RU2239656C2 - Method for preparing purine nucleoside and nucleotide, strain as producer of purine nucleoside (variants) - Google Patents

Abstract

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to a method for preparing purine nucleosides, such as inosine and xanthosine, and also to a method for preparing purine nucleotides, such as inosine 5'-phosphate, xanthosine 5'-phosphate and guanosine 5'-phosphate. Strains of microorganisms belonging both to genus Escherichia and to genus Bacillus are used as producers wherein production of purine nucleosides by indicated microorganisms can be increased due to elevating activity of protein encoding by gene rhtA (ybiF).

EFFECT: improved preparing method.

17 cl, 5 dwg, 7 tbl, 11 ex

Description

Technical field

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

State of the art

Traditionally, nucleosides are produced on an industrial scale by fermentation using strains of microorganisms auxotrophic for adenine, or these strains, which are additionally given resistance to various compounds, such as purine analogues and sulfaguanidine. Examples of such strains are strains belonging to the genus Bacillus (Japanese patent applications No. 38-23039, 54-17033, 55-2956 and 55-45199, Japanese patent application laid out No. 56-162998, Japanese patent applications 57-14160 and 57-41915 and Japanese Patent Application Laid-open No. 59-42895), to the genus Brevibacterium (Japanese patent applications No. 51-5075 and 58-17592 and Agric. Biol. Chem., 42, 399 (1978)), to the genus Escherichia (PCT application WO 9903988 ) and the like.

Obtaining these mutant strains usually consists of processing microorganisms to obtain mutations, for example, by irradiation with UV radiation or treatment with nitrosoguanidine (N-methyl-N’-nitro-N-nitrosoguanidine), followed by selection of the desired strain on a suitable breeding medium. On the other hand, the cultivation of mutant strains belonging to the genus Bacillus is also practiced (Japanese Patent Applications Laid-Open No. 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 (Japanese Patent Application Laid-open 63-248394) obtained using genetic engineering methods.

Previously, the authors of the present invention obtained, based on E. coli K12, a mutant containing the rhtA23 mutation, which confers resistance to high concentrations of threonine, homoserine and some other amino acids and amino acid analogs in a minimal nutrient medium. In addition, this rhtA23 mutation improved the production of L-threonine by the corresponding producer strain E. coli (ABSTRACTS of 17 ^th International 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). Moreover, the inventors have found that the rhtA gene is located at the 18 min position on the E. coli chromosome adjacent, and the rhtA gene is identical to the open ybiF reading frame located between the pXB and ompX genes. In addition, the authors of the present invention found that the rhtA23 mutation is a substitution of A at the G position - 1 relative to the ATG start codon. This mutation is believed to increase transport of threonine and homoserine from the cell (ABSTRACTS of 17 ^th International 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 there are currently no reports describing any exporters of purine compounds.

Description of the invention

The aim of the present invention is to increase the productivity of nucleosides by producer strains of nucleosides and to provide a method for producing nucleosides, such as inosine and xanthosine, using these strains.

This goal was achieved by establishing the fact that the rhtA23 mutation gives the microorganism resistance to the purine analog, 8-azaadenine, and increases the production of nucleoside. In addition, the natural rhtA gene encoding, as expected, a membrane protein that is not involved in the pathogen biosynthesis of purine nucleosides, also gives the microorganism resistance to the purine analogue when the natural allele of this gene is introduced into the cell on a multi-copy vector. Moreover, the natural rhtA gene can increase the production of a strain when additional copies of this gene are introduced into the corresponding nucleoside producer strains belonging to the genus Escherichia or Bacillus. Thus, the present invention has been completed.

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

In particular, the present invention provides a microorganism with an increased ability to produce purine nucleosides based on an increase in the activity of a protein involved, as expected, in the transport of purine nucleosides from a cell of said microorganism. More specifically, the present invention provides a microorganism with an increased ability to produce purine nucleosides based on increased expression of a gene encoding a protein involved in the excretion of purine nucleosides.

Further, the present invention provides a method for producing purine nucleosides by a fermentation method, comprising the steps of growing the above microorganism in a culture medium in order to produce and accumulate purine nucleosides in a culture medium and isolate purine nucleosides from the culture fluid.

The present invention further provides a method for producing purine nucleotides, such as inosine-5’-phosphate and xanthosine-5’-phosphate, comprising the steps of growing the bacteria of the present invention in a culture medium, phosphorylating the resulting and accumulated purine nucleoside, and isolating the resulting purine nucleoside.

The present invention also provides a method for producing guanosine-5’-phosphate, comprising the steps of growing the above bacteria in a nutrient medium, phosphorylating the obtained and accumulated xanthosine, aminating the obtained xanthosine-5’-phosphate, and isolating the obtained guanosine-5’-phosphate.

The present invention includes the following.

Invention 1. A bacterium belonging to the genus Escherichia or to the genus Bacillus, which is capable of producing a purine nucleoside in which the activity of the protein described in points (A) or (B) in the cell of the said bacterium is increased:

(A) a protein that is represented by the amino acid sequence shown in sequence listing number 2;

(B) a protein that is represented by an amino acid sequence including deletions, substitutions, insertions, or the addition of one or more amino acids to the amino acid sequence listed in sequence number 2, and which has an activity that confers the bacteria increased resistance to 8-azaadenine.

Invention 2. A bacterium in accordance with Invention 1, in which the activity of the proteins described in points (A) or (B) is increased by transforming the bacterium using DNA encoding the proteins described in points (A) or (B), or by changes in the regulation of expression of said DNA in said bacterium.

The invention 3. The bacterium in accordance with the Invention 2, in which the transformation is carried out using a multi-copy vector.

The invention 4. The bacterium in accordance with the Invention 1, in which the purine nucleoside is inosine.

The invention 5. The bacterium in accordance with the Invention 1, in which the purine nucleoside is xanthosine.

Invention 6. A method for producing a purine nucleoside, comprising the steps of growing a bacterium in accordance with any of Inventions 1-5 in a nutrient medium and isolating the obtained and accumulated purine nucleoside from the culture fluid.

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

Invention 8. The method in accordance with Invention 6, wherein the purine nucleoside is xanthosine.

The invention 9. The method in accordance with any of Inventions 6-8, in which the bacterium is modified to increase the expression of purine nucleoside biosynthesis genes.

The invention 10. A method for producing a purine nucleotide, comprising the steps of growing a bacterium in accordance with any of Inventions 1-5 in a nutrient medium, phosphorylating the obtained and accumulated nucleoside, and isolating the obtained 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.

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

The invention 14. A method of producing guanosine-5'-phosphate, comprising the steps of growing bacteria in accordance with the invention of 5 in a nutrient medium, phosphorylation of the obtained and accumulated xanthosine, amination of the obtained xanthosine-5'-phosphate, and isolation of the obtained and accumulated guanosine-5'- phosphate.

The invention 15. The method in accordance with the Invention 14, in which the bacterium is modified to increase the expression of purine nucleoside biosynthesis genes.

The present invention will be described in more detail below.

1. The microorganism according to the present invention.

The aforementioned bacterium according to the present invention is a bacterium belonging to the genus Escherichia or to the genus Bacillus, which is capable of producing a purine nucleoside in which the activity of the protein described in points (A) or (B) in the cell of said bacterium is increased: (A) protein which is represented by the amino acid sequence shown in the list of sequences at number 2; (B) a protein that is represented by an amino acid sequence including deletions, substitutions, insertions, or the addition of one or more amino acids to the amino acid sequence listed in sequence number 2, and which has activity that confers resistance to 8-azaadenine on the bacteria.

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

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

The term “ability to produce a purine nucleoside” as used herein means the ability to produce and accumulate a purine nucleoside in a culture medium. The term “capable of producing purine nucleoside” means that a microorganism belonging to the genus Escherichia or the genus Bacillus has the ability to produce and accumulate in the nutrient medium a purine nucleoside in an amount greater than the natural E. coli strain, such as E strains .coli W3110 and MG1655, or a natural B. subtilis strain, such as B. subtilis 168 strain, and preferably means that the microorganism is capable of producing and accumulating in the nutrient medium an amount of not less than 10 mg / l, more preferably not less than 50 mg / l inos ina, xanthosine, guanosine and / or adenosine.

The term “activity of the protein described in points (A) or (B) in the cell of said bacterium is increased” means that the number of molecules of the specified protein in the cell is increased or the activity in terms of protein is increased. The term “activity” means activity that confers resistance to 8-azaadenine on bacteria.

Proteins according to the present invention include proteins described in the following paragraphs (A) or (B):

(A) a protein that is represented by the amino acid sequence shown in sequence listing number 2;

(B) a protein that is represented by an amino acid sequence including deletions, substitutions, insertions, or the addition of one or more amino acids to the amino acid sequence listed in sequence number 2, and which has activity that confers resistance to 8-azaadenine on the bacteria.

The protein that is represented by the amino acid sequence listed in sequence number 2 is the RhtA protein. The RhtA protein is believed to be a transmembrane protein consisting of 95 amino acids and having an unknown function. The RhtA protein is encoded by the rhtA gene. The rhtA gene is located at the 18 min position on the E. coli chromosome next to the glnHPQ operon encoding the components of the glutamine transport system. The rhtA gene is identical to the open reading frame (ORF1) of the ybiF gene (nucleotides 764 to 1651 in the sequence with accession number AAA218541, gi: 440181 in the GenBank database) located between the expB and opmp genes. The region expressing the protein encoded by the indicated ORF was designated as rhtA (rht: resistance to homoserine and threonine - resistance to homoserine and threonine), since the authors of the present invention previously obtained, based on E. coli K12, a mutant containing a mutation in the rhtA gene , T-23 or thrR (also referred to as rhtA23 here), conferring resistance to high concentrations of threonine and homoserine on a minimal nutrient medium (SU Patent No.974817, Astaurova, O. B. et al., Appl. Bioch. And Microbiol. 21, 611-616 (1985)). This rhtA23 mutation improved the production of L-threonine (SU Patent No.974817, US Patent No.6165756), homoserine and glutamate (Astaurova, O.V. et al., Appl. Bioch. And Microbiol., 27, 556-561, 1991) an appropriate E. coli producer strain, such as VKPM B-3996 strain. In addition, the authors of the present invention found that the rhtA23 mutation is a substitution of A for G position - 1 relative to the ATG start codon (ABSTRACTS of 17 ^th International 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 protein (A).

The term “resistance to 8-azaadenine” means the ability of a bacterium to grow on a minimum nutrient medium containing 8-azaadenine in a concentration at which a wild-type strain or parent strain cannot grow, or the ability of a bacterium to grow on a nutrient medium containing 8-azaadenine, at a faster rate than the wild-type strain or the parent strain. The concentration of 8-azaadenine mentioned above is usually from 50 to 5000 μg / ml, preferably from 100 to 1000 μg / ml.

Methods for increasing the activity of a protein of the present invention, in particular methods for increasing the number of molecules of a given protein in a cell, include, but are not limited to methods for changing the expression sequence of the DNA encoding the protein of the present invention and methods for increasing the number of copies of a gene.

Changing the sequence that regulates the expression of the DNA encoding the protein of the present invention can be achieved by placing the DNA encoding the protein of the present invention under the control of a strong promoter. As strong promoters, for example, the lac promoter, trp promoter, trc promoter, pl lambda phage promoter are known. On the other hand, the promoter can be enhanced, for example, by introducing a mutation in the specified promoter in order to increase the level of transcription of the gene located after the promoter. Further, it is known that the replacement of several nucleotides in the region between the ribosome binding site (RBS) and the start codon, and especially in the sequence immediately before the start codon, significantly affects the translational ability of the mRNA. For example, a 20-fold change in expression level was found depending on the nature of the three nucleotides preceding the start codon (Gold et al. Annu. Rev. Microbiol. 35, 365-403, 1981; Hui et al., EMBO J., 3 623-629, 1984). As described above, the authors of the present invention have found that the rhtA23 mutation is a substitution of A at the G position - 1 relative to the ATG start codon. Therefore, it was suggested that the rhtA23 mutation enhances the expression of the rhtA gene and, as a result, increases the level of resistance to threonine, homoserine and some other substrates transported from the cell.

Moreover, a certain “enhancer" can be additionally introduced in order to increase the level of transcription of this gene. The introduction of DNA containing either a gene or a promoter into a chromosomal DNA is described, for example, in Japanese Patent Laid-open No. 1-215280.

Alternatively, the number of copies of a gene can be increased by introducing the gene into a multi-copy vector to form recombinant DNA, followed by introducing such recombinant DNA into the microorganism. Examples of vectors used to introduce recombinant DNA are plasmid vectors such as pMW118, pBR322, pUC19, pET22b and the like, phage vectors such as 11059, 1BF101, M13mp9, phage Mu (Japanese Patent Application Laid-Open No. 2-109985) and similar to them, and transposons (Berg, DE and Berg, CM, Bio / Technol., 1, 417 (1983)), such as Mu, Tn10, Tn5 and the like. In addition, enhancing gene expression can be achieved by integrating the gene into the bacterial chromosome by homologous recombination or the like.

Methods for using a strong promoter or enhancer can be combined with methods for increasing the number of copies of a gene.

To remove a microorganism belonging to the genus Escherichia or the genus Bacillus and having increased expression of the gene encoding the protein according to the present invention, the necessary sections of the genes can be obtained using PCR (polymerase chain reaction) based on already available information about the genes E. coli and B . subtilis. For example, the rhtA gene, which is believed to encode a transporter, can be cloned from the chromosomal DNA of E. coli K12 W3110 and E. coli MG1655 strains using the PCR method. The chromosomal DNA used for this can also be obtained from any other E. coli strain.

Proteins according to the present invention include mutants and variants of the RhtA protein, which may exist due to natural diversity, provided that these mutants and variants demonstrate the functional properties of the RhtA protein, at least resistance to 8-azaadenine. DNA encoding these mutants and variants can be obtained by isolating DNA that hybridizes with the rhtA gene (SEQ ID NO: 1) or a portion of the specified gene under stringent conditions and which encodes a protein that increases the production of purine nucleotides. The term “stringent conditions”, mentioned here, means the conditions under which the so-called specific hybrids are formed, and non-specific ones are not formed. For example, harsh conditions include conditions under which DNA with a high degree of homology hybridizes, for example, DNA with a homology of at least 70% relative to each other. Alternatively, stringent conditions are exemplified by conditions corresponding to Southern hybridization wash conditions, for example 60 ° C, 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS. Part of the nucleotide sequence numbered 1 can also be used as a probe for DNA encoding variants and hybridizing with the rhtA gene. A probe of this kind can be obtained by PCR using oligonucleotides obtained on the basis of the nucleotide sequence numbered as 1, and a DNA fragment containing the nucleotide sequence at number 1, as a matrix. When a DNA fragment of about 300 base pairs in length is used as a probe, the washing conditions during hybridization correspond, for example, to 50 ° C, 2 × SSC and 0.1% SDS.

The bacterium according to the present invention can be obtained by introducing the above DNA into a bacterium already possessing the ability to produce purine nucleosides. On the other hand, a bacterium according to the present invention can be obtained by giving a bacterium already containing said DNA the ability to produce purine nucleosides.

As the parent producer strain of inosine, in which the activity of the proteins of the present invention will be enhanced, E. coli strain AJ13732 (FADRaddeddyicPpgixapA (pMWKQ)) (WO 9903988) can be used. The specified strain is derived from the known strain W3110 containing mutations introduced into the purF gene encoding PRPP amidotransferase, purR gene encoding the purine biosynthesis repressor, deoD gene encoding purine nucleoside phosphorylase, purA gene encoding succinyl AMP synthase, add gene, encoding adenosine deaminase, edd gene encoding 6-phosphogluconate dehydrase, pgi gene encoding phosoglucose isomerase, harA gene encoding xanthosine phosphorylase (purF ^- , purA ^- , deoD ^- , purR ^- , add ^- , edd ^- , pgi ^- , and also charA ^- ) containing the plasmid pMWKQ - derivative of the vector p MW218, which contains the purFKQ genes encoding PRPP amidotransferase insensitive to guanosine monophosphate (GMP) (WO 9903988).

As the parent producer xanthosine strain in which the activity of the proteins of the present invention will be enhanced, E. coli strain AJ13732 guaA :: Tn10 (pMWKQ) can be used. The E. coli strain AJ13732 guaA :: Tn10 (pMWKQ) was constructed by disrupting the gene encoding GMP synthetase in strain AJ13732 (pMWKQ) (see Example 7).

As a parent strain belonging to the genus Bacillus, the producer of inosine, the strain B. subtilis KMBS16 can be used. The specified strain is derived from the known strain B. subtilis trpC2 containing mutations introduced into the purR gene encoding a purine biosynthesis repressor (purR :: spc), the purA gene encoding succinyl AMP synthase (purA :: erm), deoD gene, encoding purine nucleoside phosphorylase (deoD :: kan). As other parent strains belonging to the genus Bacillus, in which the activity of the proteins of the present invention will be increased, strains of B. subtilis AJ12707 (FERM P-12951) (Japanese patent application JP 6113876 A2), B. subtilis AJ3772 (FERM) can be used P-2555) (Japanese Patent Application JP 62014794 A2) and the like.

In order to increase the activity itself in terms of the protein according to the present invention, it is also possible to introduce a mutation into the structural part of the gene encoding the protein in order to increase the activity of the protein. In order to introduce a mutation into a gene, site-specific mutagenesis can be used (Kramer, W. and Frits, HJ Methods in Enzymology, 154, 350 (1987)), recombinant PCR methods (PCR Technology, Stockton Press (1989)), chemical synthesis of specific sections of DNA, treatment of the desired gene with hydroxylamine, treatment of microbial strains containing the desired gene with UV radiation or a chemical reagent such as nitrosoguanidine or nitrous acid, or a similar method. A microorganism in which the activity of the indicated protein is increased can be selected as a strain growing on minimal medium containing 8-azaadenine.

The bacterium of the present invention can be further improved by increasing the expression of one or more genes involved in purine biosynthesis. Examples of such genes are the pUREB-purC (orf) QLF-purMNH (J) -purD operon genes from B. subtilis (Ebbole DJ and Zalkin H., J. Biol. Chem., 262, 17, 8274-87, 1987) and pur regulatory genes from E. coli (Escherichia coli and Salmonella, Second Edition, Editor in Chief: FC Neidhardt, ASM Press, Washington DC, 1996).

Inosine production was shown to increase with the use of 8-azaguanine-resistant B. subtilis mutants with genetically modified repression of purine nucleoside synthesis enzymes (Shiio I. and Ishii K., J. Biochem., 69, 339-347, 1971). Inosine production by E. coli strain containing a mutant purF gene encoding a PRPP amidotransferase that is free from feedback inhibition of GMP and AMP was increased by inactivation of the purR gene encoding a purine biosynthesis repressor (WO 9903988).

The mechanism that increases the production of purine nucleosides by the bacterium by increasing the activity of the proteins of the present invention is, as can be expected, increased excretion of the target purine nucleoside from the bacterial cell.

2. A method of obtaining purine nucleosides.

The methods of the present invention include a method for producing a purine nucleoside, comprising the steps of growing a bacterium according to the present invention in a culture medium to produce and accumulate said purine nucleoside in a culture medium and isolate the purine nucleoside from the culture fluid. More specifically, the methods according to the present invention relates to a method for producing inosine, comprising the steps of growing a bacterium according to the present invention in a culture medium for the production and accumulation of inosine in a culture medium, and the isolation of inosine from the culture fluid. Also included in the methods of the present invention is a method for producing xanthosine, comprising the steps of growing a bacterium of the present invention in a culture medium to produce and accumulate xanthosine in the culture medium and to isolate xanthosine from the culture fluid.

According to the present invention, the cultivation, isolation and purification of purine nucleoside from a culture or similar liquid can be carried out in a manner similar to traditional fermentation methods in which the purine nucleoside is produced using a microorganism. The nutrient medium used for growing can be either synthetic or natural, provided that the medium contains sources of carbon, nitrogen, mineral additives and other necessary organic. Carbon sources include various carbohydrates such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trialose, ribose and starch hydrolyzate; alcohols such as glycerin, mannitol and sorbitol; various organic acids such as gluconic acid, fumaric acid, citric acid and succinic acid and the like. As the nitrogen source, various inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen sources such as soybean hydrolyzate can be used; ammonia gas and the like. Suitable small amounts of vitamins, such as vitamin B ₁ , and other essential substances, for example, nucleic acids, such as adenine and RNA, or yeast extract and the like, are preferably 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 preferably carried out under aerobic conditions for 16-72 hours, the temperature during cultivation is maintained in the range from 30 to 45 ° C and the pH is in the range from 5 to 8. The pH of the medium can be regulated by inorganic or organic acid or alkaline substances, as well as gaseous ammonia.

After growth, solid residues, such as cells, can be removed from the culture fluid by centrifugation or filtration through a membrane, and then the target purine nucleoside can be isolated from the culture fluid by any of the traditional methods or any combination of these methods, such as ion exchange chromatography and precipitation.

3. A method for producing purine nucleotides.

The methods of the present invention also include a method for producing a purine nucleotide, comprising the steps of growing a bacterium of the present invention in a nutrient medium, phosphorylating the obtained and accumulated purine nucleoside, and isolating the obtained and accumulated purine nucleotide. More specifically, the methods of the present invention also include a method for producing inosine-5’-phosphate, comprising the steps of growing the bacteria of the present invention in a nutrient medium, phosphorylating the obtained and accumulated inosine, and isolating the obtained and accumulated inosine-5’-phosphate. The methods of the present invention also include a method for producing xanthosine 5′-phosphate, comprising the steps of growing the bacteria of the present invention in a nutrient medium, phosphorylating the obtained and accumulated xanthosine, and isolating the obtained and accumulated xanthosine 5′-phosphate.

According to the present invention, the cultivation, isolation and purification of purine nucleoside from a culture or similar liquid can be carried out in a manner similar to traditional fermentation methods in which the purine nucleoside is produced using a microorganism. Further, according to the present invention, phosphorylation of the obtained and accumulated purine nucleoside, as well as the isolation of the obtained and accumulated purine nucleotide, can be carried out by a method similar to traditional methods in which the purine nucleotide is obtained from a purine nucleoside.

Phosphorylation of purine nucleoside can be carried out enzymatically using various phosphatases, nucleoside kinases and nucleoside phosphotransferases, or chemically using phosphorylating agents such as POCl ₃ or the like. Phosphatase capable of catalyzing the selective transfer of the phosphoryl group of pyrophosphate to the 5'-position of a nucleoside (Mihara et. Al. Phosphorylation of nucleosides by the mutated acid phosphatase from Morganella morganii. Appl. Environ. Microbiol. 2000, 66: 2811-2816 can be used). ), or acid phosphatase using polyphosphoric acids (their salts), phenylphosphoric acid (its salts) or carbamylphosphoric acid (its salts) as a phosphoric acid donor (WO 9637603 A1), or the like. Also, phosphatase capable of catalytically transferring a phosphoryl group to the 2 ', 3', 5'-position of a nucleoside using p-nitrophenyl phosphate as a substrate (Mitsugi, K. et al. Agric. Biol. Chem. 1964, 28, 586-600), inorganic phosphate (JP 42-1186), or acetyl phosphate (JP 61-41555), or the like. Guanosine inosine kinase from E. coli (Mori et al. Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the kind gene product. J. Bacteriol. 1995, 177: 4921-4926; WO 9108286) or the like. As an example of nucleoside phosphotransferase, nucleoside phosphotransferase described by Hammer-Jespersen, K. (Nucleoside catabolism, p.203-258. In A Munch-Petesen (ed.). Metabolism of nucleotides, nucleosides and nucleobases in microorganism. 1980, Academic Press, can be cited. , New York), or the like. Chemical nucleoside phosphorylation can be carried out using a phosphorylating agent such as POCl ₃ (Yoshikawa et al. Studies of phosphorylation. III. Selective phosphorylation of unprotected nucleosides. Bull. Chem. Soc. Jpn. 1969, 42: 3505-3508), or like him.

The methods of the present invention also include a method for producing guanosine-5'-phosphate, comprising the steps of growing the bacteria of the present invention in a nutrient medium, phosphorylating the obtained and accumulated xanthosine, aminating the obtained and accumulated xanthosine-5'-phosphate, and isolating the obtained and accumulated guanosine 5'-phosphate. According to the present invention, the cultivation of the bacteria according to the present invention in a nutrient medium, the phosphorylation of the obtained and accumulated xanthosine, the amination of the obtained and accumulated xanthosine 5'-phosphate, and the isolation of the obtained and accumulated guanosine-5'-phosphate can be carried out by a method similar to traditional methods, in which guanosine 5'-phosphate is derived 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 of the present invention, the bacterium of the present invention can be modified to increase expression of purine nucleoside biosynthesis genes.

Drawing captions

Figure 1 shows the structure of the plasmid pNZ16.

Figure 2 shows the structure of plasmid pRHTA3.

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

Figure 4 shows the distribution of RhtA protein in cell fractions. Lane 1 contains a pellet (15,000 g) of induced BL21 (DE3) cells not containing a plasmid (control); lanes 2–7 contain equal volumes of sediment and soluble fractions of induced BL21 cells (DE3) containing the plasmid pET22b-rhtA: lanes 2 and 3 — sediment and supernatant after centrifugation at 15,000 g, respectively; lanes 4 and 5 — sediment and supernatant after centrifugation at 180,000 g, respectively; lanes 6 and 7 — sediment and supernatant after centrifugation at 180,000 g, respectively, after treatment of cells with 1 M KCl. Molecular weight markers (in kDa) are shown in the left margin, and the position of the RhtA protein is shown by an arrow in the right margin.

Figure 5 shows the structure of the plasmid pLF-RHTA.

BEST MODE FOR CARRYING OUT THE INVENTION

Example 1. Cloning of the rhtA gene from E. coli into mini-Mu phasmid.

The rhtA gene, which was previously determined to be resistant to homoserin and threonine, was cloned in vivo using the mini-Mu d5005 phasmid (Groisman, E.A. et al. J. Bacteriol., 168, 357-364 (1986)). Strain MG442 lysogenic for phage MuCts62 (Gusyatiner et al. Genetika 14, 947-956 (1978)) was used as a donor. Freshly prepared lysates were used to infect the lysogenic MuCts phage derived strain VKPM B-513 (Hfr K10 metB). The resulting cells were plated on minimal M9 medium with glucose containing methionine (50 μg / ml), kanamycin (40 μg / ml) and homoserine (10 mg / ml). Colonies that appeared after 48 hours of cultivation were selected. Plasmid DNA was isolated from the colonies and used to transform the VKPM B-513 strain by standard methods. Transformants were selected on plates with agarized L-broth containing kanamycin and homoserine, as described above. Plasmid DNA was isolated from homoserine resistant transformants, and the structure of the inserted fragments was determined by restriction mapping. It turned out that two types of fragments belonging to different parts of the chromosome were cloned from the donor. Thus, in E. coli there are at least two different genes that give bacteria resistance to homoserine when they are present in a multi-copy plasmid. It was shown that one of the types of chromosomal fragments is a fragment from the 86 min section. In this case, the resistance phenotype was determined by increased expression of the rhtB and rhtC genes (European patent applications EP 1013765 A1 and EP 1016710 A2).

In order to select phasemids with an insertion of the 18 min chromosomal fragment, colony hybridization was used using a mixture of six ³² P-labeled recombinant phages (3D2, 24F9, 1B4, 10AB, 3E5 and 1E2) from the Kohara collection (Kohara et al. , Cell, 50, 495-508, 1987) containing the chromosome fragment 17.5-18.5 min. As a result, the plasmid pNZ4S was obtained, containing a 9.3 kb fragment, which confers cell resistance to threonine and homoserine.

Example 2. Cloning of the rhtA gene into pBlueScript KS ⁺ and pAYCTER-3 vectors.

Plasmid pNZ4S was digested with restriction enzymes XmnI and StuI, and the resulting DNA fragments were separated by electrophoresis in low melting agarose. The desired fragment containing the rhtA gene, together with its own regulatory region, was eluted and inserted in the opposite direction to the lac promoter at the recognition site of EcoRV restriction enzyme pBlueScript KS ⁺ (Promega), pre-treated with EcoRV restriction enzyme. Thus, the plasmid pNPZ16 was obtained (Figure 1). In addition, the rhtA gene was cloned from pNPZ16 at the SmaI-HindIII sites of the stable mid-copy vector pAYCTER3, a derivative of the vector pAYC32. So was obtained plasmid pRHTA3 (Figure 2). The pAYCTER3 vector is a very stable mid-copy vector constructed on the basis of the plasmid RSF1010 (Christoserdov AY, Tsygankov YD Plasmid, 1986, v.16, pp. 161-167) by introducing into the pAYC32 vector a polylinker from plasmid pUC19 and the strong terminator rrnB.

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

The 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 RhtA protein by a known method (Kyte and Doolittle, J. Mol. Biol., 157, 105-132, (1982)) showed that this protein is a hydrophobic protein containing 10 putative transmembrane segments. The hydrophobicity profile and the number of putative transmembrane segments of the RhtA protein (Figure 3, solid line) and the YdeD protein (Figure 3, dashed line) are similar to each other. This result shows their homology (Lolkema, J.S., and Slotboom, D.-J. FEMS Microbiol Rev., 22, 305-322 (1998)). The ydeD gene encodes a YdeD protein involved in the release of cysteine biosynthesis metabolites from a cell (Daβ ler et al., Mol. Microbiol. 36, 1101-1112 (2000)). Based on this, it can be assumed that the rhtA gene encodes a certain membrane protein involved in the transport of certain metabolites from the cell.

Example 4. Construction of the plasmid pET22b-rhtA and determination of the cell localization of the rhtA gene product.

The coding sequence of the rhtA gene was obtained on the basis of plasmid pNPZ16 by polymerase chain reaction (PCR) using the seeds of SEQ ID NO: 3 containing the Ndel restriction site and SEQ ID NO: 4 containing the EcoRI restriction site. The resulting PCR product was digested with restriction enzymes NdeI and EcoRI and ligated into the bacterial expression vector pET22b (Novogene) pretreated with the same restriction enzymes. The resulting plasmid, pET22b-rhtA, containing the T7 RNA polymerase promoter under the control of P _lac (Novogene) gene was used for transformation of strain E. coli BL21 (DE3). The introduction of the T7 RNA polymerase gene and the production of [ ³⁵ S] labeled with methionine protein was carried out essentially as described previously (Tabor and Richardson, Proc. Natl. Acad. Sci. USA, 82, 1074-1078 (1985)), with a slight modification. The modification was as follows: protein expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG, final concentration 2 mM) to 5 ml of culture in a logarithmic growth phase on M9 medium containing a mixture of 19 amino acids (final concentration 0.005%). Newly synthesized protein was labeled by adding 50 μ Ci [ ³⁵ S] methionine to 5 ml of cell culture. Cells were harvested by centrifugation and used for fractionation. The pellet was resuspended in disruption buffer (100 mM Tris-HCl buffer, pH 7.5, containing 1 mM EDTA, 2 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride) and the cells were sonicated. After removing cell debris by centrifugation at 15,000 g for 20 minutes, the membrane fraction was obtained by ultracentrifugation at 180,000 g for 180 minutes. The resulting precipitate (membrane fraction) was resuspended in destructive buffer containing 1 M KCl for 40 minutes at room temperature and centrifuged at 180,000 g for 180 minutes. Soluble supernatant fractions were precipitated by incubation with trichloroacetic acid (final concentration 10%) at 4 ° C for 30 min, centrifuged and washed with acetone. All sediments were resuspended in the same volume of gel buffer (Laemmli, Nature 227, 680-685 (1970)). Proteins were analyzed by polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS) (Laemmli, Nature 227, 680-685 (1970)).

The distribution of RhtA protein in cell fractions is shown in FIG. 4. The RhtA protein was coprecipitated with membrane fractions (Figure 4, lanes 4 and 6) and was absent in the soluble protein fraction (Figure 4, lane 5). The same protein distribution was observed after treatment with KCl membrane fraction (Figure 4, lanes 6, 7). These data support the view that the RhtA protein is a fully membrane protein. The electrophoretic mobility of the RhtA protein corresponds to a molecular weight of about 25 kDa instead of the predicted mass of 31.3 kDa, which may be the result of the high hydrophobicity of the RhtA protein. The YdeD protein shows a similar pattern of gel motility (Daβ ler et al., Mol. Microbiol. 36, 1101-1112 (2000)).

Example 5. The effect of rhtA23 mutation and amplification of the rhtA gene on the resistance of E. coli MG1655 strain to purine base analogs.

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

In addition, plasmids pNPZ16 and pRHTA3, as well as the corresponding vectors pBluescript KS ⁺ and pAYCTER3, were introduced into E. coli strain MG1655. Thus, strains MG1655 (pNPZ16), MG1655 (pRHTA3), MG1655 (pBluescript), MG1655 (pAYCTER3) were obtained. Then, the ability of each of these strains to grow in a minimal agarized M9 medium with glucose containing stepwise increasing concentrations of the purine base analogue was determined. Cups were seeded with 10 ⁶ to 10 ⁷ cells of an overnight culture grown on minimal medium containing 100 mg / ml ampicillin in the case of plasmid strains. Growth ability was determined after 44 hours of incubation at 37 ° C. The experimental results are presented in Table 1.

As can be seen from Table 1, the rhtA23 mutation and amplification of the rhtA gene increased the resistance of bacteria to 8-azaadenine. Based on this, it can be assumed that the rhtA gene is involved in the excretion of purine derivatives.

Example 6. The effect of rhtA23 mutation on the production of inosine by E. coli strain - producer of inosine.

Strain AJ13732 (pMWKQ), the producer of inosine, was used as the parent strain for introducing the rhtA23 mutation, as described in Example 5. Thus, strain AJ13732rhtA23 (pMWKQ) was obtained. Each of the strains was 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 fermentation medium containing 100 mg / ml ampicillin and 75 mg / l kanamycin in a 20 × 200 mm tube and incubated at 37 ° C for 72 hours on a rotary shaker.

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

Glucose 40.0

(NH ₄ ) ₂ SO ₄ 16.0

K ₂ HPO ₄ 1.0

MgSO ₄ 7H ₂ O 1.0

FeSO ₄ 7H ₂ O 0.01

MnSO ₄ 5H ₂ O 0.01

Yeast Extract 8.0

CaCO ₃ 30.0

Glucose and magnesium sulfate were sterilized separately. CaCO _{3 was} sterilized at 180 ° C for 2 hours. The pH was maintained at 7.0. The antibiotic was added to the culture medium after sterilization.

After growing, the amount 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 with water and analyzed by HPLC.

Conditions for analysis using HPLC.

Column: Luna C 18 (2) 250 × 3 mm, 5 u (Phenomenex, USA). Buffer: 2% v / v C ₂ H ₅ OH; 0.8% v / v triethylamine, 0.5% v / v acetic acid (glacial), pH 4.5. Temperature: 30 ° C. Flow rate: 0.3 ml / min. Sample volume: 5 μl. UV detector: 250 nm.

Retention Time (min):

Xanthosine 13.7

Inosine 9.6

Hypoxanthine 5.2

Guanosine 11.4

Adenosine 28.2

The results are presented in Table 2.

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

Example 7. The effect of rhtA23 mutation on the production of xanthosine by E. coli strain - producer of xanthosine.

The xanthosine producing strain was obtained from strain AJ13732 (pMWKQ), an inosine producer. Using phage P1 transduction, the guaA.:Tn10 mutation was introduced into strain AJ13732 (pMWKQ), which inactivates the GMP synthetase gene (guaA). Transductants were selected on LB medium containing 20 μg / ml tetracycline. Among them, a strain AJ13732 gw.4 :: Tn10 (pMWKQ) was found, capable of producing xanthosine. A derivative of this strain containing the rhtA23 mutation was obtained as described in Example 5 to obtain the AJ13732 strain guaA :: Tn 10rhtA23 (pMWKQ).

Each of the strains AJ13732 gua4 :: Tn10 (pMWKQ) and AJ13732 guaA :: Tn10 rhtA23 (pMWKQ) was 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 fermentation medium (see Example 3), containing 75 mg / l kanamycin and 10 mg / l tetracycline, in a test tube of 20 × 200 mm and incubated at 37 ° C for 48 h on a rotary rocking chair. After growing, the amount of 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 rhtA23 mutation improves the production of xanthosin by the strain AJ13732 guaA :: Tn10 (pMWKQ).

Example 8. The effect of amplification of the rhtA gene on the production of inosine by E. coli strain - producer of inosine.

Strain AJ13732 (pMWKQ), an inosine producer, was transformed with the pAYCTER3 vector and the plasmid pRHTA3. Thus, strains AJ13732 (pMWKQ, pAYCTER3) and AJ13732 (pMWKQ, pRHTA3) were obtained. Each of these strains was 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 fermentation medium (see Example 3) containing 100 mg / l ampicillin and 75 mg / l kanamycin, in a test tube of 20 × 200 mm and incubated at 37 ° C for 48 h at rotary rocking chair. After growing, the amount of 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, amplification of the rhtA gene improves the production of inosine by strain AJ13732 (pMWKQ).

Example 9. The effect of amplification of the rhtA gene on the production of xanthosine by E. coli strain - producer of xanthosine.

Strain AJ13732 guaA :: Tn10 (pMWKQ), the xanthosine producer described in Example 7, was transformed with the plasmid pRHTA3 or pAYCTER3 vector to obtain strains AJ13732 guaA :: Tn10 (pMWKQ, pRHTA3), AJ13732CAY pAHG332AAY pAHT3 guA. Each of these strains was 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 fermentation medium (see Example 3) containing 100 mg / l ampicillin and 75 mg / l kanamycin in a 20 × 200 mm tube and incubated at 37 ° C for 48 h on a rotary rocking chair. After growing, the amount of xanthosine accumulated in the medium was determined by HPLC as described above. The results are presented in Table 5.

As can be seen from Table 5, amplification of the rhtA gene improves the production of xanthosin by the strain AJ13732 guaA :: Tn10 (pMWKQ).

Example 10. Cloning of the rhtA gene into pLF14 vector.

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

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

Then, this design was cloned at the SacI - SphI sites into the mobilized single-replicon shuttle vector pLF14 (Shevelev et al., Plasmid, 43, 190-199, 2000), which can express various genes in Bacillus cells under the control of the Kan gene promoter. The resulting plasmid, pLF-RHTA (Figure 5), was mobilized from E. coli TG1 strain using plasmid RP4 to B. subtilis strain 168. Being expressed in Bacillus cells, the rhtA gene gave them increased resistance to homoserine during growth at a minimum M9 medium (Table 6).

The cultures were grown at 37 ° C for 44 hours on a minimal agar medium containing the indicated concentrations of homoserine: + good growth, - no growth.

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

Strain B. subtilis KMBS 16, the producer of 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) Construction of a B. subtilis mutant deficient in purR.

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

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

2) Construction of the B. subtilis mutant deficient in pURA.

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), а также 9 дополнительных нуклеотидов, содержащих сайт SolI, присоединенных к 5’-концу. Затравка №4 (29 звеньев) включает в себя последовательность с 51 по 70 нуклеотид после стоп-кодона гена риrА, а также 9 дополнительных нуклеотидов, содержащих сайт SphI, присоединенных к 5’-концу. Фрагмент, полученный в ходе ПЦР, (1.5 т.п.о.) и обработанный SalI и SphI, был вставлен по тем же сайтам рестрикции вектора pSTV28 (TaKaRa, Япония). Полученная плазмида pSTV28BSPA была расщеплена с помощью МluI и BglII, что привело к удалению внутреннего фрагмента длиной 0.4 т.п.о. из амплифицированного гена рurА, концы были затуплены с помощью фрагмента Кленова, затем в нее лигирован фрагмент гена устойчивости к эритромицину с затупленными концами (1.6 т.п.о.) из Staphylococcus anreus, вырезанный из pDG646 (Bacillus Genetic Stock Center, Ohio).PCR was performed at 94 ° C for 30 s; 55 ° C, 1 min; 72 ° C, 2 min; 30 cycles (Gene Amp PCR System Model 9600, Perkin Elmer), using the chromosomal DNA of strain B. sublilis 168 Marburg as a matrix and the following oligonucleotide seeds, No. 3 (SEQ ID NO: 9) and No. 4 (SEQ ID NO: 10 ) synthesized according to the DNA sequence in the genomic database. Seed No. 3 (29 links) includes the sequence from 137 to 118 nucleotides before the start codon of the pURA gene 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), as well as 9 additional nucleotides containing a SolI site attached to the 5'-end. Seed No. 4 (29 links) includes a sequence from 51 to 70 nucleotides after the stop codon of the rirA gene, as well as 9 additional nucleotides containing the SphI site attached to the 5'-end. A fragment obtained by PCR (1.5 kb) and processed with SalI and SphI was inserted at the same restriction sites of the pSTV28 vector (TaKaRa, Japan). The resulting plasmid pSTV28BSPA was digested with MluI and BglII, which led to the removal of the 0.4 kb internal fragment. from the amplified pURA gene, the ends were blunted using a Klenov fragment, then a fragment of the erythromycin resistance gene with blunt ends (1.6 kb) from Staphylococcus anreus cut from pDG646 (Bacillus Genetic Stock Center, Ohio) was ligated into it.

The resulting plasmid pSTV28BSPA :: erm was used to transform competent KMBS4 cells obtained by the Dubunau and Davidoff-Abelson method, as described above. Double cross mutants were tested by preparing chromosomal DNA from each erythromycin-resistant colony, followed by PCR as described above. One of the colonies, which was confirmed to be a mutant deficient in the pURA gene, was named KMBS13. As expected, KMBS13 cells were auxotrophic for adenine.

3) Construction of a mutant of B. subtilis deficient in deoD.

To obtain the 5’-terminal portion of the deoD gene and the preceding portion from B. subtilis, PCR was performed at 94 ° C for 30 s; 55 ° C, 1 min; 72 ° C, 1 min; 30 cycles (Gene Amp PCR System Model 9600, Perkin Elmer) using the following oligonucleotide seeds, No. 5 (SEQ ID NO: 11) and No. 6 (SEQ ID NO: 12), synthesized according to the DNA sequence in the genomic database . Seed No. 5 (29 links) includes a sequence from 310 to 291 nucleotides in front of the start codon of the deoD gene, as well as 9 additional nucleotides containing an EcoRI site attached to the 5’-end. Seed No. 6 (28 links) includes the sequence from 39 to 57 nucleotides after the start codon of the deoD gene, as well as 9 additional nucleotides containing the BamHI site attached to the 5’-end. A fragment obtained by PCR (0.4 kb) and processed with EcoRI and BamHI was inserted at the same restriction sites of the pSTV28 vector (TaKaRa, Japan), resulting in plasmid pSTV28DON.

To obtain the 3’-terminal portion of the deoD gene and the subsequent plot, PCR was performed at 94 ° C for 30 s; 55 ° C, 1 min; 72 ° C, 1 min; 30 cycles (Gene Amp PCR System Model 9600, Perkin Elmer) using the following oligonucleotide seeds, No. 7 (SEQ ID NO: 13) and No. 8 (SEQ ID NO: 14), synthesized in accordance with the DNA sequence in the genomic database . Seed No. 7 (29 links) includes the sequence from 321 to 302 nucleotides after the stop codon of the deoD gene, as well as 9 additional nucleotides containing the HindIII site attached to the 5’-end. Seed No. 8 (28 links) includes a sequence of 24 to 42 nucleotides in front of the stop codon of the deoD gene, as well as 9 additional nucleotides containing the BamHI site attached to the 5’-end. A fragment obtained by PCR (0.4 kb) and processed with HindIII and BamHI was inserted at the same restriction sites of the pSTV28DON vector, resulting in plasmid pSTV28DONC.

In order to amplify the kanamycin resistance gene from Streptococcus faecalis, PCR was performed at 94 ° C for 30 s; 55 ° C, 1 min; 72 ° C, 2 min; 30 cycles (Gene Amp PCR System Model 9600, Perkin Elmer) using the pDG783 DNA plasmid (Bacillus Genetic Stock Center, Ohio) as the template and the following oligonucleotide seeds, No. 10 (SEQ ID NO: 15) and No. 11 (SEQ ID NO: 16) synthesized according to the DNA sequence in the genomic database. Seed No. 10 (33 links) includes a sequence from 513 to 490 nucleotides in front of the start codon of the kanamycin resistance gene, as well as 9 additional nucleotides containing the BamHI site attached to the 5’-end. Seed No. 11 (33 links) includes the sequence from 117 to 140 nucleotides after the stop codon of the indicated gene, as well as 9 additional nucleotides containing the BamHI site attached to the 5’-end. A fragment obtained by PCR (1.5 kb) and processed by BamHI was inserted at the unique BamHII restriction site of the vector pSTV28DONC. The resulting plasmid pSTV28deoD :: kan was used to transform competent KMBS13 cells obtained by the Dubunau and Davidoff-Abelson method, as described above. Double cross mutants were tested by preparing chromosomal DNA from each kanamycin resistant colony, followed by PCR using primers No. 5 and No. 7, as described above. One of the colonies, which was confirmed to be a deoD-deficient mutant (purR :: spc purA :: erm deoD :: kan), was named KMBS16.

Example 11. The effect of amplification of the rhtA gene on the production of inosine by B. subtilis strain - producer of inosine.

Plasmid pLF-RHTA and the vector pLFH were placed in B. subtilis strain KMBS16, the producer of inosine. Thus, strains of B. subtilis KMBS16 (pLF-RHTA) and B. subtilis KMBS16 (pLF14) were obtained. Each of these strains was grown at 37 ° C for 18 h in L-broth containing 10 mg / l chloramphenicol, and 0.3 ml of the resulting culture was transferred to 3 ml of Bacillus fermentation medium containing 10 mg / l chloramphenicol, in a test tube 20 × 200 mm and incubated at 37 ° C for 72 hours on a rotary shaker.

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

Glucose 80.0

KH ₂ PO ₄ 1.0

MgSO ₄ 0.4

FeSO ₄ × 7H ₂ O 0.01

MnSO ₄ × 5H ₂ O 0.01

Mameno-TN 1.35

DL-methionine 0.3

NH ₄ Cl 32.0

Adenine 0.1

Tryptophan 0.02

CaCO ₃ 50.0

After growing, the amount 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, amplification of the rhtA gene improves the production of inosine by B. subtilis strain KMBS16.

Claims (17)

1. A method of producing a purine nucleoside, comprising the steps of growing Escherichia coli bacteria or Bacillus subtilis bacteria in a nutrient medium and isolating the obtained and accumulated purine nucleoside from the culture fluid, characterized in that Escherichia coli bacterium or Bacillus subtilis bacterium is used, the ability to produce purine the nucleoside of which is increased by increasing the activity of proteins selected from the group: (A) a protein that is represented by the amino acid sequence shown in the list of sequences under measure 2 (SEQ ID NO: 2); (B) a protein that is represented by an amino acid sequence including deletions, substitutions, insertions, or the addition of one or more amino acids to the amino acid sequence listed in sequence number 2 (SEQ ID NO: 2), and which has an activity that confers enhanced resistance to bacteria to 8-azaadenine. 2. The method according to claim 1, characterized in that the purine nucleoside is inosine. 3. The method according to claim 1, characterized in that the purine nucleoside is xanthosine. 4. The method according to any one of claims 1 to 3, characterized in that in said bacterium the expression of purine nucleoside biosynthesis genes is increased. 5. The method according to claim 2, characterized in that the Escherichia coli strain AJ13732rhtA23pMWKQ), the Escherichia coli strain AJ13732 (pMWKQ, pRHTA3) or the Bacillus subtilis KMBS16 strain (pLF-RHTA) are used as the inosine producer. 6. The method according to claim 3, characterized in that the Escherichia coli strain AJ13732 guaA :: Tnl0 rhtA23 or the Escherichia coli strain AJ13732 guaA :: Tnl0 (pMWKQ, pKNTA3) is used as a xanthosine producer. 7. A method for producing a purine nucleotide, comprising the steps of growing Escherichia coli bacteria or Bacillus subtilis bacteria in a nutrient medium, phosphorylating the obtained and accumulated nucleoside, and isolating the resulting purine nucleotide, characterized in that Escherichia coli bacterium or Bacillus subtilis bacterium, the ability to produce purine nucleoside which is increased by increasing the activity of proteins selected from the group: (A) a protein that is represented by the amino acid sequence shown in the list of sequences stey under number 2 (SEQ ID NO: 2); (B) a protein that is represented by an amino acid sequence including deletions, substitutions, insertions, or the addition of one or more amino acids to the amino acid sequence listed in sequence number 2 (SEQ ID NO: 2), and which has an activity that confers enhanced resistance to bacteria to 8-azaadenine. 8. The method according to claim 7, characterized in that the purine nucleotide is inosine-5'-phosphate. 9. The method according to claim 7, characterized in that the purine nucleotide is xanthosine 5'-phosphate. 10. The method according to any one of claims 7 to 9, characterized in that in said bacterium the expression of purine nucleoside biosynthesis genes is increased. 11. A method of producing guanosine 5'-phosphate, comprising the steps of growing Escherichia coli bacteria or Bacillus subtilis bacteria in a nutrient medium, phosphorylating the obtained and accumulated xanthosine, amination of the obtained xanthosine 5'-phosphate, and isolating the obtained guanosine 5'-phosphate the fact that they use the bacterium Escherichia coli or the bacterium Bacillus subtilis, the ability to produce a purine nucleoside of which is increased by increasing the activity of proteins selected from the group: (A) a protein that is represented by the amino acid sequence It is shown in the list of sequences under number 2 (SEQ ID NO: 2); (B) a protein that is represented by an amino acid sequence including deletions, substitutions, insertions, or the addition of one or more amino acids to the amino acid sequence listed in sequence number 2 (SEQ ID NO: 2), and which has an activity that confers enhanced resistance to bacteria to 8-azaadenine. 12. The method according to claim 11, characterized in that the said bacterium has an increased expression of purine nucleoside biosynthesis genes. 13. The strain Escherichia coli AJ13732 rhtA23 (pMWKQ) is the producer of inosine. 14. The strain Escherichia coli AJ13732 guaA :: Tnl0 rhtA23 (pMWKQ) is a producer of xanthosine. 15. The strain Escherichia coli AJ 13732 (pMWKQ, pRHTA3) - producer of inosine. 16. The strain Escherichia coli AJ 13732 guaA :: Tnl0 (pMWKQ, pKHTA3) is a producer of xanthosine. 17. The strain Bacillus subtilis KMBS16 (pLF-RHTA) - producer of inosine.

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