RU2244004C2 - Method for preparing inosine and 5'-inosinic acid, strain escherichia coli as producer of inosine - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: inosine and 5'-inosinic acid are obtained by using microorganism Escherichia coli. Production of inosine by indicated microorganism is elevated due to enhancing activity of protein encoding by gene ydeD. Invention provides elevating yield of inosine and 5'-inosinic acid.

EFFECT: improved preparing method.

8 cl, 3 dwg, 2 tbl, 4 ex

Description

The field of technology.

The present invention relates to a method for producing inosine, which is an important starting reagent for the synthesis of 5'-inosinic acid (inosine-5’-phosphate) and to a new microorganism used for its production.

State of the art

Traditionally, nucleosides are produced on an industrial scale by fermentation using strains of microorganisms auxotrophic for adenine, or these strains that are additionally resistant to various compounds, such as purine analogues and sulfaguanidine, while these strains belong to the genus Bacillus (Japanese patent application No. 38 -23039 (1963), 54-17033 (1979), 55-2956 (1980) and 55-45199 (1980), Japanese Patent Application Laid-open 56-162998 (1981), Japanese Patent Applications 57-14160 (1982) and 57- 41915 (1982), Japanese Patent Laid-open No. 59-42895 (1984), to the genus Brevibacterium (pa Japanese patent applications No. 51-5075 (1976) and 58-17592 (1983), 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, such as irradiation with UV radiation or treatment with nitrosoguanidine (N-methyl-N’-nitro-N-nitrosoguanidine), followed by selection of the desired strain in a suitable breeding medium for selection. On the other hand, the cultivation of mutant strains belonging to the genus Bacillus is also practiced (Japanese Patent Application Laid-Open No. 58-158197 (1983), 58-175493 (1983), 59-28470 (1984), 60-156388 (1985), 1- 27477 (1989), 1-174385 (1989), 3-58787 (1991), 3-164185 (1991), 5-84067 (1993) and 5-192164 (1993)) to the genus Brevibacterium (Japanese Patent Application Laid-Open 63- 248394 (1988)) and to the genus Escherichia (application WO 9903988) obtained using genetic engineering methods.

The productivity of the inosine producing strains can then be improved by increasing the ability to excrete inosine. Currently, it is generally accepted that the penetration of metabolites through the cytoplasmic membrane usually occurs with the participation of more or less specific transport proteins that release them (Rao et al, 1998, Microbiol. Mol. Biol. Rev., 62, 1-34; Paulsen et al , 1998, J. Mol. Biol., 277, 573-592; Saier et al, 1999, J. Mol. Microbiol. Biotechnol., 1, 257-279). Previously, the authors of the present invention showed that strains producing inosine or xanthosine belonging to the genus Escherichia and to the genus Bacillus and having increased activity of the RhtA protein encoded by the rhtA gene (ybiF) produced more inosine or xanthosine than the parent strains (patent application RF No. 2002101666).

It was previously shown that the ydeD gene is involved in the release of cysteine biosynthesis pathways from metabolite cells (DaBler et al., Mol. Microbiol., 36, 1101-1112, 2000; US 5972663). The ydeD gene is known as the putative transmembrane subunit encoding a protein with unknown function (nucleotides 1618262 to 1619062 in sequence with accession number NC_000913, gi: 16129492 in the GenBank database).

Description of the invention

The aim of the present invention is to increase the productivity of inosine by producer strains of inosine and to provide a method for producing inosine and 5’-inosinic acid using these strains.

This goal was achieved by establishing the fact that the ydeD gene, which supposedly encodes a membrane protein that is not involved in the pathogen biosynthesis of purine nucleosides, confers resistance to the purine base analog, 8-azaadenine, when the natural allele of this gene is introduced into the cells of the strain on multicopy plasmid. Moreover, the ydeD gene can increase nucleoside production when additional copies of it are introduced into the cells of the corresponding inosine producers belonging to the genus Escherichia. Thus, the present invention has been completed.

Thus, the present invention provides a microorganism belonging to the genus Escherichia, with the ability to produce inosine.

In particular, the present invention provides a microorganism with an increased ability to produce inosine 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 increased ability to produce inosine based on increased expression of a gene encoding a protein involved in the process of excretion of purine nucleosides.

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

Further, the present invention provides a method for producing 5′-inosinic acid, comprising the steps of growing a bacterium according to the present invention in a nutrient medium, phosphorylating the obtained and accumulated inosine and isolating the obtained and accumulated 5’-inosinic acid.

The present invention includes the following:

The invention 1. A bacterium belonging to the genus Escherichia, having the ability to produce inosine, in which the activity of the protein described in paragraphs (A) or (B) in the cell of 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 gives bacteria enhanced resistance to 8-azaadenine (here and further, the proteins described in points (A) and (B) are referred to as “proteins according to the present invention”).

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 the chromosome of 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.

Invention 4. A method for producing purine nucleoside, comprising the steps of growing a bacterium in accordance with any of Inventions 1-3 in a nutrient medium and isolating the obtained and accumulated inosine from the culture fluid.

Invention 5. The method in accordance with Invention 4, in which the bacterium has increased expression of purine nucleoside biosynthesis genes.

Invention 6. A method for producing 5′-inosinic acid, comprising the steps of growing a bacterium in accordance with any of Inventions 1-3 in a nutrient medium, phosphorylating the obtained and accumulated inosine and isolating the obtained and accumulated 5’-inosinic acid.

The invention 7. The method in accordance with the Invention 6, 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 bacterium according to the present invention.

The above bacterium according to the present invention is a bacterium belonging to the genus Escherichia, which is capable of producing inosine, 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 given 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” means that the bacterium belongs to the genus Escherichia in accordance with the classification known to the person skilled in the field of microbiology. As an example of a microorganism belonging to the genus Escherichia used in the present invention, the bacterium Escherichia coli (E. coli) may be mentioned.

The term “inosine production ability” as used herein means the ability to produce and store inosine in a culture medium. The term "possessing the ability to produce inosine" means that a microorganism belonging to the genus Escherichia has the ability to produce and accumulate in the nutrient medium inosine in an amount greater than the natural E. coli strain, such as E. coli strains W3110 and MG1655, and preferably means that the microorganism is capable of production and accumulation in a nutrient medium in an amount of not less than 10 mg / l, more preferably not less than 50 mg / l of inosine.

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 YdeD protein. Protein YdeD is a highly hydrophobic protein consisting of 266 amino acids, containing 10 putative transmembrane segments and having unknown function. The YdeD protein is encoded by the ydeD gene. The ydeD gene (nucleotides from 1618262 to 1619062 in the sequence with accession number NC_000913, gi: 16129492 in the GenBank database) is located on the E. coli chromosome between the tarB and ydeF genes. The ydeD gene encodes a protein with unknown function.

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 27, 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, P _L 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. It is further known that the replacement of several nucleotides in the region between the binding site of the ribosome (RBS) and the start codon, and especially in the sequence immediately before the start codon, significantly affects the mRNA translatability. For example, a 20-fold change in expression level was found depending on the nature of the three nucleotides prior to the start of the codon (Gold et al., Annu. Rev. Microbiol, 35, 365-403, 1981; Hui et al, EMBO J, 3, 623 -629, 1984).

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 (1989).

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 the introduction of such recombinant DNA into the microorganism. Examples of vectors used to introduce recombinant DNA are plasmid vectors such as pMW118, pBR322, pUC19, pBluescript KS ⁺ pACYC177, pACYC184, pAYC32, pMW119, pET22b and the like, phage vectors such as 11059, MuF9, 1BF1 (Japanese Patent Application Laid-open No. 2-109985) and the like, and transposons (Berg, DE and Berg, CM, Bio / Technol., 1417 (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.

Methods for obtaining chromosomal DNA, hybridization, polymerase chain reaction (PCR), obtaining plasmid DNA, DNA cleavage and ligation, transformation, selection of oligonucleotides as seeds and similar methods can be traditional methods well known to those skilled in the art. These methods are described in Sambrook, J. and Russell D.'s "Molecular Cloning A Laboratory Manual, Third Edition", Cold Spring Harbor Laboratory Press (2001) and other similar publications.

To remove a microorganism belonging to the genus Escherichia and having increased expression of the gene encoding the protein of the present invention, the necessary regions of the genes can be obtained by PCR based on already available information about the E. coli genes. For example, the ydeD gene, which is believed to encode a transporter, can be cloned from the chromosomal DNA of E. coli K 12 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 YdeD protein, which may exist due to natural diversity, provided that these mutants and variants demonstrate the functional properties of the YdeD protein, at least resistance to 8-azaadenine. DNA encoding these mutants and variants can be obtained by isolating DNA that hybridizes with the ydeD gene (SEQ ID NO: 1) or part 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, stringent 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 the washing conditions for Southern hybridization, for example 60 ° C, 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS. Part of the nucleotide sequence number 1 can also be used as a probe for DNA encoding the variants and hybridizing with the ydeD gene. A probe of this kind can be obtained by PCR using oligonucleotides obtained on the basis of the nucleotide sequence number 1 as seeds. and a DNA fragment containing the nucleotide sequence at number 1, as a matrix. When a DNA fragment with a length of about 300 base pairs 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 strain, producers of inosine, in which the activity of the proteins of the present invention will be increased, 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 a purine biosynthesis repressor, deoD gene encoding purine nucleoside phosphorylase, pURA gene encoding succinyl AMP synthase, add encoding adenosine deaminase, edd gene encoding 6-phosphogluconate dehydrase, pgi gene encoding phosphoglucose isomerase, harA gene encoding xanthosine phosphorylase (purF ^- , purA ^- , deoD ^- , purR ^- , add ^- , edd ^- , pgi ^- , and also charA ^- ) containing plasmid pMWKQ - derivative from vector pMW218, in which genes are purFKQ, coding for PRPP amidotransferase insensitive to guanosine monophosphate (GMP) (WO 9903988).

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 to increase the activity of the protein. In order to introduce a mutation into the gene, site-specific mutagenesis (Kramer, W. and Frits, HJ, Methods in Enzymology, 154, 350 (1987)), recombinant PCR methods (PCR Technology, Stockton Press (1989)) can be used. 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 E. coli riggulon genes (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F.C. Neidhardt, ASM Press, Washington D.C., 1996). Described by E. coli strain, an inosine producer containing a mutant purF gene encoding a PRPP amidotransferase free from feedback inhibition of GMP and AMP, an inactivated 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 inosine, comprising the steps of growing the bacteria of the present invention in a culture medium to produce and accumulate inosine in the culture medium and isolate inosine 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, inorganic ions and other necessary organic components. 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, ammonium solution and the like. Suitable small amounts of vitamins, such as vitamin Bi, and other essential substances, for example, nucleic acids, such as adenine and RNA, or yeast extract and the like, are preferably present in the culture 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 5’-inosinic acid.

The methods of the present invention also include a method for producing 5'-inosinic acid, comprising the steps of growing a bacterium according to the present invention in a nutrient medium, phosphorylating the obtained and accumulated inosine, and isolating the obtained and accumulated 5'-inosinic acid.

According to the present invention, the cultivation, isolation and purification of inosine from a culture or similar liquid can be carried out in a manner similar to traditional fermentation methods in which inosine is produced using a microorganism. Further, according to the present invention, the phosphorylation of the obtained and accumulated inosine, as well as the isolation of the obtained and accumulated 5'-inosinic acid, can be carried out by a method similar to traditional methods in which purine nucleotides, such as 5'-inosinic acid, are obtained from a purine nucleoside, such as inosine.

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-nitrophenylphosphate as a substrate (Mitsugi, K., et al, Agric. Biol. Chem 1964, 28, 586-600), inorganic phosphate (JP42-1186), or acetyl phosphate (JP61-41555), 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 purified gene product. J. Bacteriol. 1995. 177: 4921-4926 can be cited as an example of nucleoside kinase. ; WO9108286) or the like. As an example of a nucleoside phosphotransferase, the 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.

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. Scheme of the chromosomal DNA fragment contained in pAZA10 phasemid, which conferred resistance to 8-azaadenine on the cells.

Figure 2 shows the structure of plasmid pYDED1.

Figure 3 shows the structure of plasmid pYDED3.

BEST MODE FOR CARRYING OUT THE INVENTION

Example 1. Cloning of genes that confer resistance to purine base analogs from E. coli to mini-Mu phasmid.

Genes from E. coli, which cause resistance to purine base analogs, were initially cloned in vivo using the mini-Mu d5005 phasmid (Groisman, E. A., et al., J. Bacteriol., 168, 357-364 (1986)). Strain MG1655, lysogenic for phage MuCts62, was used as a donor. Freshly prepared lysates were used to infect a lysogenic MuCts phage derivative of this strain. The resulting cells were plated on minimal M9 medium with glucose containing kanamycin (40 μg / ml) and 8-azaadenine (200 mg / ml). Colonies that appeared after 48 hours of cultivation were selected, plasmid DNA was isolated from the colonies and used to transform strain MG1655 by standard methods. Transformants were selected on plates with agarized L-broth containing kanamycin, as described above. Plasmid DNA was isolated from transformants resistant to 200 μg / ml 8-azaadenine. The nucleotide sequence of the ends of the chromosomal DNA inserts was determined using the seeds shown in the List of sequences numbered 3 and 4 (SEQ ID NO: 3 and 4), respectively, for the left and right sections of the attachment of phage Mi. The complete nucleotide sequence of the genome of the E. coli K-12 strain has been determined (Science, 277, 1453-1474, 1997). Therefore, the obtained nucleotide sequence data was compared with information from GenBank and thus cloned fragments were identified.

It turned out that several types of inserts belonging to different parts of the chromosome were cloned. One of them, AZA-10, which is a chromosome insert (8861 kb) in the pAZA10 phasmid, is shown in Figure 1.

Example 2. Identification of the ydeD gene conferring resistance to 8-azaadenine.

Cloning of the ydeD gene into pAYCTER3 vector.

The AZA-10 fragment contained at least 10 genes. In order to identify the gene conferring resistance to 8-azaadenine, each of the genes was subcloned into the pUC21 multi-copy vector and tested for its ability to confer resistance to the indicated reagent. More specifically, a ydeD gene containing about 250 bp before the proposed start codon, it was excised from the AZA-10 fragment using the PvuII and VspI restriction enzymes and inserted into the pUC21 vector pretreated with the restriction enzymes Eco22I and NdeI. Thus, the plasmid pYDED1 containing the ydeD gene was obtained (Figure 2). E. coli cells containing this multi-copy plasmid did not grow well on minimal medium. Therefore, the ydeD gene was cloned from the pUC21 plasmid at the SmaI and XbaI sites of the pAYCTER3 vector derived from pAYC32. Thus, the plasmid pYDEDS was obtained (Figure 3). The pAYCTER3 vector is a very stable mid-copy vector constructed on the basis of the plasmid RSF1010 (Christoserdov A.Y., Tsygankov Y.D, Plasmid, 1986, v.16, pp. 161-167) by introducing into the pAYC32 plasmid a polylinker from plasmid pUC19 and the strong terminator of the rrb gene.

Example 3. The effect of amplification of the ydeD gene on the resistance of E. coli TG1 strain to 8-azaadenine.

Plasmid pYDED3, as well as the vector pAYCTER3, were introduced into the E. coli TG1 strain. Thus, strains of TG1 (pYDED3) and TG1 (pAYCTER3) were obtained. Then, the ability of these strains to grow in the presence of 8-azaadenine on plates with minimal agarized M9 medium with glucose containing stepwise inhibitor concentrations was determined. The cups were seeded with cells (from 10 ⁵ to 10 ⁶ ) of an overnight culture grown on minimal medium containing 100 mg / ml ampicillin in the case of plasmid strains. The degree of growth was evaluated after incubation for 44 hours at 37 ° C. The results are presented in Table 1.

Table 1 Strain Growth on minimal environment containing 8-azaadenine, mcg / ml not containing 100 300 500 1000 additives Tg1 + - - - - TG1 (pAYCTER3) + - - - - TG1 (pYDED3) + + + + ± Explanations +: good growth; ±: poor growth; -: lack of growth.

As can be seen from Table 1, amplification of the ydeD gene significantly increased cell resistance to 8-azaadenine. The ydeD gene is known to encode a protein involved in the release of cysteine biosynthesis pathways from cells of metabolites (DaBler et al., Mol. MicrobioL, 36, 1101-1112, 2000; US 5972663). Therefore, it can be assumed that the ydeD gene has broad specificity and is also involved in the excretion of purine derivatives.

Example 4. The effect of amplification of the ydeD 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 pYDED3. Thus, strains AJ13732 (pMWKQ, pAYCTER3) and AJ13732 (pMWKQ, pYDED3) were obtained. Each of these strains was grown at 37 ° C for 18 hours in L-broth containing 100 mg / l ampicillin and 75 mg / l kanamycin, and 0.3 ml of the resulting culture was transferred to 3 ml of fermentation medium containing 100 mg / l of ampicillin and 75 mg / l of kanamycin, in vitro 20 × 200 mm and incubated at 37 ° C for 72 hours on a rotary shaker.

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

Glucose 40.0

(NH ₄ ) ₂ SO ₄ 16.0

KN ₂ PO 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 manganese sulfate were sterilized separately. CaCO _{3 was} sterilized by heating at 180 ° C for 2 hours. pH was maintained at 7.0. Antibiotics were added to the 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 minutes, the supernatant was diluted 4 times with water and analyzed by HPLC.

Conditions for analysis using HPLC:

Column: Luna C18 (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.

Table 2. Strain OD ₅₄₀ Inosine, g / l AJ13732 (pMWKQ) 5.5 6.3 AJ13732 (pMWKQ, pAYCTER3) 5.6 5.8 AJ13732 (pMWKQ, pYDED3) 5.3 6.7

As can be seen from Table 2, amplification of the ydeD gene increased inosine production by strain AJ13732 (pMWKQ).

Claims (8)

1. A method of producing inosine, comprising the steps of growing Escherichia coli bacteria in a nutrient medium and isolating the obtained and accumulated inosine from the culture fluid, characterized in that they use Escherichia coli bacterium, whose ability to produce inosine is increased by increasing the activity of proteins selected from group (A) a protein that is represented by the amino acid sequence 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 the sequence number 2 (SEQ ID NO: 2), and which has an activity that gives bacteria enhanced resistance to 8-azaadenine. 2. The method according to claim 1, characterized in that 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 the chromosome of said bacterium. 3. The method according to claim 2, characterized in that the transformation is carried out using a multi-copy vector. 4. The method according to claim 1, characterized in that the said bacterium has an increased expression of the inosine biosynthesis genes. 5. The method according to claim 1, characterized in that the Escherichia coli strain AJ3732 (pMWKQ, pYDED3) is used as the producer of inosine. 6. A method of producing 5'-inosinic acid, comprising the steps of growing Escherichia coli bacteria in a nutrient medium, phosphorylating the obtained and accumulated inosine and isolating the obtained 5'-inosinic acid, characterized in that they use Escherichia coli bacterium, the ability of which to produce inosine is increased over by increasing the activity of proteins selected from group (A) a protein that is represented by the amino acid sequence 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 addition of one or more amino acids to the amino acid sequence shown in the list of sequences under number 2 (SEQ ID NO: 2), and which has an activity that gives bacteria increased resistance to 8-azaadenine. 7. The method according to claim 6, characterized in that the said bacteria have increased expression of inosine biosynthesis genes. 8. The strain Escherichia coli AJ13732 (pMWKQ, pYDED3) - producer of inosine.

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