-γ-glutamylphosphate reductase, N-acetylornithin aminotransferase, N-acetylornithine deacetylase, ornithinecarbamoyltransferase, arginine-argon-argon-argon-argon-argon-argon-arginase-argon-arginase , respectively. The acetyl group, which is removed by N-acetylornithine deacetylase (ArgE) from N-acetylornithine to form ornithine, binds to coenzyme A (HS-CoA, CoA) to form acetylated coenzyme A (AcS-CoA, acetyl-CoA), which acts as a donor of the acetyl group in the acylation reaction of L-glutamine, catalyzed by amino acid-N-acetyltransferase (argA).

The cyclic biosynthesis pathway of L-arginine has been found in prokaryotes, such as Corynebacterium glutamicum (Udaka S. and Kinoshita S., J. Gen. Appl. Microbiol., 1958, 4: 272-282; Sakanyan V. et al., Microbiology, 1996, 142: 99-108), Bacillus species (Sakanyan V. et al., J. Gen. Microbiol., 1992, 138: 125-130), Thermotoga neapolitana (Marc F. et al., Eur. J. Biochem ., 2000, 267 (16): 5217-5226), and eukaryotes (De Deken RH, Biochim. Biophys. Acta., 1962, 78: 606-616). In contrast to the linear pathway, in the cyclic pathway, the transfer of the acetyl group from N-acetylornithine to L-glutamate is catalyzed by bifunctional ornithine acetyltransferase / N-acetylglutamate synthase (EC 2.3.1.35/2.3.1.1), encoded by the argJ gene.

In addition to the bifunctional enzyme encoded by the argJ gene, the ArgJ protein encoded by the argJ gene and exhibiting exclusively ornithine acetyltransferase activity is known (Haas D. et al., Eur. J. Biochem., 1972, 31: 290-295; Marc F. et al., Eur. J. Biochem., 2000, 267 (16): 5217-5226). Monofunctional and bifunctional ArgJ enzymes can be distinguished in two ways: (i) enzymatic analysis using two acetyl donors, such as N-acetylornithine and AcS-CoA; and (ii) a complementation test using argE and argA mutants of E. coli with cloning of the argJ gene. The monofunctional enzyme ArgJ transfers the acetyl group from N-acetylornithine to L-glutamate and complements the argE mutant, while the bifunctional enzyme ArgJ transfers the acetyl group from N-acetylornithine and AcS-CoA and complements argE and argA mutants.

A new biosynthetic pathway of L-arginine was recently discovered in Xanthomonas campestris, where N-acetylornithine is converted to N-acetylcytrulline by the action of acetylornithinecarbamoyltransferase encoded by the argF 'gene and citrulline is formed from N-acetylcytrulline Sh by D. alg E.g. Biol. Chem., 2005, 280: 14366-14369).

The known E. coli bacterium, which originally had a linear pathway, was modified so that it contains the argJ gene from Bacillus stearothennophilus or Thermotoga neapolitana (T. neapolitana), which encodes the bifunctional enzyme ArgJ, to initiate a less energy-intensive cyclic pathway and, thus, increase L-arginine production through a modified bacterium (US Pat. No. 6,897,048 B2).

In microorganisms that initially use a linear biosynthetic pathway or have a modified linear pathway that functions as a cyclic pathway, amino acid N-acetyltransferase (N-acetyl-L-glutamate synthetase) (ArgA) may be necessary to initiate and maintain the biosynthesis of L-arginine through delivery N-acetylglutamate. Thus, to increase the production of L-arginine using a recombinant E. coli strain, the number of copies of the argA gene can be increased by the cloned wild-type argA gene on plasmid vectors and their inclusion in a strain having the cloned argJ gene (US Patent No. 6,897,048 B2). On the other hand, the argA gene encoding a mutant amino acid-N-acetyltransferase with resistance to inhibition by the feedback principle of L-arginine (Eckhardt T. et al., Mol. Gen. Genet, 1975, 138: 225-232) can be introduced into the E. coli strain to enhance the production of L-arginine (EP 1170361 A2).

Despite the fact that there is a positive effect on the production of L-arginine from the use of the bifunctional ArgJ protein, additional copies of the argA gene and / or mutant N-acetyl-L-glutamate synthetase (mutant ArgA), etc., it is clearly established approaches for further increases in L-arginine production by microorganisms are not obvious.

Currently, there is no data describing the effect of the disrupted argE gene so that the activity of N-acetylornithine deacetylase (ArgE) is reduced or absent on the production of L-arginine by modified bacterial strains of the Enterobacteriaceae family with the heterologous argJ gene.

Disclosure of the invention

The authors of the present invention have suggested that in microorganisms, for example, bacteria, having a heterologous argJ gene that encodes a monofunctional enzyme ornithine acetyltransferase or a bifunctional enzyme ornithine acetyltransferase / N-acetylglutamate synthase, weakening the expression of the argE gene or inactivating the gene process can be a source of arg with the transfer of the acetyl group from N-acetylornithine to L-glutamate, thus promoting the production of L-arginine using a modified strain as compared to the parent strain. The authors of the present invention have suggested that the ArgJ enzyme, having mono- or double activity and encoded by the argJ gene cloned from a donor microorganism, can restore the biosynthesis of L-arginine in strains mutant for the argE or argA and argE genes. From this point of view, the artificial cyclic pathway, being initiated by the first biosynthetic reaction to produce N-acetylglutamate, can function by direct acylation of L-glutamate with an acetyl group derived from N-acetylornithine rather than from the more energy-rich AcS-CoA.

The purpose of the present invention is the provision of a bacterium belonging to the Enterobacteriaceae family, which may belong to the genus Escherichia, more specifically to the species Escherichia coli, which has the activity of ornithine acetyl transferase or ornithine acetyl transferase / N-acetylglutamate synthase (ArgJ) and in which N-acetyl acetyl zin reacts ArgE activity is reduced.

Another objective of the present invention is the provision of a method for producing L-amino acids, such as L-arginine, using bacteria of the Enterobacteriaceae family as described herein.

These goals were achieved thanks to the discovery that the inactivation or attenuation of the expression of the argE gene in bacteria of the Enterobacteriaceae family with the heterologous argJ gene leads to increased production of L-arginine.

An object of the present invention is to provide an L-arginine producing bacterium belonging to the Enterobacteriaceae family having a recombinant DNA that contains the argJ gene encoding an enzyme having at least ornithine acetyltransferase activity, characterized in that the bacterium is modified so that it contains N-acetylornithine deacetylase with impaired activity.

Another objective of the present invention is the provision of a bacterium described herein, wherein the argJ gene is derived from a microorganism having an enzyme having ornithine acetyltransferase or ornithine acetyltransferase / N-acetylglutamate synthase activity.

Another objective of the present invention is the provision of a bacterium described herein, characterized in that the argJ gene is derived from a microorganism belonging to a family selected from the group consisting of the families Thermotogaceae, Bacillaceae and Methanocaldococcaceae.

Another objective of the present invention is the provision of a bacterium described herein, wherein the argJ gene is derived from Thermotoga neapolitana.

Another objective of the present invention is the provision of the bacteria described here, characterized in that the argJ gene encodes a protein selected from the group consisting of:

(A) a protein having the amino acid sequence shown in SEQ ID NO: 2, which has an ornithine acetyltransferase / N-acetylglutamate synthase activity;

(B) a variant of a protein having the amino acid sequence shown in SEQ ID NO: 2, but which contains the substitution, deletion, insertion and / or addition of one or more amino acid residues and has the activity of ornithine acetyltransferase / N-acetylglutamate synthase in accordance with the amino acid sequence represented by in SEQ ID NO: 2;

and

(C) combinations thereof.

Another objective of the present invention is the provision of a bacterium described herein, characterized in that N-acetylornithine deacetylase is a protein selected from the group consisting of:

(D) a protein having the amino acid sequence shown in SEQ ID NO: 4;

(E) a variant of a protein having the amino acid sequence shown in SEQ ID NO: 4, but which contains a substitution, deletion, insertion and / or addition of one or more amino acid residues and has N-acetylornithine deacetylase activity in accordance with the amino acid sequence shown in SEQ ID NO: 4; and

(F) combinations thereof.

Another objective of the present invention is the provision of the bacteria described herein, characterized in that the activity of N-acetylornithine deacetylase is reduced.

Another objective of the present invention is the provision of the bacteria described herein, characterized in that the activity of N-acetylornithine deacetylase is absent.

Another objective of the present invention is the provision of the bacteria described herein, characterized in that the bacterium belongs to the genus Escherichia.

Another objective of the present invention is the provision of a bacterium described herein, characterized in that the bacterium belongs to the species Escherichia coli.

Another objective of the present invention is the provision of the bacteria described herein, characterized in that the bacterium belongs to the genus Pantoea.

Another objective of the present invention is the provision of the bacteria described herein, characterized in that the bacterium belongs to the species Pantoea ananatis.

The purpose of the present invention is the provision of a method for producing L-arginine or its salt, including:

(i) growing the bacteria described above in a nutrient medium;

(ii) the accumulation of L-arginine or its salt in bacteria or culture fluid, or both; and if necessary

(iii) isolation of L-arginine or its salt from a bacterium or culture fluid.

The present invention is described in detail below.

Brief Description of the Figures

Figure 1 shows a diagram of the integration of the argJ gene into the chromosome of E. coli strain MG1655ΔargAΔargR.

Figure 2 shows the construction scheme of the E. coli strain MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJargEm24 :: Cm.

BEST MODE FOR CARRYING OUT THE INVENTION

1. Bacteria

The term "bacterium-producer of L-amino acids" can mean a bacterium of the Enterobacteriaceae family that is capable of producing, excreting or secreting and causing the accumulation of L-amino acids in the culture fluid or bacterial cells when the bacterium is grown (cultivated) in a nutrient medium.

The term “ability of a bacterium to produce an L-amino acid” may mean the ability of a bacterium to produce, excrete or secrete and cause the accumulation of an L-amino acid in a culture fluid or bacterial cells, resulting in an accumulation of an L-amino acid in quantities sufficient to isolate it from a culture fluid or bacterial cells when the specified bacterium is grown (cultivated) in a nutrient medium.

The term "L-amino acid" may mean L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine L-glutamic acid, L-glutamine, 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.

A bacterium may have the ability to produce amino acids initially, in accordance with its natural characteristics, or may be modified by mutations (mutagenesis) or recombinant DNA technologies so that it receives the ability to produce amino acids.

A bacterium belonging to the Enterobacteriaceae family can be selected from bacteria belonging to the genera of this family, such as Enterobacter, Erwinia, Escherichia, Klebsiella, Morganella, Pantoea, Photorhabdus, Providencia, Salmonella, Yersinia, etc., and capable of producing L-amino acid. More specifically, bacteria classified as Enterobacteriaceae can be used according to the taxonomy used in the NCBI database (National Center for Biotechnology Information) (https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/ wwwtax.cgi? id = 543). Bacteria belonging to the genus Escherichia, Enterobacter or Pantoea are preferred for modifications according to the invention.

The selection of bacterial strains belonging to the genus Escherichia that can be modified in the present invention is not limited in any way, however, as examples, bacteria of the genus Escherichia described in Neidhardt et al. (Bachmann, BJ, Derivations and genotypes of some mutant derivatives of E. coli K-12, p. 2460-2488. In FC Neidhardt et al. (Ed.), E. coli and Salmonella: cellular and molecular biology, 2 ^nd ed. ASM Press, Washington, DC, 1996) may be included among the bacteria of the present invention. As a specific example, E. coli. Strains, such as E. coli W3110 (ATCC 27325), E. coli MG1655 (ATCC 47076), etc., which originate from the wild-type parent strain, i.e. E. coli K-12 strain. This strain can be obtained, in particular, from the American Type Culture Collection (American Type Culture Collection, (ATCC) "(PO Box 1549, Manassas, VA 20108, United States of America). Each strain is assigned an individual registration number, and strains can be ordered according to the registration number (see link www.atcc.org). Registration numbers of strains are in the list of the catalog of the American Type Culture Collection, American Type Culture Collection, ATCC.

Examples of Enterobacter bacteria include Enterobacter agglomerans, Enterobacter aerogenes, etc. Examples of Pantoea bacteria include Pantoea ananatis, etc. Recently, some Enterobacter agglomerans species have been reclassified as Pantoea agglomerans, Pantoea ananatis, or Pantoea stewartii based on analysis of the 16S rRNA nucleotide sequence and other evidence. Bacteria belonging to the genus Enterobacter or Pantoea can be used in accordance with the present invention, as they belong to the Enterobacteriaceae family. Genetic engineering strains of Pantoea ananatis, such as Pantoea ananatis AJ13355 strain (PERM BP-6614), strain AJ13356 (PERM BP-6615), strain AJ13601 (PERM BP-7207) and their derivatives can be used in accordance with this invention. These strains were classified as Enterobacter agglomerans upon isolation, and they were deposited as Enterobacter agglomerans. However, they were later classified as Pantoea ananatis based on analysis of the 16S rRNA nucleotide sequence and other evidence.

The term “L-arginine producing bacterium" may mean a bacterium that is capable of producing, excreting or secreting and causing the accumulation of L-arginine in culture fluid or bacterial cells in amounts greater than the wild-type strain or parent E. coli strain such as E .coli K-12 when the bacterium is grown in a culture medium. The ability to produce L-arginine may mean the ability of a bacterium to produce, excrete or secrete and cause the accumulation of L-arginine in the culture medium in an amount of at least 0.5 g / l or not less than 1.0 g / l, so that L-arginine can be isolated from the culture fluid and / or bacterial cells when the specified bacterium is grown (cultivated) in a nutrient medium.

Examples of parental strains that can be used to produce L-arginine producing bacteria 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 derived strains having mutant N-acetylglutamate synthase (EP 1170361 A2), E. coli strain 382 (VKPM B-7926) (EP1170358 A1) and a strain producing L-arginine having the introduced argA gene (EP1170361 A1) , and the like.

The Enterobacteriaceae family L-arginine producing bacterium can be further modified to contain the heterologous argJ gene, which encodes the monofunctional ornithine acetyltransferase enzyme or the bifunctional ornithine acetyltransferase / N-acetylglutamate synthase (ArgJ) enzyme. For example, a bacterium of the Enterobacteriaceae family may have the argJ gene encoding the mono- or bifunctional ArgJ enzyme that is resistant to feedback inhibition by L-arginine. The argJ gene can be isolated from a thermophilic microorganism, such as a microorganism belonging to the Thermotogaceae family, and more specifically, to the Thermotoga genus, for example, T. pearo-Shapa (T. neapolitana). The argJ gene can also be isolated from other bacterial sources, such as Bacillus stearothermophUus (or Geobacillus stearothermophilus) of the Bacillaceae family or archaea, such as Methanocaldococcus jannaschii (M. jannaschii) (formerly Methanococcus jannaschii) of the family Methanocaldococaceae. However, various bacterial sources of the argJ gene can be used provided that the argJ gene encodes an enzyme having at least ornithine acetyltransferase activity.

The Enterobacteriaceae family L-arginine producing bacterium can be further modified so that it contains an inactivated argE gene or an expression-reduced argE gene, so that N-acetylornithine deacetylase (ArgE) is inactive in the modified bacterium, or N-acetylornithine deacetylase activity is reduced compared to unmodified a bacterium.

The term “L-arginine producing bacterium” may mean a bacterium of the Enterobacteriaceae family having the argA gene encoding the wild type or mutant amino acid N-acetyltransferase (ArgA). The enzyme ArgA, resistant to feedback inhibition by the principle of L-arginine, can be called as a mutant amino acid-N-acetyltransferase. For example, an ArgA protein mutant that is resistant to feedback inhibition by L-arginine can be obtained from the wild-type ArgA protein by replacing the amino acid sequence at positions 15 to 19 with another amino acid sequence, as indicated in EP 1170361 A2. A mutant ArgA protein containing, in the amino acid sequence, substitution, division, insertion and / or addition of one or more amino acid residues at one or more positions other than positions 15-19, may also be referred to as a mutant ArgA protein, provided that the tertiary structure of the mutant the amino acid N-acetyltransferase is slightly changed compared to the wild-type protein or the activity of the mutant enzyme has not deteriorated and there is resistance to inhibition by the feedback principle of L-arginine.

The term “L-arginine producing bacterium” may also mean a bacterium of the Enterobacteriaceae family, as described above, having an amplified, attenuated and / or inactivated argA gene encoding a wild type or amino acid-N-acetyltransferase mutant.

The term “bacterium-producer of L-arginine” can also mean a bacterium of the Enterobacteriaceae family, further modified so that it lacks ArgR-dependent transcriptional repression. The DNA-binding transcriptional double regulator of ArgR, which in microorganisms is involved in the negative control of the biosynthetic pathway of L-arginine, can be inactivated, for example, by inactivation of the argR gene encoding ArgR.

The term “wild-type protein” can mean a native protein naturally produced by a wild-type strain or a parent bacterial strain of the Enterobacteriaceae family, for example, a wild-type strain of E. coli MG1655. The wild-type protein can be encoded by the wild-type gene, or by an unmodified gene naturally found in the genome of wild-type bacteria.

The term “bacterium modified in such a way that it contains the argJ gene” may mean that a bacterium belonging to a first-type bacterium that naturally does not contain the argJ gene, and thus referred to as a recipient bacterium, is modified to contain one or more recombinant DNA molecules having the argJ gene synthesized and / or derived and introduced from a bacterium belonging to the second species, referred to as a donor bacterium, which is different from a bacterium of the first species. An example of a modification for the introduction of recombinant DNA can be the expression of a heterologous gene. For example, the recipient bacterium may belong to the Enterobacteriaceae family, for example, a species of E. coli; the donor bacterium may belong to a thermophilic bacterium, for example, the species T. neapolitana.

The term "bacterium modified in such a way that it contains recombinant DNA" may mean that the bacterium is modified in the usual way so that it contains exogenous DNA. Conventional methods include, for example, transformation, transfection, infection, conjugation and mobilization. Transformation, transfection, infection, conjugation, or mobilization of a bacterium by a DNA molecule encoding a protein can impart the ability of a bacterium to synthesize a protein encoded by a DNA molecule. Methods of transformation, transfection, infection, conjugation and mobilization include any of the previously known methods. For example, a method is known for efficiently transforming and transfecting DNA by treating Escherichia coli K-12 recipient cells with calcium chloride to increase cell permeability for DNA (Mandel M. and Higa A., Calcium-dependent bacteriophage DNA infection, J. Mol. Bioi, 1970, 53: 159-162). Methods of specialized and / or general transduction are described (Morse ML et al., Transduction in Escherichia coli K-12, Genetics, 1956, 41 (1): 142-156; Miller JH, Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor La. Press, 1972). Other methods of randomly and / or directionally integrating DNA into the host genome may be used, for example, “Mu-dependent integration / amplification” (Akhverdyan et al., Appl. Microbiol. Biotechnol., 2011, 91: 857-871), “Red / ET-dependent "or" λRed / ET-dependent integration "(Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA 2000, 97 (12): 6640-45; Zhang Y., et al., Nature Genet, 1998, 20: 123-128). Moreover, for multiple insertions of desired genes in addition to Mu-dependent replicative transposition (Akhverdyan et al., Appl. Microbiol. Biotechnol., 2011, 91: 857-871) and chemically induced chromosome evolution based on recA-dependent homologous recombination leading to amplification of the desired genes (Tuo KEJ et al., Nature Biotechnol., 2009, 27: 760-765), other approaches based on various combinations of transposition, site-directed and / or homologous Red / ET-dependent recombination can be used , and / or P1-dependent total transduction (see, for example, Minaeva N. et al., IUD B iotechnology, 2008, 8:63; Koma D. et al., Appl. Microbiol. Biotechnol., 2012, 93 (2): 815-829).

Heterologous expression of the argJ gene in host microorganisms can be achieved by replacing rare (rarely used in the host body) codons with synonymous medium or frequently used codons, and the term “use of a codon” can be defined as the number of times (frequency) of codon translation in an organism’s cell per unit time or as the average frequency of codon occurrence in sequenced protein-coding reading frames (Zhang SP et al., Gene, 1991, 105 (1): 61-72). The use of codons in the body can be found in the Codon Usage Database, which is an extended web version of the CUTG (Codon Usage Tabulated from GenBank) (https://www.kazusa.or.jp/codon/; Nakamura Y. et al., Codon usage tabulated from the international DNA sequence databases: status for the year 2000, Nucl. Acids Res., 2000, 28 (1): 292). In E. coli, such mutations may include, but are not limited to, replacing the rare Arg codons AGA, AGG, CGG, CGA with CGT or CGC; rare Ile codon ATA on the ATC or ATT; the rare Leu codon STA on CTG, STS, CTT, TTA or TTG; rare Pro-codon CCC to CCG or CCA; the rare Ser codon TCG on TST, TCA, TCC, AGC or AGT; rare Gly codons GGA, GGG on GGT or GGC; etc. Replacing less-used codons with synonymous often-used codons is preferred. Replacing rare and / or poorly used codons with synonymous medium or frequently used codons can be combined with co-expression of genes encoding tRNAs that recognize rare codons.

The term “bacterium modified in such a way that it contains the argE gene with impaired expression” may mean that the bacterium is modified in such a way that the expression of the argE gene is impaired in the modified bacterium or the argE gene is inactivated.

The term "argE gene expression is attenuated" may mean that the amount of ArgE protein in a modified bacterium in which the argE gene expression is attenuated is reduced compared to an unmodified bacterium, for example, a wild-type strain or a parent strain.

The term "argE gene expression is attenuated" may also mean that the modified bacterium contains a modified argE gene that encodes a mutant ArgE protein having reduced activity compared to the wild-type ArgE protein, or a region functionally linked to the gene that includes sequences that control gene expression, such as promoters, enhancers, attenuators, ribosome binding sites (RBS), etc., modified to reduce the level of expression of the argE gene, and other examples (see, for example, WO95 / 34672; Carrier TA and Keasling JD, Biotech nol. Prog., 1999, 15: 58-64).

Similar definitions can be given for the terms “argR gene expression is attenuated” and “argA gene expression is attenuated”.

The expression of the argE, argA, and / or argR genes can be attenuated by replacing the sequence that controls gene expression, such as a chromosomal DNA promoter, with a weaker one. The strength of the promoter is determined by the frequency of acts of initiation of RNA synthesis. Examples of methods for evaluating promoter strength are described in Goldstein et al., Prokaryotic promoters in biotechnology, Biotechnol. Annu. Rev. 1995, 1: 105-128, etc. In addition, it is also possible to introduce a nucleotide substitution into the promoter region of the target gene in such a way as to weaken the strength of the promoter, as described in International Patent Publication WO00 / 18935. In addition, it is known that the replacement of several nucleotides in the region between the Shine-Dalgarno (SD) sequence and the start codon in the ribosome-binding region (RBS), in particular, in the sequence located above and immediately after the start codon, is significantly affects the translation efficiency of mRNA. A similar modification of RBS can be combined with a decrease in the level of transcription of the argE, argA and / or argR genes.

Gene expression of argE, argA, and / or argR can also be attenuated by inserting a transposon or IS factor into the coding region of a gene (US Pat. No. 5,175,107), or into a region that controls gene expression, or into a nearby or remote region with respect to the structural part gene argE, argA and / or argR, or by conventional methods such as mutagenesis by UV radiation or nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine). In addition, the mutation can be site-directed introduced using known methods for changing the chromosome, based, for example, on λRed / ET-dependent recombination.

The terms “enzymatic activity of ArgE is reduced”, “enzymatic activity of ArgA is reduced” and “enzymatic activity of ArgR are reduced” may mean that the enzymatic activity of N-acetylornithine deacetylase (ArgE), amino acid-N-acetyltransferase (ArgA) and / or regulator ArgR, respectively, lower than in an unmodified strain, for example, a wild-type strain of a bacterium belonging to the Enterobacteriaceae family, more specifically, to the species E. coli. For example, the enzymatic activity of N-acetylornithine deacetylase (ArgE), the amino acid-N-acetyltransferase and / or ArgR regulator can be reduced by inactivation of the corresponding gene.

The enzymatic activity of the enzyme encoded by the gene (s) argE, argA and / or argR can also be reduced by introducing a mutation into the gene on the chromosome so that the intracellular activity of the protein encoded by the gene (s) argE, argA and / or. argR was reduced compared to the unmodified strain. Such a mutation in the gene (s) or in front of the gene in the structure of the operon may be the replacement of one or more bases, leading to the replacement of the amino acid residue in the protein encoded by the gene (s) (missense mutation), the introduction of a stop codon (nonsense mutation), deletion of one or more bases, leading to a reading frame shift, insertion of a gene reporting antibiotic resistance, partial or complete removal of genes in the genome (Qiu Z. and Goodman MF, J. Biol. Chem. 1997, 272: 8611-8617; Kwon DH et al., J. Antimicrob. Chemother. 2000, 46: 793-796).

In a modified bacterium, the amount of ArgE protein encoded by the argE gene can be reduced to such a level that the residual ArgE activity is less than or about 3%, or less than or about 2%, or less than or about 1% of the original activity, but higher than zero compared to unmodified bacteria.

In a modified bacterium, the specific activity of ArgE encoded by the argE gene can be reduced to such a level that the residual specific activity of ArgE is not less than 1000 nmol / mg min, 750 nmol / mg min, 500 nmol / mg min, 250 nmol / mg min, 100 nmol / mg min, 50 nmol / mg min, 25 nmol / mg min, 10 nmol / mg min, 5 nmol / mg min or about 3 nmol / mg min according to the scatter of experimental data. The specific activity of bacterial ArgE in terms of 1 mg of crude protein can be determined in a crude extract of ultrasound-destroyed bacterial cells in the manner described (Takahara K. et al. FEBS J., 2005, 272: 5353-5364). The concentration of the crude protein can be determined by the Bradford method (Bradford M.M., Anal. Biochem., 1976, 72: 248-254) using bovine serum albumin as standard.

The enzymatic activity of N-acetylornithine deacetylase (ArgE) can be reduced by changing fermentation conditions, such as nutrient acidity (pH), concentration of cofactor (s), such as metal ions, temperature, ionic strength, etc.

The term "argE gene is inactivated" may mean that the modified gene encodes a completely inactive or non-functional protein. It is also possible that the modified portion of the DNA is not capable of naturally expressing the gene due to deletion of part of the gene or the whole gene, shift of the reading frame of the gene, insertion of the missense / nonsense mutation (s) or modification of the adjacent portion of the gene, inclusion of sequences that control gene expression, such as a promoter (s), enhancer (s), attenuator (s), ribosome binding site (s), etc. Gene inactivation can also be carried out by conventional methods, such as mutagenesis, by treating microorganisms, for example, UV radiation or nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, disruption of the gene structure using homologous recombination and / or mutagenesis by insertion deletion (Yu D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 5978-83; Datsenko KA and Wanner BL, Proc. Natl. Acad Sci. USA 2000, 97 (12): 6640-45), also referred to as “λRed / ET-dependent integration”.

Similar definitions can be given for the terms “argR gene inactivated” and “argA gene inactivated”.

The term “ArgE activity is absent” is equivalent to the term “ArgE protein is inactivated” and may also mean that ArgE activity is completely absent, that is, equal to zero, or ArgE activity is below a certain level when activity is measured using the method described in Takahara K. et al. FEBS J., 2005, 272: 5353-5364. As a comparison control, for example, wild-type Enterobacteriaceae bacteria, including E. coli strain MG1655, and the like, can serve. In a modified bacterium, the specific activity of ArgE encoded by the argE gene can be reduced to such a level that the residual specific activity of argE is zero or not more than 0.01 nmol / mg min, 0.05 nmol / mg min, 0.1 nmol / mg min or about 0.5 nmol / mg min according to the scatter of experimental data and when determining the level of activity as described above.

As described above, the activity of ArgE protein (N-acetylornithine deacetylase) may be reduced and / or absent. Therefore, the term "ArgE activity is impaired" can be understood as "ArgE activity is reduced" and / or "there is no ArgE activity," as described above.

The term “increased expression of the argA gene” may mean that the total enzymatic activity of the corresponding gene protein, ArgA, is increased, for example, by the introduction and / or increase in the number of copies of the argA gene in the bacterial genome or by an increase in activity per protein molecule (can be called as specific activity) encoded by this gene compared to an unmodified strain, such as a wild-type strain or a parent strain. The bacterium can be modified so that the activity of the ArgA protein in terms of cell is increased to 150% or more, 200% or more, 300% or more of the activity of the protein in the unmodified strain. As an example of an unmodified comparison strain, wild-type microorganism strains can be cited. Enterobacteriaceae family, such as E. coli strain MG1655 (ATCC 47076), strain W3110 (ATCC 27325), Pantoea ananatis strain AJ13335 (PERM BP-6614), etc.

The term "increased expression of the argA gene" may also mean that the level of expression of the argA gene in a modified strain is higher than the expression level in an unmodified strain, for example, a wild-type strain or a parent strain.

Methods that can be used to increase the expression of the argA gene include, but are not limited to these examples, increasing the number of copies of the argA gene in the bacterial genome (on the chromosome and / or autonomously replicating plasmid) and / or introducing the argA gene into the vector so that it becomes able to increase the number of copies and / or level of expression of the argA gene in bacteria of the Enterobacteriaceae family in accordance with genetic engineering methods known to specialists in this field.

Examples of such vectors include, but are not limited to, vectors with a wide range of hosts (broad-host-range vectors), for example, pCM110, pRK310, pVK101, pBBR122, pBHR1 and the like. Multiple copies of the argA gene can be introduced into the chromosomal DNA of a bacterium through, for example, homologous recombination, Mu-dependent integration, or similar methods. Homologous recombination can be carried out using a multiple copy sequence in chromosomal DNA. Multiple sequences in chromosomal DNA include, but are not limited to, repeating portions of DNA or reverse repeats at the ends of a moving genetic element. It is also possible to introduce the argA gene into a transposon to introduce several copies of the argA gene into the chromosomal DNA. When using Mu-dependent integration, more than 3 copies can be introduced into chromosomal DNA in one act (Akhverdyan V.Z. et al., Biotechnol. (Russian), 2007, 3: 3-20).

An increase in the level of expression of the argA gene can also be achieved by modifying the regulatory region adjacent to the argA gene, or by introducing native and / or modified foreign regulatory regions. Examples of regulatory regions or sequences include promoters, enhancers, attenuators, transcription termination and anti-termination signals, ribosome binding sites (RBS), and other expression control elements (e.g., sites to which repressors or inducers bind, and / or binding sites of transcriptional and translational regulatory proteins, for example, as part of mRNA). Such regulatory sequences are described, for example, in Sambrook J., Fritsch EF and Maniatis T., "Molecular Cloning: A Laboratory Manual", 2 ^nd ed., Cold Spring Harbor Laboratory Press (1989). Modifications of regions that control the expression of gene (s) can be combined with an increase in the number of copies of the modified gene (s) in the bacterial genome using known methods (see, for example, Akhverdyan VZ et al., Appl. Microbiol. Biotechnol., 2011, 91 : 857-871; Tyo KEJ et al., Nature Biotechnol., 2009, 27: 760-765).

Strong promoters can be examples of promoters that enhance expression of the argA gene. For example, the lac promoter, trp promoter, trc promoter, tac promoter, P _R or P _L phage λ promoters are known as strong promoters. Strong promoters providing a high level of gene expression in bacteria belonging to the Enterobacteriaceae family can be used. Also, the influence of the promoter can be enhanced, for example, by introducing a mutation in the region of the argA promoter of the gene in order to obtain a promoter with a stronger function, thus leading to an increase in the level of transcription of the argA gene located under the promoter. In addition, it is known that the replacement of several nucleotides in a 3D sequence, and / or in the region between the SD sequence and the start codon, and / or in the sequence immediately before and / or after the start codon in the ribosome-binding site, significantly affects the translational ability of mRNA. For example, a 20-fold variation in the level of gene expression was found depending on the nature of the three nucleotides preceding the start codon (Gold L. et al., Annu. Rev. Microbiol., 1981, 35: 365-403; Hui A. etal., EMBO J., 1984, 3: 623-629).

The expression level of the heterologous argJ gene can be increased using the approaches described above for the argA gene.

The number of copies, the presence or absence of an operon gene and / or genes can be measured, for example, by restriction of chromosomal DNA followed by Southern blotting using a probe selected on the basis of the nucleotide sequence of the gene, in situ fluorescence hybridization hybridization, FISH) and similar methods. The level of gene expression can be determined by measuring the amount of transcribed mRNA using well-known methods, including, for example, Northern blotting, quantitative RT-PCR (RT-PCR), etc. The amount of proteins encoded by the gene can be determined by known methods, including SDS-PAGE (SDS-PAGE) followed by Western blotting (Western blotting), or mass spectrometric analysis of protein samples, etc.

Methods for working with a recombinant DNA molecule, cleavage, molecular cloning and heterologous gene expression, such as the preparation of plasmid DNA, cleavage, ligation and transformation of DNA, the choice of oligonucleotides as primers, etc. can be carried out by conventional methods known to the person skilled in the art. These methods are described, for example, in Sambrook J., Fritsch EF and Maniatis T., “Molecular Cloning: A Laboratory Manual, 2 ^nd ed.”, Cold Spring Harbor Laboratory Press (1989). Molecular cloning and expression of heterologous genes are described in Bernard R. Glick, Jack J. Pasternak and Cheryl L. Patten, Molecular Biotechnology: principles and applications of recombinant DNA, 4 ^th ed., Washington, DC: ASM Press (2009) ; Evans Jr., TC and Xu M.-Q., “Heterologous gene expression in E. soi,” 1 ^st ed., Humana Press (2011).

The argJ gene encodes a monofunctional protein, ornithine acetyltransferase ArgJ (KEGG, Kyoto Encyclopedia of Genes and Genomes, Kyoto Encyclopedia of Genes and Genomes, entry No. MJ_0186; UniProtKB / Swiss-Prot, Protein Knowledgebase, entry No. Q57645). The argJ gene (GenBank accession number NC_000909.1; nucleotides complementary to the nucleotides at position: 184215 to 185423; Gene ID: 1451033) is located between the MJ_0187 gene on the same molecular chain and the MJ_0185 gene on the opposite molecular chain of M. jannaschii DSM 2661 chromosome. The nucleotide sequence of the argJ gene and the amino acid sequence of the monofunctional ArgJ protein encoded by the argJ gene are shown in SEQ ID NO: 29 and SEQ ID NO: 30, respectively.

The argJ gene encodes a bifunctional protein, ornithine acetyltransferase / N-acetylglutamate synthase ArgJ (KEGG, entry No. CTN_1181; UniProtKB / Swiss-Prot, entry No. Q9Z4S1). The argJ gene (GenBank inventory no. NC_011978.1; nucleotides complementary to nucleotides at position: 1146678 to 1147871; Gene ID: 7377498) are located between the argC and CTN_1180 genes on the T. neapolitana DSM 4359 chromosome. The nucleJ sequence of the argJ gene and the amino acid sequence of the bifunctional ArgJ encoded by the argJ gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The argE gene encodes an ArgE N-acetylornithine deacetylase protein (KEGG, entry No. b3957). The argE gene (GenBank inventory no. NC_000913.2; nucleotides complementary to nucleotides at position: 4151719 to 4152870; Gene ID: 948456) is located between the ppc gene on the same molecular chain and the argC gene on the opposite molecular chain of the E. coli K- strain chromosome 12. The nucleotide sequence of the argE gene and the amino acid sequence of the ArgE protein encoded by the argE gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

The argA gene encodes an ArgA amino acid-N-acetyltransferase protein (KEGG, entry No. b2818). The argA gene (GenBank inventory no. NC_000913.2; nucleotide positions: 2947264 to 2948595; Gene ID: 947289) are located between the amiC and recD genes, both on the opposite chromosome chain of E. coli strain K-12. The nucleotide sequence of the argA gene and the amino acid sequence of the ArgA protein encoded by the argA gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

The argR gene encodes a DNA-binding transcriptional double regulator that binds L-arginine, ArgR (KEGG, entry No. b3237). The argR gene (GenBank inventory no. NC_000913.2; nucleotide positions: 3382725 to 3383195; Gene ID: 947861) is located between the yhcN gene on the same molecular chain and the mdh gene on the opposite molecular chain of the E. coli K-12 strain chromosome. The nucleotide sequence of the argR gene and the amino acid sequence of the ArgR protein encoded by the argR gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

Due to the fact that there may be some differences in DNA sequences between genera or strains of the families Methanocaldococcaceae, Thermotogaceae, and Enterobacteriaceae, the genes argJ, argE, argA, and argR are not limited to the genes shown in SEQ ID NOs: 1, 3, 5, 7, and 29 but may include genes that are variant nucleotide sequences or homologous to nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, and 29, encoding variants of ArgJ, ArgE, ArgA, and ArgR proteins.

The term "protein variant" may mean a protein that contains one or more changes in the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8 or 30, which may be substitutions, deletions, insertions and / or additions of one or more amino acid residues, provided that the activity of such a protein is the same as that of ArgJ, ArgE, ArgA, and ArgR proteins, respectively, or the tertiary structure of ArgJ, ArgE, ArgA, and ArgR proteins has not changed significantly compared to the wild-type protein or unmodified protein. The number of changes in protein variants depends on the type or position of the amino acid residue in the tertiary structure of the protein. It can be, but is not strictly limited, from 1 to 30, in another example from 1 to 15, in another example from 1 to 10, in another example from 1 to 5 in SEQ ID NO: 2, 4, 6, 8 and thirty.

An example of substitution, deletion, insertion and / or addition of one or more amino acid residues can be a conservative mutation (s), provided that the activity and properties of the protein variant are preserved and are the same as those of ArgJ, ArgE, ArgA and ArgR proteins. An example of a conservative mutation is a conservative substitution. Conservative substitutions may be reciprocal substitutions between Phe, Trp and Tyr, if the substitution site is an aromatic amino acid; between Ala, Leu, Ile and Val, if the substitution site is a hydrophobic amino acid; between Glu, Asp, Gln, Asn, Ser, His and Thr if the substitution site is a hydrophilic amino acid; between Gln and Asn if the substitution site is a polar amino acid; between Lys, Arg and His, if the substitution site is a basic amino acid; between Asp and Glu, if the substitution site is an acidic amino acid; and between Ser and Thr, if the substitution site is an amino acid having a hydroxyl group. Examples of conservative substitutions include replacing Ser or Thr with Ala, replacing Asn, Glu or Gln with Asp, replacing Ser or Ala with Cys, replacing Asn, Glu, Lys, His, Asp or Arg with Gln, replacing Asn, Gln, Lys or Asp with Glu, replacing Pro with Gly, replacing Asn, Lys, Gln, Arg or Tyr with His, replacing Leu, Met, Val or Phe with Ile, replacing Ile, Met, Val or Phe with Leu, replacing Asn, Glu, Gln, His or Arg with Lys, replacing Ile, Leu, Val or Phe with Met, replacing Trp, Tyr, Met, Ile or Leu with Phe, replacing Thr or Ala with Ser, replacing Ser or Ala with Thr, replacing Phe or Tyr with Trp replacing His, Phe or Trp with Tyr and replacing Met, Ile or Leu with Val.

An example of the substitution, deletion, insertion and / or addition of one or more amino acid residues can also be a non-conservative (s) mutation (s), provided that this (s) mutation (s) are compensated by one or more mutations in different positions of the amino acid sequence, so that the activity and properties of the protein variant are preserved and are the same as those of ArgJ, ArgE, ArgA, and ArgR proteins.

The degree of homology of the protein or DNA can be determined using several well-known approaches, for example, the computer algorithms BLAST and FASTA and the ClustalW method. The BLAST algorithm (Basic Local Alignment Search Tool, www.ncbi.nlm.nih.gov/BLAST/), which allows for hierarchical search, is embedded in the programs blastp, blastn, blastx, megablast, tbiastn and tbiastx; these programs assign significance to found objects using the statistical methods described in Samuel K. and Altschul S.F. ("Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc. Natl. Acad. Sci. USA, 1990, 87: 2264-2268; "Applications and statistics for multiple high-scoring segments in molecular sequences" (Proc. Natl. Acad. Sci. USA, 1993, 90: 5873-5877). The BLAST algorithm computes three parameters: the number of amino acid residues, identity, and similarity. The FASTA search algorithm is described in Pearson W.R. ("Rapid and sensitive sequence comparison with FASTP and FASTA," Methods Enzymol., 1990, 183: 63-98). The ClustalW method is described in Thompson J.D. et al. (“CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice”, Nucleic Acids Res., 1994, 22: 4673-4680).

Moreover, the genes argJ, argE, argA, and argR may be variants of nucleotide sequences. The term "nucleotide sequence variant" may mean a nucleotide sequence that encodes a "protein variant" using any synonymous amino acid codons in accordance with the table of the standard genetic code (see, for example, Lewin B., Genes VIII, 2004, Pearson Education, Inc. , Upper Saddle River, NJ 07458). The term “nucleotide sequence variant” can also mean, but not limited to this example, a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to that presented in SEQ ID NOs: 1, 3, 5, 7, and 29, or with a probe which can be synthesized based on these nucleotide sequences, provided that it encodes a functional protein. "Harsh conditions" means those conditions under which a specific hybrid is formed, for example, a hybrid having an identity of at least 70%, at least 80%, at least 90%, at least 95%, at least 96% , not less than 97%, not less than 98% or not less than 99%, and a nonspecific hybrid is not formed, for example, a hybrid having a homology lower than that indicated above. A practical example of harsh conditions can be a single or multiple washing, or, alternatively, two or three washing at a salt concentration of 1 × SSC (standard sodium citrate or standard sodium chloride) and 0.1% SDS (sodium dodecyl sulfate) or, in another case, 0.1 × SSC and 0.1% SDS at 60 ° C or 65 ° 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 a Hybond ™ -N + positive charge nylon membrane (GE Healthcare) under severe conditions is 15 minutes. Rinsing can be done two or three times. As a probe, a portion of the sequence complementary to that shown in SEQ ID NOs: 1, 3, 5, 7, and 29 can be used. A similar probe can be obtained using the PCR method (polymerase chain reaction, White TJ et al., Trends Genet , 1989, 5: 185-189) using oligonucleotide primers prepared based on the sequences shown in SEQ ID NOs: 1, 3, 5, 7 and 29 and a DNA fragment containing the nucleotide sequence as a template. The recommended probe length should be> 50 bp, it can be selected depending on hybridization conditions and is usually from 100 to 1000 bp For example, when using a DNA fragment of about 300 bp in length as a probe washing conditions can be as follows: 2 × SSC, 0.1% SDS at 50 ° C, or at 60 ° C, or at 65 ° C.

Genes encoding ArgJ proteins of the species M. jannaschii and T. neapolitana, and ArgE, ArgA and ArgR proteins of the E. coli species are known (see description above); therefore, genes encoding variants of the ArgJ, ArgE, ArgA, and ArgR proteins can be obtained using the PCR method (polymerase chain reaction described in White TJ et al., Trends Genet, 1989, 5: 185-189) using primers synthesized based on the nucleotide sequence of the argJ, argE, argA, and argR genes, or chemically synthesized like full-sized genes. Genes encoding ArgJ, ArgE, ArgA, and ArgR proteins or variants thereof from other microorganisms can be obtained in a similar manner.

Ornithine acetyltransferase activity (monofunctional enzyme ArgJ) (EU: 2.3.1.35; synonyms: glutamate-N-acetyltransferase, N-acetyl-L-glutamate synthetase, etc.) means activity in the catalysis of the following reaction: N2-acetyl-L-ornithine + L-glutamate ↔ L-ornithine + N-acetyl-L-glutamate.

The activity of ornithine acetyltransferase / N-acetylglutamate synthase (bifunctional enzyme ArgJ) (EC: 2.3.1.35/2.3.1.1) means activity in the catalysis of the following reaction: N2-acetyl-L-ornithine + L-glutamate ↔ L-ornithine + N-acetyl- L-glutamate and acetyl-CoA + L-glutamate ↔ CoA + N-acetyl-L-glutamate.

N-acetylornithine deacetylase (ArgE) activity (EC: 3.5.1.16; synonyms: 2-N-acetyl-L-ornithinamidohydrolase, N-acetylornithinase) means activity in the catalysis of the following reaction: N2-acetyl-L-ornithine + H ₂ O ↔ acetate + L-ornithine.

The activity of amino acid-N-acetyltransferase (ArgA) (EC: 2.3.1.1; synonyms: N-acetylglutamate synthase, N-acetyl-L-glutamate synthetase) means activity in the catalysis of the following reaction: acetyl-CoA + L-glutamate ↔ CoA + N-acetyl -L-glutamate.

The term “operably linked to a gene” may mean that one or more regulatory sequences are linked to the nucleotide sequence of a nucleic acid molecule or a given gene in such a way that expression is possible (eg, enhanced, increased, constitutive, basal, attenuated, reduced or repressive expression) a nucleotide sequence, preferably expression of a gene product encoded by said nucleotide sequence.

The term “enzyme having at least ornithine acetyltransferase activity” may mean that the enzyme has ornithine acetyltransferase activity (monofunctional ArgJ enzyme) or ornithine acetyltransferase / N-acetylglutamate synthase activity (bifunctional ArgJ enzyme), as described above.

The bacterium described in the present invention can be obtained by introducing the aforementioned DNA into a bacterium initially capable of producing an L-amino acid. In addition, the bacterium described herein can be obtained by reporting the ability to produce the L-amino acid of a bacterium already containing said DNA.

In addition to the properties mentioned above, a bacterium can also have other specific properties, without going beyond the scope of this invention, such as: need various nutrients, have sensitivity, resistance and dependence on antibiotics.

2. The method of obtaining L-amino acids

A method for producing an L-amino acid may include the steps of growing a bacterium in a nutrient medium in order to obtain an L-amino acid, excretion and accumulation in the culture fluid and isolate the L-amino acid from the culture fluid and / or bacterial cells.

The cultivation of the bacteria according to the present invention, the accumulation and purification of the L-amino acid or its salt from the environment, etc. can be carried out in a manner similar to traditional fermentation methods, characterized in that said amino acid is produced using a microorganism. The nutrient medium for producing the L-amino acid may be a typical medium that contains a carbon source, a nitrogen source, inorganic ions and other necessary organic components. Sugars such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose and starch hydrolysates can be used as a carbon source; alcohols such as glycerin, mannitol and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid, malic acid and succinic acid and the like. Inorganic ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate can be used as a nitrogen source; organic nutrients such as soybean hydrolysates; ammonia gas; aqueous ammonia and the like Vitamins, such as vitamin B1, essential substances, for example, organic nutrients, such as nucleic acids, adenine and RNA, or yeast extract and the like, may be present in appropriate amounts or in the form of traces. In addition to the above, small amounts of calcium phosphate, magnesium sulfate, iron ions, manganese ions and the like can be added if necessary.

The cultivation is preferably carried out under aerobic conditions from 16 to 96 hours or from 48 to 72 hours, the temperature of the cultivation, in the range of which the cultivation can be controlled, from 30 to 45 ° C, or in the range from 30 to 37 ° C; medium acidity (pH) is maintained in the range of 5-8, or in the range of 6.5-7.2. The acidity of the medium is maintained using inorganic or organic acidic or alkaline substances, such as ammonia gas. Typically, growing for 1 to 5 days leads to the accumulation of the target L-amino acid in the culture fluid.

After growth, solid particles such as cells and cell debris can be removed from the liquid medium by centrifugation or membrane filtration, and then the target L-amino acid can be isolated from the enzymatic mixture by a combination of known methods, such as concentration, ion exchange chromatography, crystallization, etc. .

Examples

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

Example 1

Obtaining a mutant E. coli strain MG1655 having the deleted argE gene and containing the plasmid pJ-T carrying the argJ gene from T. neapolitana

First, a mutant E. coli strain MG1655 having the deleted argR gene was obtained using a known method (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 6640-6645) (Example 1.1 ) Then, the argE gene was removed using the same method (Example 1.2). The chloramphenicol resistance gene (cat, Cm ^R marker) was used to mark the ΔargR mutation, and the kanamycin resistance gene (kan, Km ^R marker) was used to mark the ΔargE mutation. Cells were cured of the Cm ^R marker as described (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 6640-6645). Plasmid pJ-T containing the argJ gene from T. neapolitana (US patent No. 6897048) was introduced into the obtained mutant E. coli strains MG1655ΔargR and ΔargRΔargE :: Km by electroporation. Electroporation was carried out using a Bio-Rad electroporator (USA) (No. 165-2098, version 2-89) in accordance with the manufacturer's instructions. Thus, mutant E. coli strains MG1655ΔargR / pJ-T and ΔargRΔargE :: Km / pJ-T were obtained.

Example 1.1.

ArgR gene deletion

The E. coli strain MG1655ΔargR was obtained by the λRed-dependent integration method developed by Datsenko KA and Wanner BL [Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 6640-6645). A DNA fragment containing a chloramphenicol resistance marker (Cm ^R ) was obtained by PCR using primers P1 (SEQ ID NO: 9) and P2 (SEQ ID NO: 10) and plasmid pMW118-attL-Cm-attR as a matrix ( WO2005010175 A1). Primer P1 comprises a region complementary to the region located at the 5 ′ end of the argR gene and a region complementary to the attR region. Primer P2 contains a region complementary to the region located at the 3 ′ end of the argR gene, and a region complementary to the attL region. The conditions for PCR were as follows: initial denaturation - 3 min at 95 ° C; profile for the initial two cycles: 1 min at 95 ° C, 30 sec at 50 ° C, 40 sec at 72 ° C; profile for the final 25 cycles: 30 sec at 95 ° C, 30 sec at 54 ° C, 40 sec at 72 ° C; final elongation - 5 min at 72 ° C. The product obtained by PCR (about 1,600 bp) was purified by agarose gel electrophoresis and used for electroporation into E. coli strain MG1655 containing plasmid pKD46 with a temperature-sensitive replicon. Plasmid pKD46 (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 6640-6b45) contains a phage λ DNA fragment of 2.154 nucleotides in length (nucleotide positions 31088 to 33241, GenBank accession no. : J02459), including the λRed genes of the homologous recombination system (γ, β, exo genes) under the control of the arabinose-induced P _araB promoter. Plasmid pKD46 is necessary for the integration of the product obtained by PCR into the chromosome of E. coli strain MO 1655 (ATCC 47076). The E. coli strain MG1655 containing the recombinant plasmid pKD46 can be obtained from E. coli Genetic Stock Center, Yale University, New Haven, USA, accession number CGSC7669.

Electrocompetent cells were prepared as follows: E. coli strain MG1655 / pKD46 was grown at 30 ° C. overnight in LB medium (Luria-Bertani medium, also called lysogenic medium, as described in Sambrook, J. and Russell, DW (2001) Molecular Cloning: A Laboratory Manual (3 ^{rd ed.) Cold Spring Harbor Laboratory} Press), containing ampicillin (150 mg / l).. The cell culture was dissolved 100 times with 5 ml of SOB medium (Sambrook J. and Russell DW, Molecular Cloning:. A Laboratory Manual ( 3 ^{rd ed), Cold Spring Harbor Laboratory} Press, 2001) containing ampicillin (150 mg / l) and L-arabinose (1 mM). Cells were grown with aeration (250 rpm) at 30 ° C to OD ₆₀₀ ≈0.6. Electrocompetent cells were prepared by concentrating 100 times and washing three times with deionized cold water. Electroporation was performed using 70 μl of cells and ≈100 ng of PCR product. After electroporation cells were incubated in 1 ml of SOC medium (Sambrook J. and Russell DW, Molecular Cloning:. A Laboratory Manual ( 3 ^{rd ed), Cold Spring Harbor Laboratory} Press, 2001) at 37 ° C for 2.5 h, placed in L-agar containing chloramphenicol (25 mg / L) and grown at 37 ° C to select Cm ^R recombinants. To remove the pKD46 plasmid, cells were plated twice on L-agar containing chloramphenicol (25 mg / L) at 42 ° C and the resulting colonies were tested for sensitivity to ampicillin. The Cm ^R marker was removed using the pMW-Int / Xis helper plasmid (WO2005010175 A1), which was electroporated into the selected non-plasmid integrants using the technique described above for electroporation of the PCR fragment. After electroporation, the cells were placed in L-agar containing 0.5% glucose and ampicillin (150 mg / L) and incubated at 37 ° C overnight to induce the synthesis of Int / Xis proteins. Clone growth was performed on L-agar without chloramphenicol to select Cm ^S (chloramphenicol-sensitive) variants. Thus, the E. coli strain MG1655ΔargR was obtained.

Example 1.2

ArgE gene divisions

The E. coli strain MG1655ΔargR having the deleted argE gene was obtained by the λRed-dependent integration method originally developed by Datsenko KA and Wanner BL (Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 6640-6645). A DNA fragment containing the Km ^R marker was obtained by PCR using primers P3 (SEQ ID NO: 11) and P4 (SEQ ID NO: 12) and plasmid pMW118-attL-Km-attR as a template (Katashkina Zh.I. et al., Mot Biol. (Mosk.), 2005, 39 (5): 823-831). The E. coli strain MG1655ΔargR / pKD46 was used for electroporation of the obtained DNA fragment (Example 1.1). Antibiotic resistant cells were selected using kanamycin (25 mg / L) and the resulting Km ^R recombinant was cured of the plasmid pKD46 as described in Example 1.1. Thus, E. coli MG1655ΔargRΔargE :: Km was obtained.

Example 2

Obtaining L-arginine using a modified E. coli strain MG1655 having the deleted argE gene and containing the pJ-T plasmid carrying the argJ gene from T. neapolitana

Eight independent colonies of each strain obtained (E. coli MG1655ΔargR / pJ-T and ΔargRΔargE :: Km / pJ-T) were selected to evaluate L-arginine production. The obtained strains were grown in L-agar plates at 37 ° C overnight, then a loop from each of the obtained cultures was introduced into 2 ml of fermentation medium in 20 × 200 mm tubes and cultivated at 32 ° C for 72 hours on a rotary shaker ( 220-230 rpm).

The content of the fermentation medium (g / l):

Glucose fifty (NH ₄ ) ₂ SO ₄ 35 K ₂ HPO ₄ 2.0 MgSO ₄ × 7H ₂ O 1.0 Thiamine hydrochloride 0.002 Yeast extract 1.0-5.0 CaCO ₃ twenty

Glucose and magnesium sulfate were sterilized separately. CaCO _{3 was} stylized with dry heat at 180 ° C for 2 hours. The pH value was maintained at 7.0. Antibiotics were introduced into the medium after sterilization.

The amount of L-arginine accumulated in the medium was determined by thin layer chromatography (TLC) or paper chromatography using the mobile phase: butanol: acetic acid: water = 4: 1: 1 (v / v). A solution of ninhydrin (2%) in acetone was used as a coloring reagent. A spot containing L-arginine was excised, L-arginine was eluted with a 0.5% aqueous solution of CdCl _2, and the amount of L-arginine was evaluated spectrophotometrically at 540 nm. The results of 8 independent fermentation tubes (as an average value) are presented in Table 1. As can be seen from Table 1, the modified E. coli strain MG1655ΔargR / pJ-T causes a greater accumulation of L-arginine compared to the parent E. coli strain MG1655ΔargRΔargE :: Km / pJ-T.

Example 3

Obtaining a mutant E. coli strain MG1655 having the deleted argE gene and argJ gene from T. neapolitana introduced into the chromosome

The argJ gene from T. neapolitana was inserted into the chromosome of E. coli strain MG1655ΔargR to get rid of the instability effect of plasmid pJ-T carrying the argJ gene. To enhance the expression of the argJ gene, an efficient artificial P _nlp8φ10 promoter was _obtained that was placed in front of the argJ gene (Example 3.1).

In order to exclude the natural production of L-arginine, the argA gene was removed from the chromosome (Example 3.1).

The E. coli strain MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJ was used in further genetic operations. The ΔargE mutation was labeled with the kanamycin resistance gene (kan) (Example 1.2) and introduced into the chromosome of E. coli strain MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJ using the well-known P1 transduction method (Miller JH, Experiments in molecular genetics, Cold Spring Harbor Laboratory Press, 1972). Colonies of Km-resistant transductants were selected on L-agar containing kanamycin (25 mg / L) (Example 1). Thus, the E. coli strain MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJΔargE :: Km was obtained.

Example 3.1

Obtaining strain E. coli MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJ

The E. coli strain MG1655 containing the deleted argA gene labeled with the cat gene was obtained by the λRed-dependent integration method, originally developed by Datsenko K.A. and Wanner BL (Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 6640-6645) (Example 1.1). The DNA fragment containing the Cm ^R marker was obtained by PCR using primers P5 (SEQ ID NO: 13) and P6 (SEQ ID NO: 14) and plasmid pMW118-attL-Cm-attR as a template (WO2005010175 A1). The E. coli strain MG1655Δarg / pKD46 was used for electroporation of the obtained DNA fragment. The chloramphenicol resistance marker (Cm ^R ) was removed using the plasmid pMW-Int / Xis (WO2005010175 A1) as described in Example 1.1. Thus, the E. coli strain MG1655ΔargAΔargR was obtained.

The DNA fragment containing the nlpD gene promoter from E. coli was obtained by PCR using primers P7 (SEQ ID NO: 15) and P8 (SEQ ID NO: 16) and E. coli chromosomal DNA MG1655 as a template. The conditions for PCR were as follows: initial denaturation - 3 min at 95 ° C; profile for the initial 2 cycles: 1 min at 95 ° C, 30 sec at 50 ° C, 40 sec at 72 ° C; profile for the final 25 cycles: 20 sec at 94 ° C, 20 sec at 55 ° C, 15 sec at 72 ° C; final elongation - 5 min at 72 ° C. The resulting DNA fragment (about 200 bp) was purified by agarose gel electrophoresis and treated with endonucleases PaeI and SalI (Fermentas) (Sambrook J., Fritsch EF and Maniatis T., “Molecular Cloning: A Laboratory Manual”, 2 ^nd ed., Cold Spring Harbor Laboratory Press (1989); manufacturer's instructions). The resulting DNA fragment was ligated with plasmid pMIV-5JS (Russian patent application No. 2006132818 and EP 1942183), which was pretreated with endonuclease Pael and SalI, following the manufacturer's instructions. The ligation mixture was incubated at 4 ° C. overnight and then used to transform E. coli strain MG1655 (ATCC 47076) by electroporation as described in Example 1. The resulting transformants were placed on L-agar containing ampicillin (50 mg / L), and plates were incubated at 37 ° C. overnight until individual colonies were visible. Plasmids were isolated from the obtained transformants and analyzed by restriction method using Pael and SalI nucleases. Thus, the plasmid pMIV-PnlpD containing the native P _nlpD promoter of the _nlpD gene from E. coli was obtained.

A -10 region of the P _nlp D promoter was _randomized and a P _nlp8 promoter was _selected . The 3'-end of the P _nlpD promoter was obtained by PCR using primers P7 (SEQ ID NO: 15) and P9 (SEQ ID NO: 17) and plasmid pMIV-PnlpD as template. Primer P9 contains random nucleotides that are designated in sequence SEQ ID NO: 17 as "n", where n is A, G, C or T. Conditions for PCR were as follows: initial denaturation - 3 min at 95 ° C; profile for the initial 2 cycles: 1 min at 95 ° C, 30 sec at 50 ° C, 40 sec at 72 ° C; profile for the final 25 cycles: 20 sec at 94 ° C, 20 sec at 60 ° C, 15 sec at 72 ° C; final elongation - 5 min at 72 ° C. The 5'-end of the P _nlpD promoter was obtained by PCR using primers P8 (SEQ ID NO: 16) and P10 (SEQ ID NO: 18) and plasmid pMIV-PnlpD as template. Primer P10 contains random nucleotides that are designated in sequence SEQ ID NO: 18 as “n”, where n is A, G, C, or T. Conditions for PCR were as follows: initial denaturation - 3 min at 95 ° C; profile for the initial 2 cycles: 1 min at 95 ° C, 30 sec at 50 ° C, 40 sec at 72 ° C; profile for the final 25 cycles: 20 sec at 94 ° C, 20 sec at 60 ° C, 15 sec at 72 ° C; final elongation - 5 min at 72 ° C.

Both obtained DNA fragments were purified by agarose gel electrophoresis, treated with BglII endonuclease (Fermentas) and ligated in equimolar ratios (Sambrook J., Fritsch EF and Maniatis T., Molecular Cloning: A Laboratory Manual, 2 ^nd ed., Cold Spring Harbor Laboratory Press, 1989). The ligation mixture was incubated at 4 ° C. overnight. The obtained DNA fragment was obtained by PCR using primers P7 (SEQ ID NO: 15) and P8 (SEQ ID NO: 16) and the obtained DNA fragment as a matrix. The conditions for PCR were as follows: initial denaturation - 3 min at 95 ° C; profile for the initial 2 cycles: 1 min at 95 ° C, 30 sec at 50 ° C, 40 sec at 72 ° C; profile for the final 12 cycles: 20 sec at 94 ° C, 20 sec at 60 ° C, 15 sec at 72 ° C; final elongation - 5 min at 72 ° C. The resulting DNA fragment (about 200 bp) was purified by agarose gel electrophoresis.

The purified DNA fragment was treated with a Klenov fragment (Fermentas) and ligated in equimolar ratio with the plasmid pMW118-λattL-Km ^R- λattR (Russian Patent Application No. 2006134574), which was pretreated with XbaI endonuclease (Fermentas) and Klenov fragment. The ligation mixture was incubated at 4 ° C. overnight and then used to transform E. coli strain MG1655 by electroporation as described in Example 1. The resulting transformants were placed on L-agar containing kanamycin (20 mg / L), and plates were incubated at 37 ° C overnight until individual colonies are visible. Plasmids were isolated from the obtained transformants and analyzed by restriction method using PstI and HindIII (Fermentas) nucleases (Sambrook J., Fritsch EF and Maniatis T., Molecular Cloning: A Laboratory Manual, 2 ^nd ed., Cold Spring Harbor Laboratory Press ( 1989); manufacturer's manual). Thus, the plasmid pMW-Km-Pnlp8 containing the P _nlp8 promoter was _obtained .

The DNA fragment containing the argJ gene was obtained by PCR using primers P11 (SEQ ID NO: 19) and P12 (SEQ ID NO: 20) and plasmid pJ-T (US patent 6897048) as a template. The conditions for PCR were as follows: initial denaturation - 1 min at 95 ° C; profile for 25 cycles: 1 min at 95 ° C, 30 sec at 55 ° C, 1 min at 72 ° C; final elongation - 2 min at 72 ° C. The resulting DNA fragment (about 1,200 bp) was purified by agarose gel electrophoresis.

A DNA fragment containing the λattL-Km-λattR-P _{nlp8φ10 cassette} was obtained by PCR using primers P13 (SEQ ID NO: 21) and P14 (SEQ ID NO: 22) and plasmid pMW-Km-Pnlp8 as a template. The conditions for PCR were as follows: initial denaturation - 1 min at 95 ° C; profile for 25 cycles: 1 min at 95 ° C, 30 sec at 55 ° C, 1 min 40 sec at 72 ° C; final elongation 5 min at 72 ° C. The resulting DNA fragment (about 1.700 bp) (Figure 1) was purified by agarose gel electrophoresis. Primers P11 (SEQ ID NO: 19) and P14 (SEQ ID NO: 22) contained overlapping portions.

The obtained DNA fragment (Figure 1) with overlapping regions was obtained by PCR using primers P12 (SEQ ID NO: 20) and P13 (SEQ ID NO: 21) and the obtained DNA fragment as a template. The conditions for PCR were as follows: initial denaturation - 2 min at 95 ° C; profile for 30 cycles: 30 sec at 94 ° C, 30 sec at 55 ° C, 2 min at 72 ° C; final elongation - 5 min at 72 ° C. The resulting DNA fragment (about 2.900 bp) was purified by agarose gel electrophoresis.

The resulting DNA fragment contains the Km ^R marker and the argJ gene under the control of the P _nlp8φ10 promoter. This fragment was integrated instead of a cluster of artPIQMJ genes in the chromosome of E. coli strain MG1655ΔargAΔargR by λRed-dependent integration, as described above. Thus, the E. coli strain MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJ was obtained.

Example 4

Obtaining L-arginine using a modified E. coli strain MG1655 having the deleted argE gene and the argJ gene from T.neapolitana introduced into the chromosome

Six independent colonies of each of the obtained strains (E. coli MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJ and MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJΔargE :: Km) were selected to evaluate the production of L-arginine. L-arginine production was determined as described in Example 2. The results of 6 independent fermentation tubes (as an average result) are shown in Table 2. As can be seen from Table 2, the modified E. coli strain MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJΔargE :: Km causes a greater accumulation of L-arginine in comparison with the parent E. coli strain MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJ.

Example 5

Obtaining a mutant E. coli strain MG1655 having a weakened argE gene and an argJ gene from T. neapolitana introduced into the chromosome

The E. coli strain MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJ was used as the parent strain to obtain a strain having the reduced expression expression argE gene. First, a Cm ^R marker was introduced into the intergenic region between the ppc and argE genes of strain MG1655 by the λRed-dependent integration method (Example 1.1). The DNA fragment containing the Cm ^R marker encoded by the cat gene was obtained by PCR using primers P15 (SEQ ID NO: 23) and P16 (SEQ ID NO: 24) and plasmid pMW118-attL-Cm-attR as a matrix ( WO2005010175 A1). Primer P15 contains a region complementary to a region located below the argE gene and a region complementary to attR. Primer P16 contains a plot complementary to the plot located below the ppC gene and a plot complementary to attL. The conditions for PCR were as follows: initial denaturation - 3 min at 95 ° C; profile for the initial 2 cycles: 1 min at 95 ° C, 30 sec at 50 ° C, 40 sec at 72 ° C; profile for the initial 25 cycles: 30 sec at 95 ° C, 30 sec at 54 ° C, 40 sec at 72 ° C; final elongation - 5 min at 72 ° C. The PCR product (about 1,600 bp) was purified by agarose gel electrophoresis and used for electroporation of the E. coli strain MG1655 / pKD46, as described in Example 1.1. After electrotransformation of E. coli strain MG1655 / pKD46 with the obtained DNA fragment containing the cat gene, several colonies were placed on plates with L-agar containing chloramphenicol (20 mg / L). Electrocompetent cells were prepared, electroporation was performed, and Cm ^R recombinants were selected as described in Example 1.1. Thus, the E. coli strain MG1655-Cm-argE was obtained.

Secondly, L76K substitution in ArgE was obtained. The first DNA fragment containing the Cm ^R marker and the distal argE portion encoding the L76K mutation was obtained by PCR using primers P17 (SEQ ID NO: 25) and P18 (SEQ ID NO: 26) containing overlapping regions and chromosomal DNA E. coli strain MG1655-Cm-argE as a matrix. The conditions for PCR were as follows: initial denaturation - 1 min at 95 ° C; profile for 25 cycles: 1 min at 95 ° C, 30 sec at 55 ° C, 2 min at 72 ° C; final elongation - 5 min at 72 ° C. The obtained first DNA fragment (about 2.800 bp) (Figure 2) was purified by agarose gel electrophoresis.

The second DNA fragment containing the proximal part of argE encoding the L76K mutation was obtained by PCR using primers P19 (SEQ ID NO: 27) and P20 (SEQ ID NO: 28) and chromosomal DNA of E. coli strain MG1655-Cm-argE c as a matrix. The conditions for PCR were as follows: initial denaturation - 1 min at 95 ° C; profile for 25 cycles: 1 min at 95 ° C, 30 sec at 55 ° C, 30 sec at 72 ° C; final elongation - 5 min at 72 ° C. The obtained second DNA fragment (about 300 bp) (Figure 2) was purified by agarose gel electrophoresis.

The third DNA fragment was obtained by PCR using primers P17 (SEQ ID NO: 25) and P20 (SEQ ID NO: 28) and the obtained first and second DNA fragments with overlapping regions (Figure 2) as a template. The conditions for PCR were as follows: initial denaturation - 2 min at 95 ° C; profile for 30 cycles: 30 sec at 94 ° C, 30 sec at 55 ° C, 2 min 20 sec at 72 ° C; final elongation - 5 min at 72 ° C. The obtained third DNA fragment (about 3.100 bp) was purified by agarose gel electrophoresis. Thus, a third DNA fragment was obtained containing the Cm ^R marker and the argE gene with substitutions T226A and T227A in the nucleotide sequence of SEQ ID NO: 3, resulting in a mutation L76K. The mutant argE gene was named as the argEm24 gene.

Finally, the third DNA fragment was integrated instead of the native argE gene into the chromosome of E. coli strain MG1655ΔargAΔargRΔartP-J :: P _nlpφ10 argJ by the λRed-dependent integration method, as described above. Thus, the E. coli strain MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJargEm24 :: Cm was obtained.

Example 6

Production of L-arginine using a modified E. coli strain MG1655 having a weakened argE gene and an argJ gene from T. neapolitana introduced into the chromosome

Six independent colonies of each of the obtained strains (E. coli MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJargEm24 :: Cm and MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJ) were selected to evaluate the production of L-arginine. The production of L-arginine was evaluated as described in Example 2. The results of 6 independent fermentation tubes (as an average value) are presented in Table 3. As can be seen from Table 3, the modified E. coli strain MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJΔargEm24 :: Cm causes a greater accumulation of L-arginine in comparison with the parent E. coli strain MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJ.

Example 7

Enzymatic activity of the mutant protein ArgE ^L76K

The enzymatic activity of the control protein and the mutant ArgE ^L76K protein encoded by the argEm24 mutant gene was measured as described in K. Takahara et al. FEBS J., 2005, 272: 5353-5364, using crude protein extracts obtained after cell disruption by ultrasound. The E. coli strains MG1655, MG1655ΔargE, and MG1655argEm24 were used as protein sources to evaluate the level of attenuation of ArgE activity due to L76K mutation. The E. coli strains MG1655ΔargE and MG1655argEm24 were obtained as described in Supplementary Example 1. As can be seen from Table 4, the ArgE ^L76K protein showed a specific activity of less than about 1% compared to the unmodified ArgE protein.

Additional example 1.

The E. coli strain MG1655 ΔargE :: Km was obtained according to the procedure described in Example 1.2 for the E. coli strain MG1655ΔargRΔargE :: Km. The Km ^R marker was removed using the plasmid pMW-Int / Xis (WO2005010175 A1) as described in Example 1.1 for the Cm ^R marker. Thus, the E. coli MG1655ΔargE strain was obtained.

The E. coli strain MG1655argEm24 :: Cm was obtained as described in Example 5 for E. coli strain MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJ argEm24 :: Cm. The Cm ^R marker was removed using the plasmid pMW-Int / Xis (WO2005010175 A1) as described in Example 1.1. Thus, the E. coli strain MG1655ΔargEm24 was obtained.

Table 1 Effect of ΔargE mutation on L-arginine production by E. coli strain containing the pJ-T plasmid carrying the argJ gene from T. neapolitana Strain OD ₅₄₀ L-Arginine, g / l MG1655ΔargR / pJ-T 14.6 ± 0.7 1.8 ± 0.1 MG1655ΔargRΔargE :: Km / pJ-T 16.9 ± 0.6 2.8 ± 0.3

table 2 Effect of ΔargE mutation on L-arginine production by E. coli strain containing the argJ gene introduced into the chromosome Strain OD ₅₄₀ L-Arghinn, g / l MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJ 24.7 ± 0.5 0.9 ± 0.1 MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJ ΔargE :: Km 23.0 ± 1.3 3.0 ± 0.6

Table 3 Effect of the argEm24 mutation on L-arginine production by E. coli strain having the argJ gene introduced into the chromosome Strain OD ₅₄₀ L-Arginine, g / l MG1655ΔargAΔargRΔartP-J :: P _nlp8φ10 argJ 24.7 ± 0.5 0.9 ± 0.1 MG1655ΔargAΔargRΔartP-J :: P _nlpφ10 argJ argEm24 :: Cm 24.5 ± 3.4 9.3 ± 0.9

Table 4 The effect of the argEm24 mutation on the activity of N-acetylornithine deacetylase (ArgE) (in nmol / mg min) in E. coli strain MG1655. Values are given as mean ± SD (n = 4), where SD is the standard deviation Strain The specific activity of ArgE, nmol / mg min MG1655 (control) 750 MG1655ΔargE <0.5 MG1655argEm24 3.0

Claims (10)

1. The bacterium-producer of L-arginine, belonging to the genus Escherichia, having recombinant DNA, which contains the argJ gene encoding an enzyme having at least ornithine acetyltransferase activity, characterized in that the bacterium is modified so that it contains N- acetylornithine deacetylase with impaired activity. 2. The bacterium according to claim 1, characterized in that said argJ gene is derived from a microorganism having an enzyme having the activity of ornithine acetyltransferase or ornithine acetyltransferase / N-acetylglutamate synthase. 3. The bacterium according to claim 1 or 2, characterized in that said argJ gene is derived from a microorganism belonging to a family selected from the group consisting of the families Thermotogaceae, Bacillaceae and Methanocaldococcaceae. 4. The bacterium according to claim 3, characterized in that said argJ gene originates from the species Thermotoga neapolitana. 5. The bacterium according to claim 1 or 4, characterized in that said argJ gene encodes a protein selected from the group consisting of:
(A) a protein having the amino acid sequence shown in SEQ ID NO: 2, which has an ornithine acetyltransferase / N-acetylglutamate synthase activity;
(B) a variant of a protein having the amino acid sequence shown in SEQ ID NO: 2, but which includes the substitution, deletion, insertion and / or addition of one or more amino acid residues and has the activity of ornithine acetyltransferase / N-acetylglutamate synthase in accordance with the amino acid sequence represented by in SEQ ID NO: 2. 6. The bacterium according to claim 1, characterized in that N-acetylornithine deacetylase is a protein selected from the group consisting of:
(C) a protein having the amino acid sequence shown in SEQ ID NO: 4;
(D) a variant of a protein having the amino acid sequence shown in SEQ ID NO: 4, but which includes the substitution, deletion, insertion and / or addition of one or more amino acid residues and the activity of N-acetylornithine deacetylase in accordance with the amino acid sequence shown in SEQ ID NO: 4. 7. The bacterium according to claim 1, characterized in that the indicated activity of N-acetylornithine deacetylase is reduced. 8. The bacterium according to claim 1, characterized in that the indicated activity of N-acetylornithine deacetylase is absent. 9. The bacterium according to claim 1, characterized in that said bacterium belongs to the species Escherichia coli. 10. A method of obtaining L-arginine or its salt, including:
(i) growing bacteria according to any one of paragraphs. 1-9 in a nutrient medium;
(ii) the accumulation of L-arginine or its salt in bacteria or culture fluid, or both; and if necessary
(iii) isolation of L-arginine or its salt from a bacterium or culture fluid.

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