The method of obtaining purine nucleosides and nucleotides, producing strains of purine nucleosides (options)

 

The invention relates to biotechnology and is a way to obtain purine nucleosides such as inosine and xanthosine, as well as a method of obtaining a purine nucleotides, such as inosine-5'-phosphate, xanthosine-5'-phosphate and guanosine-5'-phosphate, as producers use strains of bacteria, as belonging to the genus Escherichia and the genus Bacillus, in which the production of purine nucleosides these bacteria increased by increasing the activity of the proteins encoded by the genome rhtA (ybiF). 8 N., and 9 C.p. f-crystals, 7 tab., 5 Il.

The technical field

The present invention relates to a method for producing purine nucleosides such as inosine and xanthosine, which is an important source reagents for the synthesis of inosine-5’-phosphate, xanthosine-5’-phosphate and guanine-5’-phosphate, and to a new microorganism used for their products.

Prior art

Traditionally nukes get on an industrial scale by fermentation using strains of microorganisms, auxotrophic for adenine, or these strains, which are additionally given the resistance to various compounds such as purine analogues the research Institute No. 38-23039, 54-17033, 55-2956 and 55-45199 laid patent application of Japan No. 56-162998, patent application Japan 57-14160 and 57-41915 and laid patent application of Japan No. 59-42895), to the genus Brevibacterium (patent application of Japan No. 51-5075 and 58-17592 and Agric. Biol. Chem., 42, 399 (1978)), to the genus Escherichia (PCT application WO 9903988) and the like.

Getting these mutant strains usually consists of treatment of microorganisms with the aim of obtaining mutations, for example, by irradiation of UV-radiation or treatment with nitrosoguanidine (N-methyl-N’-nitro-N-nitrosoguanidine) with subsequent selection of the desired strain on a suitable nutrient medium for selection. On the other hand, also practiced the cultivation of the mutant strains belonging to the genus Bacillus (lined patent application Japan№58-158197, 58-175493, 59-28470, 60-156388, 1-27477, 1-174385, 3-58787, 3-164185, 5-84067 and 5-192164) and to the genus Brevibacterium (lined patent application Japan 63-248394), obtained using genetic engineering techniques.

Previously the authors of the present invention has been based on E. coli K12 mutant containing a mutation rhtA23, giving resistance to high concentrations of threonine, homoserine and some other amino acids and analogs of amino acids on a minimal nutrient medium. In addition, this mutation rhtA23 improved production of L-trione the Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California, August 24-29, 1997, abstract No.457). Moreover, the authors of the present invention found that the rhtA gene is located at position 18 min on the E. coli chromosome is near, and rhtA gene is identical to an open reading frame ybiF located between genes rehv and ompX. In addition, the authors of the present invention have found that the mutation rhtA23 is a replacement And the G position 1 relative to the start codon ATG. It is assumed that this mutation increases the transport of threonine and homoserine of cells (ABSTRACTS of the 17thInternational Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California, August 24-29, 1997, abstract No.457).

But currently there are no reports describing some exporters purine compounds.

Description of the invention

The aim of the present invention is to increase the productivity of nucleosides strains-producers of nucleosides and the provision of a method of producing nucleosides such as inosine and xanthosine using these strains.

This goal was achieved by establishing the fact that the mutation rhtA23 gives the microorganism resistance to purine analog, 8-asadinho, and increases the production of the nucleoside. In addition, natural rhtA gene, encoding as predpoll is the resistance to purine analog, when natural allele of the specified gene is introduced into the cell on mnogoseriynom vector. Moreover, natural rhtA gene can increase the production of strain when in the appropriate strains-producers of nucleosides belonging to the genus Escherichia or Bacillus, introduced additional copies of the specified gene. Thus was accomplished the present invention.

Thus, the present invention provides a microorganism belonging to the genus Escherichia or Bacillus, with the ability to production of purine nucleosides.

In particular, the present invention provides a microorganism with a high potential for the production of purine nucleosides based on the increased activity of a protein involved are expected in the transport of purine nucleosides from the cell of the specified microorganism. More specifically, the present invention provides a microorganism with a high potential for the production of purine nucleosides based on the increased expression of the gene encoding a protein involved in excretion of purine nucleosides.

Further, the present invention provides a method of obtaining a purine nucleosides by fermentation, comprising the stage of growth Titelnoj environment, and excretion of purine nucleosides from the culture fluid.

Further, the present invention provides a method of obtaining a purine nucleotides, such as inosine-5’-phosphate and xanthosine-5’-phosphate, comprising the stage of growth of bacteria according to the present invention in a nutrient medium, phosphorylation received and accumulated purine nucleoside, and secretions obtained purine nucleoside.

The present invention also provides a method of producing guanosine-5’-phosphate, comprising the stage of growth of the above bacteria in a nutrient medium, phosphorylation received and accumulated xanthosine, amination received xanthosine-5’-phosphate, and secretions obtained guanosin-5’-phosphate.

The present invention includes the following.

The invention 1. The bacterium belonging to the genus Escherichia or the genus Bacillus, with the ability to products of purine nucleoside in which the activity of the protein, as described in paragraphs (A) or (B) in the cell mentioned bacteria raised:

(A) a protein that presents the amino acid sequence listed in sequence number 2;

(B) a protein that presents the amino acid sequence is an explicit sequence listed sequence number 2, and which has activity, giving bacteria more resistant to 8-asadinho.

The invention 2. The bacterium according to the Invention 1, in which the activity of the proteins described in paragraphs (A) or (B) is increased by transformation of bacteria with DNA that encodes a protein, as described in paragraphs (A) or (B), or by changing the regulation of expression of the indicated DNA in the above-mentioned bacteria.

The invention 3. The bacterium in accordance with the Invention 2, in which the transformation is carried out using mnogoopytnogo vector.

The invention 4. The bacterium according to the Invention 1, in which the purine nucleoside is inosine.

The invention 5. The bacterium according to the Invention 1, in which the purine nucleoside is xanthosine.

The invention 6. A method of obtaining a purine nucleoside, comprising the stage of growth of bacteria in accordance with any one of Inventions 1 through 5 in a nutrient medium and a selection from the culture fluid obtained and accumulated therein purine nucleoside.

The invention 7. The method in accordance with the Invention 6, in which the purine nucleoside is inosine.

The invention 8. --- What about in accordance with any one of the Inventions 6-8, in which the bacterium is modified to increase the expression of genes of the biosynthesis of purine nucleosides.

The invention 10. A method of obtaining a purine nucleotide, including the stage of growth of bacteria in accordance with any one of Inventions 1 through 5 in a nutrient medium, phosphorylation received and accumulated nucleoside, and the selection is received and accumulated purine nucleotide.

The invention 11. The method in accordance with the Invention 10, in which the purine nucleotide is inosine-5’-phosphate.

The invention 12. The method in accordance with the Invention 10, in which the purine nucleotide is xanthosine-5’-phosphate.

Invention 13. The method in accordance with any one of the Inventions 10-12, in which the bacterium is modified to increase the expression of genes of the biosynthesis of purine nucleosides.

The invention 14. The method of obtaining guanosin-5’-phosphate, comprising the stage of growth of bacteria in accordance with the Invention 5 in a nutrient medium, phosphorylation received and accumulated xanthosine, amination received xanthosine-5’-phosphate, and the selection is received and accumulated guanosin-5’-phosphate.

The invention 15. The method in accordance with the Invention 14, in which the bacterium modable will be described in detail below.

1. The microorganism according to the present invention.

The above bacterium according to the present invention is a bacterium belonging to the genus Escherichia or the genus Bacillus, with the ability to products of purine nucleoside in which the activity of the protein, as described in paragraphs (A) or (B) in the cell mentioned bacteria is increased: (A) a protein that presents the amino acid sequence listed in sequence number 2; (C) a protein that presents the amino acid sequence comprising deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence listed in sequence No. 2, and which has the activity that gives the bacteria resistance to 8-asadinho.

The term “bacterium belonging to the genus Escherichia or the genus Bacillus” means that the bacterium belongs to the genus Escherichia or the genus Bacillus in accordance with the classification known to a specialist in the field of Microbiology. As examples of the microorganism belonging to the genus Escherichia, used in the present invention, may be mentioned the bacterium Escherichia coli (E. coli). As examples of the microorganism belonging

The term “purine nucleoside” includes inosine, xanthosine, guanosine and adenosine.

Used here, the term “capacity for the production of purine nucleoside” refers to the ability to the production and accumulation of purine nucleoside in a nutrient medium. The term “capable of production of purine nucleoside” means that the microorganism belonging to the genus Escherichia or the genus Bacillus, has the ability to produce and accumulate in the medium purine nucleoside more than a natural strain of E. coli, such as E. coli W3110 and MG1655, or natural strain of B. subtilis, such as a strain of B. subtilis 168, and preferably means that the microorganism is able to produce and accumulate in the medium, the amount of not less than 10 mg/l, more preferably not less than 50 mg/l of inosine, xanthosine, guanosine or adenosine.

The term “protein activity, as described in paragraphs (A) or (B) in the cell mentioned bacteria increased” means that the number of molecules of a specified protein in the cell is increased or the activity in terms of protein increased. The term “activity” means an activity that gives the bacteria resistance to 8-asadinho.

To proteins according to the present isolates sequence, see the list of sequences under number 2;

(B) a protein that presents the amino acid sequence comprising deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence listed in sequence number 2, and which has the activity that gives the bacteria resistance to 8-asadinho.

A protein that presents the amino acid sequence listed in sequence number 2, is a protein RhtA. Protein RhtA is, as expected, a transmembrane protein, consisting of 95 amino acids and have an unknown function. Protein RhtA encoded rhtA gene. RhtA gene is located at position 18 min on the E. coli chromosome near the operon glnHPQ coding for components of the system of transport of glutamine. RhtA gene is identical to an open reading frame (RF1) gene ybiF (nucleotides from 764 in 1651 in sequence with inventory number AAA, gi:440181 in GenBank), located between genes rehv and Omr. Plot expressing the protein encoded by the specified ORF was designated as rhtA (rht: resistance to homoserine and threonine - resistant homoserine and threonine), as earlier authors present invention, the floor is non resistance to high concentrations of threonine and homoserine on a minimal nutrient medium (SU Patent No.974817, Astaurova, O. C. et al., Appl. Bioch. And Environ., 21, 611-616 (1985)). This mutation rhtA23 improved production of L-threonine (SU Patent No.974817, US Patent No.6165756), homoserine and glutamate (Astaurova, O. C. et al., Appl. Bioch. And Environ., 27, 556-561, 1991) the corresponding strain-producing E. coli, such as strain VKPM B-3996. In addition, the authors of the present invention have found that the mutation rhtA23 is a replacement And the G position 1 relative to the start ATG codon (ABSTRACTS of the 17thInternational Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California, August 24-29, 1997, abstract No.457).

The number of “several” amino acids varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein. It can be from 2 to 30, preferably from 2 to 15 and more preferably from 2 to 5 for the protein (A).

The term “resistance to 8-asadinho” refers to the ability of bacteria to grow on minimal medium containing 8-azadani in concentration at which the wild-type strain or a parent strain cannot grow, or the ability of bacteria to grow on a nutrient medium containing 8-azadani, with greater rate than the wild-type strain or a parent strain. The above-mentioned concentration of 8-asadena is usually from 50 to dobritoiu, in particular, methods of increasing the number of molecules of a specified protein include methods for modifying the sequence that regulates the expression of DNA that encodes a protein according to the present invention, and methods of increasing the number of copies of the gene, but are not limited to.

A change in the sequence that regulates the expression of DNA that encodes a protein according to the present invention can be achieved by placing the DNA that encodes a protein according to the present invention, under the control of a strong promoter. As strong promoters are known, for example, lac promoter, trp promoter, trc promoter, pl promoter of phage lambda. On the other hand, the promoter can be enhanced, for example, by introducing mutations in the indicated promoter to increase the level of transcription of a gene located after the promoter. Further, it is known that substitution of several nucleotides in the area between the binding site of the ribosome (RBS) and the start-codon, and in particular, in the sequence immediately before the start-codon, has a significant impact on broadcast ity mRNA. For example, it was found 20-fold change of expression level depending on the nature of three nucleotides preceding the start-codon (Gold et al. Annu. Rev. MicrhtA23 is a replacement And the G position 1 relative to the start codon ATG. Therefore, it has been suggested that the mutation rhtA23 enhances the rhtA gene expression and, as a consequence, increases the level of resistance to threonine, homoserine and some other substrates transported out of the cell.

Moreover, an “enhancer” can be added to increase the transcription level of the specified gene. Introduction DNA containing either the gene or the promoter in the chromosomal DNA, as described, for example, in patent application laid Japan No. 1-215280.

Alternatively, the number of copies of a gene can be increased by introduction of a gene in mnogoopytny vector with the formation of recombinant DNA, followed by the introduction of such recombinant DNA in a microorganism. Examples of vectors used for the introduction of recombinant DNA, are plasmid vectors such as pMW118, pBR322, pUC19, pET22b and the like, phage vectors, such as 11059, 1BF101, M13mp9, phage Mu (lined patent application of Japan No. 2-109985) and the like, and the transposon (Berg, D. E. and Berg, C. M., Bio/Technol., 1, 417 (1983)), such as Mu, Tn10, Tn5, and the like. In addition, amplification of gene expression can be achieved by integration of the gene into a bacterial chromosome by the method of homologous recombination or the like.

MPI gene.

For removing microorganism belonging to the genus Escherichia or the genus Bacillus and having enhanced expression of the gene encoding the protein according to the present invention, the necessary parts of genes can be obtained by PCR (polymerase chain reaction) on the basis of already available information about genes of E. coli and B. subtilis. For example, rhtA gene, it is believed that encodes the Transporter, may be cloned from chromosomal DNA of strains of E. coli K12 W3110 and E. coli MG1655 using the method of PCR. Chromosomal DNA used for this purpose, can also be obtained from any other strain of E. coli.

To proteins according to the present invention include mutants and variants of the protein RhtA that may exist due to natural diversity, provided that these mutants and variants demonstrate the functional properties of the protein RhtA at least resistance to 8-asadinho. DNA encoding these mutants and variants can be obtained by DNA extraction, which hybridizes rhtA gene (SEQ ID NO:1) or part of a specified gene in stringent conditions and which encodes a protein that increases the production of purine nucleotides. The term “stringent conditions” referred to here means the conditions under which the s are terms, in which hybridize with DNA having high homology, for example, DNA having a homology of at least 70% relative to each other. Alternatively, an example of the stringent conditions are conditions that match the conditions of washing by hybridization to Southern, for example 60°, 1× SSC, 0,1% SDS, preferably 0.1 to× SSC, 0,1% SDS. As probes for DNA encoding options and hybridization with the rhtA gene, can also be used a part of the nucleotide sequence at number 1. The probe of this kind can be obtained from PCR using as a nucleating oligonucleotides derived from the nucleotide sequence under number 1, and the DNA fragment containing the nucleotide sequence at number 1 as the matrix. In the case when the probe is a DNA fragment with a length of about 300 base pairs, conditions of washing in hybridization correspond to, for example, 50° C, 2× SSC and 0.1% SDS.

The bacterium according to the present invention can be obtained by introducing the above-mentioned DNA in a bacterium already having the ability to production of purine nucleosides. On the other hand, the bacterium according to the present invention can be obtained by the ve parent strain-producer of inosine, in which the activity of the proteins according to the present invention will be raised, can be used a strain of E. coli AJ13732 (FADRaddeddyicPpgixapA(pMWKQ)) (WO 9903988). The specified strain is derived from a known strain W3110 containing the mutations introduced in the purF gene encoding PRPP amidotransferase, purR gene encoding the repressor of the biosynthesis of purines, deoD gene encoding phosphorylase purine nucleosides, gene Riga, encoding succinyl AMR synthase, add gene encoding adelaideans, edd gene encoding 6-phosphoglyceraldehyde, pgi gene encoding phsopohdeisterase, gene Hara encoding xanthocephalus (purF-, purA-, deoD-, purR-, add-, edd-, pgi-, Hara-), as well as containing plasmid pMWKQ is derived from the vector pMW218, in which genes are purFKQ encoding PRPP amidotransferase, insensitive to guanosin monophosphate (GMP) (WO 9903988).

As the parent strain-producer xanthosine in which the activity of the proteins according to the present invention will be raised, can be used a strain of E. coli AJ13732 guaA::Tn10 (pMWKQ). The strain E. coli AJ13732 guaA::Tn10 (pMWKQ) designed by destroying the gene encoding the GMP synthetase, strain AJ13732 (pMWKQ) (see Example 7).

As the parent strain is derived from a known strain Century subtilis trpC2 containing mutations introduced in the purR gene encoding the repressor of the biosynthesis of purines (purR::spc), purA gene encoding succinyl AMR synthase (purA::erm), deoD gene encoding phosphorylase purine nucleosides (deoD::kan). As the other parent strains belonging to the genus Bacillus, in which the activity of the proteins according to the present invention will be raised, can be used strains of B. subtilis AJ12707 (FERM P-12951) (patent application of Japan JP 6113876 A2), B. subtilis AJ3772 (FERM P-2555) (patent application of Japan JP 62014794 A2), and the like.

To increase the activity in terms of protein according to the present invention, it is also possible to introduce a mutation in the structural part of the gene encoding the protein to increase the protein activity. In order to introduce a mutation into a gene, can be used site-specific mutagenesis (Kramer, W. and Frits, H. J. Methods in Enzymology, 154, 350 (1987)), the methods of recombinant PCR (PCR Technology, Stockton Press (1989)), the chemical synthesis of specific DNA segments, the processing of the desired gene using hydroxylamine, treatment of microbial strains containing the desired gene, using UV radiation or a chemical reagent, such as nitrosoguanidine or nitrous acid, or similar method. The microorganism in which the activity of the indicated b the Oia according to the present invention can be further improved by increasing the expression of one or more genes, involved in the biosynthesis of purines. Examples of such genes are the genes of the operon of Rigaku-purC(orf)QLF-purMNH(J)-purD from B. subtilis (Ebbole, D. J. and H. Zalkin, J. Biol. Chem., 262, 17, 8274-87, 1987) genes rig regulon from E. coli (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D. C., 1996).

It was shown that the production of inosine increased when using resistant 8-azaguanine mutants of B. subtilis with genetically modified repression of enzyme synthesis of purine nucleosides (Shiio I. and Ishii, K., J. Biochem., 69, 339-347, 1971). Products inosine strain of E. coli containing mutant purF gene encoding PRPP amidotransferase, free from inhibition of GMP and AMR type of feedback has been increased by inactivation of the purR gene encoding the repressor of the biosynthesis of purines (WO 9903988).

The mechanism that increases the production of purine nucleosides bacteria by increasing the activity of the proteins according to the present invention, is, as one might expect, increased excretion target purine nucleoside of the bacterium cell.

2. The method of obtaining purine nucleosides.

The methods according to the present invention is a method of obtaining a purine nucleoside, comprising the stage of growth of bacteria according to the present invention in paatelainen nucleoside from the culture fluid. More specifically, to methods according to the present invention is a method of producing inosine, including the stage of growth of bacteria according to the present invention in a nutrient medium for the purpose of production and accumulation of inosine in a nutrient medium, and the selection of inosine from the culture fluid. Also to methods according to the present invention is a method of obtaining xanthosine, including the stage of growth of bacteria according to the present invention in a nutrient medium to produce and accumulate xanthosine in a nutrient medium, and the selection xanthosine from the culture fluid.

According to the present invention, the cultivation, isolation and purification of purine nucleoside from the culture or similar fluid may be carried out in a manner similar to traditional methods of fermentation, in which the purine nucleoside is produced using a microorganism. The nutrient medium used for cultivation, can be both synthetic and natural, provided that the medium contains sources of carbon, nitrogen, mineral supplements and other necessary organic. The carbon sources include various carbohydrates such as glucose, lactose, gala is Rin, mannitol and sorbitol; and organic acids such as gluconic acid, fumaric acid, citric acid and succinic acid and the like. As the nitrogen source can be used various inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen sources such as soybean hydrolysate; gaseous ammonia and similar compounds. It is desirable that suitable small amounts of vitamins, such as vitamin B1and other necessary substances, such as nucleic acids, such as adenine and RNA, or yeast extract and similar compounds were present in the nutrient medium as organic nutrients. In addition, small amounts of calcium phosphate, magnesium sulfate, iron ions, manganese ions, and similar compounds can be added, if necessary.

The cultivation is carried out preferably under aerobic conditions for 16-72 h, the temperature during cultivation is maintained in the range from 30 to 45° C and a pH in the range from 5 to 8. pH can be adjusted inorganic or organic acidic or alkaline substances as well as gaseous ammonia.

After exp the Finance or by filtration through a membrane, and then the target purine nucleoside can be isolated from the culture fluid by any of the conventional methods or any combination of these methods, such as ion exchange chromatography and precipitation.

3. The method of obtaining purine nucleotides.

The methods according to the present invention also includes a method of obtaining a purine nucleotide, including the stage of growth of bacteria according to the present invention in a nutrient medium, phosphorylation received and accumulated purine nucleoside, and the selection is received and accumulated purine nucleotide. More specifically, to methods according to the present invention also concerns a method for production of inosine-5’-phosphate, comprising the stage of growth of bacteria according to the present invention in a nutrient medium, phosphorylation received and accumulated inosine, and the selection is received and accumulated inosine-5’-phosphate. Also to methods according to the present invention is a method of obtaining xanthosine-5’-phosphate, comprising the stage of growth of bacteria according to the present invention in a nutrient medium, phosphorylation received and accumulated xanthosine, and the selection is received and accumulated xantos cultural or similar fluid may be carried out in a manner, similar to traditional methods of fermentation, in which the purine nucleoside is produced using a microorganism. Further, according to the present invention phosphorylation received and accumulated purine nucleoside, and the selection is received and accumulated purine nucleotide can be carried out by a method similar to the traditional methods, in which a purine nucleotide is derived from a purine nucleoside.

Phosphorylation of purine nucleoside can be carried out enzymatically with the use of various phosphatases, nucleotidases and nucleotidyltransferase or chemically using fosfauriliruetsa agents, such as l3or similar. Can be used phosphatase capable of selective catalysis of the transfer of the phosphoryl group of pyrophosphate in the 5’-position of the nucleoside (Mihara et. al. Phosphorylation of nucleosides by the mutated acid phosphatase from Morganella morganii. Appl. Environ. Environ. 2000, 66:2811-2816), or acid phosphatase, using polyphosphoric acid (salt), phenylphosphino acid (salt) or carbamylphosphate acid (salt) as a donor of phosphoric acid (WO 9637603 A1), or the like. As an example, the phosphatase may be given phosphatase capable is substrate p-nitrophenylphosphate (Mitsugi, K. et al. Agric. Biol. Chem. 1964, 28, 586-600), inorganic phosphate (JP 42-1186), or acetylmorphine (JP 61-41555), or similar. As an example nucleosidase can be given guanosin-insinking from E. coli (Mori et al. Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product. J. Bacteriol. 1995, 177:4921-4926; WO 9108286), or similar. As an example nucleotidyltransferase can be given nucleotidyltransferase described Hammer-Jespersen, K. (Nucleoside catabolism, p.203-258. In A Munch-Petesen (ed.). Metabolism of nucleotides, nucleosides and nucleobases in microorganism. 1980, Academic Press, New York), or similar. Chemical phosphorylation of nucleosides can be accomplished using fosforiliruyusciye agent, such as l3(Yoshikawa et al. Studies of phosphorylation. III. Selective phosphorylation of unprotected nucleosides. Bull. Chem. Soc. Jpn. 1969, 42:3505-3508), or similar.

Also to methods according to the present invention is a method of producing guanosine-5’-phosphate, comprising the stage of growth of bacteria according to the present invention in a nutrient medium, phosphorylation received and accumulated xanthosine, amination received and accumulated xanthosine-5’-phosphate, and the selection is received and accumulated guanosin-5’-phosphate. According to the present invention, the cultivation of bacteria according to inromania received and accumulated xanthosine-5’-phosphate, and the selection is received and accumulated guanosin-5’-phosphate can be carried out by a method similar to the traditional methods, in which guanosin-5’-phosphate is obtained from xanthosine-5’-phosphate.

Amination of xanthosine-5’-phosphate can be carried out enzymatically using, for example, GMP synthetase from E. coli (Fujio et al. High level of expression of XMP aminase in Escherichia coli and its application for the industial production of 5’-guanylic acid. Biosci. Biotech. Biochem. 1997, 61:840-845; EP 0251489 B1).

In the method according to the present invention, the bacterium according to the present invention can be modified to increase the expression of genes of the biosynthesis of purine nucleosides.

Captions to drawings

In Fig.1 shows the structure of plasmid pNZ16.

In Fig.2 shows the structure of plasmid R3.

In Fig.3 shows a comparison of the hydrophobicity profiles of proteins RhtA and YdeD.

In Fig.4 shows the distribution of protein RhtA in cell fractions. In track 1 is the sediment (15000 g) induced cells BL21(DE3) containing no plasmid (control); lanes 2-7 are equal volumes of sediment and soluble fraction of induced cells BL21(DE3), soderdahl plasmid pET22b-rhtA: tracks 2 and 3 precipitate and supernatant after centrifugation at 15000 g, respectively; lanes 4 and the donkey centrifugation at 180000 g, respectively, after treatment cells 1 M KCl. The molecular weight markers (in kDa) are shown on the left margin, and the position of the protein RhtA shown by the arrow on the right margin.

In Fig.5 shows the structure of plasmid pLF-RHTA.

The best way of carrying out the invention

Example 1. Cloning rhtA gene from E. coli in the mini-Mu phasmida.

RhtA gene, which, as has been previously set, causes the resistance to homoserine and threonine, was cloned in vivo using phasmida mini-Mu d5005 (Groisman, E. A. et al. J. Bacteriol., 168, 357-364 (1986)). The strain MG442 lysogeny on phage MuCts62 (Gustine and other Genetics, 14, 947-956 (1978)), was used as donor. Freshly prepared lysates were used to infect Lisovenko on phage MuCts derived strain VKPM B-513 (Hfr K10 metB). The obtained cells were cultivated in minimal medium M9 with glucose containing methionine (50 µg/ml), kanamycin (40 μg/ml) and homoserine (10 mg/ml). Were selected colonies that appeared after 48 h of cultivation. From the colonies was isolated plasmid DNA and used to transform strain VKPM B-513 standard methods. Transformants were selected on plates with agar L-broth containing kanamycin and homoserine, as described above. From transformants resistant to homoserine, was the separation of the e l e C what donor were cloned two types of fragments belonging to different parts of the chromosome. Thus, in E. coli there are at least two different genes that make bacteria resistant to homoserine when they are present in mnogostadiinoi plasmid. It was shown that one of the types of chromosomal fragments is a fragment of the plot 86 min. In this case, the resistance phenotype was caused by the increased expression of genes rhtB and rht (European patent application EP 1013765 A1 and EP 1016710 A2).

In order to select fasmily with the insertion of a chromosomal fragment 18 min, hybridization was used in the colonies, using as a probe a mixture of six32P-labeled recombinant phages (3D2, 24F9, V, AV, E and E) from the collection Kohara (Kohara et al., Cell 50, 495-508, 1987), containing the chromosomal fragment 17.5 - 18.5 min. The result was obtained plasmid pNZ4S containing fragment length 9.3 kb, giving a resistance of cells to threonine and homoserine.

Example 2. Cloning rhtA gene in the vector pBlueScript KS+and pAYCTER-3.

Plasmid pNZ4S was treated restrictase He and StuI, and the resulting DNA fragments were separated by electrophoresis in low-melting agarose. The desired fragment containing the rhtA gene together with the in the recognition sites of the restriction enzyme EcoRV vector pBlueScript KS+(Promega), pre-treated with the restriction enzyme EcoRV. Thus was obtained a plasmid pNPZ16 (Fig.1). In addition, rhtA gene was periglomerular of pNPZ16 sites SmaI-HindIII stable srednekamennogo vector pAYCTER3 derived vector pAYC32. Thus was obtained a plasmid R3 (Fig.2). Vector pAYCTER3 is very stable srednekovoi vector, constructed on the basis of plasmid RSF1010 (Christoserdov A. Y., Tsygankov Y. D. Plasmid, 1986, v.16, pp.161-167) by introducing the vector pAYC32 of polylinker of plasmids pUC19 and strong terminator ggpv.

Example 3. The study of the homology of the rhtA gene product with the product ydeD gene, involved in the excretion of cysteine derivatives.

RhtA gene (SEQ ID NO:1) encodes a protein consisting of 295 amino acid residues, with a molecular weight of 31.3 kDa. Sequence analysis of the protein RhtA known method (Kyte and Doolittle, J. Mol. Biol., 157, 105-132, (1982)) showed that this protein is a hydrophobic protein containing the projected 10 transmembrane segments. The hydrophobicity profile and the projected number of transmembrane segments of the protein RhtA (Fig.3, solid line) and YdeD protein (Fig.3, dashed line) are similar to each other. This result shows homology (Lolkema, J. S., and Slotboom, D.-J. FEMS Rev Environ., 22, 305-322 (1998)). YdeD gene encodes a protein YdeD, the ol. 36, 1101-1112 (2000)). Based on this we can assume that the rhtA gene encodes a membrane protein involved in the transport of cells of some metabolites.

Example 4. Construction of the plasmid pET22b-rhtA and determining the cellular localization of the rhtA gene product.

The coding sequence of the rhtA gene was obtained on the basis of plasmids pNPZ16 by polymerase chain reaction (PCR) using nucleating SEQ ID NO:3 containing the Ndel restriction site, and SEQ ID NO:4 that contains the restriction site EcoRI. The resulting PCR product was treated with restrictase NdeI and EcoRI and Legerova in bacterial expressing vector pET22b (Novogene), pre-treated with the same restrictases. The obtained plasmid pET22b-rhtA, containing the gene for T7 RNA polymerase under control of the promoter Plac(Novogene), was used to transform E. coli strain BL21(DE3). The introduction of the gene T7 RNA polymerase and getting labeled with [35S]methionine of the protein was performed essentially as described previously (Tabor and Richardson, Proc. Natl. Acad. Sci. USA, 82, 1074-1078 (1985)), with slight modification. The modification was as follows: the expression of protein was induced by adding isopropyl--D-thiogalactopyranoside (IPTG, final concentration 2 mm) to 5 ml kochanie newly synthesized protein was performed by adding 50Ci [35S]methionine to the 5 ml culture of cells. Cells were collected by centrifuging and used for fractionation. Sediment resuspendable in destroying buffer (100 mm Tris-Hcl buffer, pH 7.5, containing 1 mm EDTA, 2 mm dithiothreitol and 1 mm phenylmethylsulfonyl) and the cells were destroyed by ultrasound. After removal of cell debris by centrifugation at 15000 g for 20 min, the membrane fraction was obtained by ultracentrifugation at 180000 g for 180 minutes the precipitate (membrane fraction) was resuspendable in destroying buffer containing 1M KCl, for 40 min at room temperature and centrifuged at 180000 g within 180 minutes of the Soluble fraction of the supernatant were precipitated by incubation with trichloroacetic acid (final concentration 10%) at 4° C for 30 min, centrifuged and washed with acetone. All sediments were resuspendable in the same volume of buffer to cover the gel (Laemmli, Nature 227, 680-685 (1970)). Proteins were analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate (SDS) (Laemmli, Nature 227, 680-685 (1970)).

The distribution of protein RhtA in cell fractions shown in Fig.4. Protein RhtA was soocarcasse with membrane Francesca was observed after treatment of KCl membrane fraction (Fig.4, tracks 6, 7). These data confirm the point of view that protein RhtA is fully membrane protein. Electrophoretic mobility of the protein RhtA corresponds to a molecular mass of approximately 25 kDa instead of the predicted mass of 31.3 kDa, which may be a result of the high hydrophobicity of the protein RhtA. YdeD protein shows a similar nature of mobility in the gel (Daler et al., Mol. Environ. 36, 1101-1112 (2000)).

Example 5. The effect of mutations rhtA23 and amplification rhtA gene for resistance strain E. coli MG1655 to analogues of purine bases.

The strain E. coli MG1655rhtA23 was constructed by introducing mutations rhtA23 from strain VKPM B-3996 (U.S. patent 5976843) in the strain E. coli MG1655 method transduction using phage P1. Transductant were selected on minimal M9 medium (Miller, 1972) containing 10 mg/ml of homoserine. Thus was obtained a pair of isogenic strains of E. coli MG1655rhtA+and E. coli MG1655rhtA23.

In addition, plasmids pNPZ16 and pRHTA3, and the corresponding vectors pBluescript KS+and pAYCTER3 were introduced into the strain E. coli MG1655. So were the strains MG1655(pNPZ16), MG1655(pRHTA3), MG1655(pBluescript), MG1655(pAYCTER3). Then determined the ability of each of these strains to grow on minimal agar medium M9 with glucose, stupas containing the>cells overnight culture grown in minimal medium containing 100 mg/ml ampicillin in the case of strains with plasmids. Capacity for growth was determined after 44 h of incubation at 37° C. the results of the experiments are presented in Table 1.

As can be seen from Table 1, mutation rhtA23 and the rhtA gene amplification was increased bacterial resistance to 8-asadinho. Based on this we can assume that the rhtA gene is involved in excretion of purine derivatives.

Example 6. The effect of mutations rhtA23 products inosine strain of E. coli - producing inosine.

Strain AJ13732(pMWKQ) producing inosine was used as the parent strain for the introduction of mutations rhtA23 as described in Example 5. So was the resulting strain AJ13732rhtA23(pMWKQ). Each of the strains were grown at 37° C for 18 h in L-broth containing 100 mg/l ampicillin and 75 mg/l kanamycin. 0.3 ml of the obtained culture was transferred to 3 ml of culture medium for fermentation, containing 100 mg/ml ampicillin and 75 mg/l kanamycin, in vitro 20× 200 mm and were incubated at 37° C for 72 h on a rotary shaker.

The composition of the medium for fermentation (g/l):

Glucose 40.0

(NH4)2SO416.0

To2NRA41.0

330.0

Glucose and magnesium sulfate were sterilized separately. Caso3sterilized at 180° C for 2 hours the pH was maintained in the area of 7.0. The antibiotic was added to the culture medium after sterilization.

After growing the number of inosine accumulated in the medium was determined by HPLC. A sample of the culture fluid (500 μl) was centrifuged at 1500 rpm for 5 min, the supernatant was diluted 100 times and analyzed by HPLC.

Conditions for analysis by HPLC.

Column: Luna 18(2) 250× 3 mm, 5 u (Phenomenex, USA). Buffer: 2% v/v C2H5OH; 0,8% v/v triethylamine, and 0.5% v/v acetic acid (glacial), pH 4.5. Temperature: 30° C. flow Rate: 0.3 ml/min Volume of sample: 5 µl. UV detector: 250 nm.

Retention time (min):

Xanthosine 13.7

Inosine 9.6

Gipoksantin 5.2

Guanosin 11.4

Adenosine 28.2

The results are presented in Table 2.

As can be seen from Table 2, the mutation rhtA23 improves production of inosine strain AJ13732.

Example 7. The effect of mutations rhtA23 products xanthosine strain of E. coli - producer xanthosine.

The strain producing xanthosine, was obtained from strain AJ13732(pMWKQ) - producer inosine. Using transduction is octante were selected on LB medium, containing 20 μg/ml tetracycline. Among them was found the strain AJ13732 gw.4::Tn10(pMWKQ) capable products xanthosine. Derived from this strain that contains a mutation rhtA23, was obtained as described in Example 5, to obtain the strain AJ13732 guaA::Tn 10rhtA23(pMWKQ).

Each of the strains AJ13732 gua4::Tn10(pMWKQ) and AJ13732 guaA::Tn10 rhtA23(pMWKQ) were grown at 37° C for 18 h in L-broth containing 10 mg/l kanamycin and 10 mg/l tetracycline. 0.3 ml of the obtained culture was transferred to 3 ml of culture medium for fermentation (see Example 3) containing 75 mg/l kanamycin and 10 mg/l tetracycline, in vitro 20× 200 mm and were incubated at 37° C for 48 h on a rotary shaker. After growing the number xanthosine accumulated in the medium was determined by HPLC as described above. The results are presented in Table 3.

As can be seen from Table 3, the mutation rhtA23 improves production xanthosine strain AJ13732 guaA::Tn10(pMWKQ).

Example 8. The effect of amplification rhtA gene products inosine strain of E. coli - producing inosine.

Strain AJ13732(pMWKQ) producing inosine was transformed vector pAYCTER3 and plasmid R3. So were the strains AJ13732(pMWKQ, pAYCTER3), AJ13732(pMWKQ, pRHTA3). Each of these strains were grown at 37° C for 18 the nutrient medium for fermentation (see Example 3) containing 100 mg/l ampicillin and 75 mg/l kanamycin, in vitro 20× 200 mm and were incubated at 37° C for 48 h on a rotary shaker. After growing the number xanthosine accumulated in the medium was determined by HPLC as described above. The results are presented in Table 4.

As can be seen from Table 4, the rhtA gene amplification improves the production of inosine strain AJ13732(pMWKQ).

Example 9. The effect of amplification rhtA gene products xanthosine strain of E. coli - producer xanthosine.

Strain J13732 guaA::Tn10(pMWKQ) - producer xanthosine described in Example 7, was transformed with plasmid pRHTA3 or vector pAYCTER3 obtaining strains AJ13732 guaA::Tn10(pMWKQ, pRHTA3), AJ13732 guaA::Tn10(pMWKQ, pAYCTER3). Each of these strains were grown at 37° C for 18 h in L-broth containing 100 mg/l ampicillin and 75 mg/l kanamycin. 0.3 ml of the obtained culture was transferred to 3 ml of culture medium for fermentation (see Example 3) containing 100 mg/l ampicillin and 75 mg/l kanamycin, in vitro 20× 200 mm and were incubated at 37° C for 48 h on a rotary shaker. After growing the number xanthosine accumulated in the medium was determined by HPLC as described above. The results are presented in Table 5.

Example 10. Cloning rhtA gene in the vector pLF14.

Based on the information obtained by analyzing the genomic database, were synthesized 51-tier seed (SEQ ID NO:5) and 19-tier seed (SEQ ID NO:6).

First the seed contains the sequence to 30 by 1 nucleotide before the start-codon of the gene Riga from B. subtilis and the sequence from the start codon to 21 nucleotide rhtA gene from E. coli. The sequence from 30 to 1 nucleotide before the start-codon of the gene Riga contains the binding site of the ribosome from the gene in Riga, together with the adjacent spacer elements of land. The second seed is a sequence complementary to the sequence of 122 105 nucleotides after the stop codon of the gene rhtA. PCR was performed at 94° C, 30 s; 55° C, 1 min; 72° C, 2 min; 30 cycles (Gene Amp PCR System Model 9600, Perkin Elmer) using chromosomal DNA from strain E. coli MG1655 as the template. The obtained DNA fragment containing the rhtA gene, fused with a binding site of the ribosome from gene rig E and its adjacent areas from B. subtilis was cloned in the vector pGEM-T (Promega).

Then this design was periglomerular sites SacI - SphI raised in dnorepository Shuttle vector pLF14 (Shevelev et al., Plasmid, 43, 190-199, 2000), which can Express a variety of genes in cells of Bacillus is Yu plasmid RP4 in the strain C. subtilis 168. Being expressed in cells of Bacillus, rhtA gene gave them an increased resistance to homoserine during growth on minimal medium M9 (table 6).

The culture was grown at 37° C for 44 hours on minimal agar media containing the indicated concentrations of homoserine: + good growth, - no growth.

Example-1. Construction of a strain of B. subtilis KMBS 16 - producer inosine.

The strain of B. subtilis KMBS 16 producing inosine, which is a mutant containing insertion-deletion mutations in the genes purR, purA and deoD, was obtained from a strain of Bacillus subtilis 168 Marburg.

1) Constructing a mutant of B. subtilis, deficient purR.

PCR was performed at 94° C, 30 s; 55° C, 1 min; 72° C, 1 min; 30 cycles; Gene Amp PCR System Model 9600, Perkin Elmer) using chromosomal DNA of strain B. subtilis 168 Marburg as a matrix, and the following oligonucleotide seed No. 1 (SEQ ID NO:7) and # 2 (SEQ ID NO:8), synthesized according to the DNA sequence in the genome data Bank. Seed No. 1 (28 links) comprises a sequence with 246 228 nucleotide before the start-codon of the gene purR from B. subtilis (M. Weng, P. L. Nagy and H. Zalkin. Identification of the Bacillus subtilis pur operon repressor. Proc. Natl. Acad. Sci. USA. 1995, 92:7455-7459), and 9 additional is the selected from 57 to 75 nucleotide after the stop codon of the gene purR, as well as 9 additional nucleotides containing PstI attached to the 5’-end. The fragment obtained by PCR (0.9, etc., O.) and treated HindIII and > PST, was inserted in the same restriction sites of the vector pHSG398 (TaKaRa, Japan) with the formation of plasmid pHSG398BSPR. EcoRV - HincII fragment (0.3, etc., O.) in the inner part of the amplified purR gene was removed from plasmid pHSG398BSPR and replaced by the gene of resistance to spectinomycin (1.2, etc., O.) from Enterococcu faecalis cut from pDG1726 (Bacillus Genetic Stock Center, Ohio).

The obtained plasmid pHSG398purR::spc was used to transform competent cells of B. subtilis 168 Marburg, obtained by the method of Dubunau and Davidoff-Abelson (Dubnau, D. and R. Davidoff-Abelson. Fate of transforming DNA following uptake by competent Bacillus subtilis. J. Mol. Biol. 1971, 56:209-221). Double crossover mutants were tested by preparation of chromosomal DNA from each colony resistant to spectinomycin, with subsequent PCR as described above. One of the colonies, which, as has been confirmed is a mutant, deficient in the gene purR, was named KMBS4.

2) Construction of a mutant of B. subtilis, deficient in Riga.

PCR was performed at 94° C, 30 s; 55° C, 1 min; 72° C, 2 min; 30 cycles (Gene Amp PCR System Model 9600, Perkin Elmer) using chromosomal DNA of strain Century sublilis 168 Marburg as a matrix and sledujushih in the genomic database. Seed No. 3 (29 links) comprises a sequence with 137 118 nucleotide before the start-codon of the gene Riga from B. subtilis (P.and H. Zalikin. Cloning and sequence of Bacillus subtilis purA and guaA, involved in the conversion of IMP to AMP and GMP. J. Bacteriol. 1992, 174:1883-1890), and 9 additional nucleotides containing the website SolI attached to the 5’-end. Seed No. 4 (29 links) comprises a sequence with 51 70 nucleotide after the stop codon of the gene Riga, as well as 9 additional nucleotides containing the SphI site, attached to the 5’-end. The fragment obtained by PCR, (1.5, etc., O.) and processed SalI and SphI, was inserted in the same restriction sites of the vector pSTV28 (TaKaRa, Japan). The obtained plasmid pSTV28BSPA was split using luI and BglII, which led to the deletion of an internal fragment length 0.4 T. p. O. of the amplified gene Riga, the ends were blunted using fragment maple, then it Legerova fragment of the gene of resistance to erythromycin with blunt ends (1.6 T. p. O.) from Staphylococcus anreus, carved from pDG646 (Bacillus Genetic Stock Center, Ohio).

The obtained plasmid pSTV28BSPA::erm was used to transform competent cells KMBS4 obtained by the method of Dubunau and Davidoff-Abelson, as described above. Double crossover mutants were tested the ANO above. One of the colonies, which, as has been confirmed is a mutant, deficient in the gene Riga, was named KMBS13. As expected, cells KMBS13 were auxotrophic for adenine.

3) Constructing a mutant of B. subtilis, deficient deoD.

To obtain the 5’end of the gene deoD and the previous site of the B. subtilis PCR was performed at 94° C, 30 s; 55° C, 1 min; 72° C, 1 min; 30 cycles (Gene Amp PCR System Model 9600, Perkin Elmer) using the following oligonucleotide seed, No. 5 (SEQ ID NO:11) and # 6 (SEQ ID NO:12), synthesized according to the DNA sequence in the genome data Bank. Seed No. 5 (29 links) comprises a sequence with 310 on 291 nucleotides before the start-codon of the gene deoD, as well as 9 additional nucleotides containing an EcoRI site, attached to the 5’-end. Seed No. 6 (28 links) comprises a sequence 39 57 nucleotide after the start-codon of the gene deoD, as well as 9 additional nucleotides containing the website Wamn attached to the 5’-end. The fragment obtained by PCR (0.4 T. p. O.) and treated EcoRI and BamHI was inserted in the same restriction sites of the vector pSTV28 (TaKaRa, Japan), which has resulted in plasmids pSTV28DON.

To obtain the 3’end of the gene deoD and subsequent land PCR was carried out by gonucleotide seed, No. 7 (SEQ ID NO:13) and No. 8 (SEQ ID NO:14), synthesized according to the DNA sequence in the genome data Bank. Seed No. 7 (29 links) comprises a sequence with 321 302 on the nucleotide after the stop codon of the gene deoD, as well as 9 additional nucleotides containing a HindIII site, attached to the 5’-end. Seed No. 8 (28 links) comprises a sequence from 24 to 42 nucleotide before the stop codon of the gene deoD, as well as 9 additional nucleotides containing a BamHI site, attached to the 5’-end. The fragment obtained by PCR (0.4 T. p. O.) and treated HindIII and BamHI was inserted in the same restriction sites of the vector pSTV28DON that resulted in plasmids pSTV28DONC.

In order to amplify the gene of resistance to kanamycin from Streptococcus faecalis, PCR was performed at 94° C, 30 s; 55° C, 1 min; 72° C, 2 min; 30 cycles (Gene Amp PCR System Model 9600, Perkin Elmer) using DNA plasmids pDG783 (Bacillus Genetic Stock Center, Ohio) as template and the following oligonucleotide seed, No. 10 (SEQ ID NO:15) and # 11 (SEQ ID NO:16), synthesized according to the DNA sequence in the genome data Bank. Seed No. 10 (33 level) comprises a sequence with 513 in 490 nucleotides before the start-codon of the gene of resistance to kanamycin, as well as 9 additional Alnost with 117 140 nucleotide after the stop codon of the specified gene, as well as 9 additional nucleotides containing the website Wamn attached to the 5’-end. The fragment obtained by PCR (1.5 T. p. O.) and processed Wamn, was inserted at the unique restriction site UMN vector pSTV28DONC. The obtained plasmid pSTV28deoD::kan was used to transform competent cells KMBS13 obtained by the method of Dubunau and Davidoff-Abelson, as described above. Double crossover mutants were tested by preparation of chromosomal DNA from each colony resistant to kanamycin, with subsequent PCR, using seed # 5 and # 7, as described above. One of the colonies, which, as has been confirmed is a mutant, deficient in the gene deoD, (purR::spc purA::erm deoD::kan) was named KMBS16.

Example 11. The effect of amplification rhtA gene products inosine strain of B. subtilis - producer inosine.

Plasmid pLF-RHTA and vector pLFH were placed in a strain of B. subtilis KMBS16 producing inosine. So were the strains of B. subtilis KMBS16(pLF-RHTA) and B. subtilis KMBS16(pLF14). Each of these strains were grown at 37° C for 18 h in L-broth containing 10 mg/l of chloramphenicol, and 0.3 ml of the obtained culture was transferred to 3 ml of culture medium for fermentation of Bacillus containing 10 mg/l of chloramphenicol, in vitro 20× 200 mm and were incubated at 37°

KH2PO41.0

MgSO40.4

FeSO4×7H2O 0.01

MnSO4×5H2O 0.01

Mameno-TN 1.35

DL-methionine 0.3

NH4Cl 32.0

Adenine 0.1

Tryptophan 0.02

Caso350.0

After growing the number of inosine accumulated in the medium was determined by HPLC as described above. The results are presented in Table 7.

As can be seen from Table 7, the rhtA gene amplification improves the production of inosine strain of B. subtilis KMBS16.

Claims

1. A method of obtaining a purine nucleoside, comprising the stage of growth of the bacteria Escherichia coli or bacteria Bacillus subtilis in a nutrient medium and a selection from the culture fluid obtained and accumulated therein purine nucleoside, wherein using the bacterium Escherichia coli and the bacterium Bacillus subtilis, the ability to products of purine nucleoside which is increased by increasing the activity of b is the sequence 2 (SEQ ID NO:2); (B) a protein that presents the amino acid sequence comprising deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence listed in sequence 2 (SEQ ID NO:2), and which has activity, giving bacteria more resistant to 8-asadinho.

2. The method according to p. 1, wherein the purine nucleoside is inosine.

3. The method according to p. 1, wherein the purine nucleoside is xanthosine.

4. The method according to any of paragraphs.1-3, characterized in that the indicated bacteria the expression of genes of the biosynthesis of purine nucleosides raised.

5. The method according to p. 2, characterized in that as a producer of inosine using a strain of Escherichia coli AJ13732rhtA23pMWKQ), Escherichia coli AJ13732(pMWKQ, pRHTA3) or a strain of Bacillus subtilis KMBS16(pLF-RHTA).

6. The method according to p. 3, characterized in that as a producer xanthosine using a strain of Escherichia coli AJ13732 guaA::Tnl0 rhtA23 or Escherichia coli AJ13732 guaA::Tnl0 (pMWKQ, RCNT).

7. A method of obtaining a purine nucleotide, including the stage of growth of the bacteria Escherichia coli or bacteria Bacillus subtilis in a nutrient medium, phosphorylation received and accumulated nucleoside, and the allocation of the received perinatally purine nucleoside which increased by increasing the activity of proteins, selected from the group of: (A) a protein that presents the amino acid sequence listed in sequence 2 (SEQ ID NO:2); (B) a protein that presents the amino acid sequence comprising deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence listed in sequence 2 (SEQ ID NO:2), and which has activity, giving bacteria more resistant to 8-asadinho.

8. The method according to p. 7, wherein the purine nucleotide is inosine-5'-phosphate.

9. The method according to p. 7, wherein the purine nucleotide is xanthosine-5'-phosphate.

10. The method according to any of paragraphs.7-9, characterized in that the indicated bacteria the expression of genes of the biosynthesis of purine nucleosides raised.

11. The method of obtaining guanosin-5'-phosphate, comprising the stage of growth of the bacteria Escherichia coli or bacteria Bacillus subtilis in a nutrient medium, phosphorylation received and accumulated xanthosine, amination received xanthosine-5'-phosphate, and secretions obtained guanosin-5'-phosphate characterized in that using the bacterium Escherichia coli and the bacterium Bacillus subtilis, the ability of s: (A) protein, which presents the amino acid sequence listed in sequence 2 (SEQ ID NO:2); (B) a protein that presents the amino acid sequence comprising deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence listed in sequence 2 (SEQ ID NO:2), and which has activity, giving bacteria more resistant to 8-asadinho.

12. The method according to p. 11, characterized in that the indicated bacteria the expression of genes of the biosynthesis of purine nucleosides raised.

13. The strain Escherichia coli AJ13732 rhtA23 (pMWKQ) producing inosine.

14. The strain Escherichia coli AJ13732 guaA::Tnl0 rhtA23 (pMWKQ) - producer xanthosine.

15. The strain Escherichia coli AJ 13732 (pMWKQ, pRHTA3) producing inosine.

16. The strain Escherichia coli J 13732 guaA::Tnl0 (pMWKQ, RCNT) - producer xanthosine.

17. The strain of Bacillus subtilis KMBS16 (pLF-RHTA) producing inosine.



 

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