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

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to a method for preparing inosine and 5'-inosinic acid that are prepared by using microorganism Escherichia coli. Production of inosine by indicated microorganisms is elevated due to the enhanced activity of protein encoded by gene yijE. Invention provides increasing yield of inosine and 5'-inosinic acid.

EFFECT: improved preparing method, valuable properties of strain.

8 cl, 3 dwg, 2 tbl, 3 ex

 

The technical field

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

Prior art

Traditionally nukes get on an industrial scale by fermentation using strains of microorganisms, auxotrophic for adenine, or these strains, cotrim additionally, given the resistance to various compounds, such as analogs of purines and sulfaguanidine, these strains belong to the genus Bacillus (patent application of Japan No. 38-23039 (1963), 54-17033 (1979), 55-2956 (1980) and 55-45199 (1980), laid patent application Japan 56-162998 (1981), patent application Japan 57-14160 (1982) and 57-41915 (1982), laid patent application Japan 59-42895 (1984), to the genus Brevibacterium (patent application of Japan 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.

Getting these mutant strains usually consists of treatment of microorganisms with the aim of obtaining mutations, such as exposure to UV radiation or treatment nitrosoguanidine (N-methyl-N’-nitro-N-nitrosoguanidine) with subsequent selection of the desired strain on a suitable nutrient medium for selection. On the other hand, also practiced the cultivation of the mutant strains, p is engliash to the genus Bacillus (lined patent application of Japan 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 (lined patent application Japan 63-248394 (1988)), and to the genus Escherichia (application WO 9903988), obtained using genetic engineering techniques.

The productivity of strains producing inosine - might be further improved by increasing the capacity for excretion of inosine. Currently, it is recognized that the penetration of metabolites through the cytoplasmic membrane usually occurs with the participation of more or less specific transport proteins, carrying out their release (RAO et al, 1998, Environ. Mol. Biol. Rev., 62, 1-34; Paulsen et al, 1998, J. Mol. Biol., 277, 573-592; Saier et al, 1999, J. Mol. Environ. Biotechnol., 1, 257-279). Previously, the authors of the present invention showed that strains - producers of inosine or xanthosine belonging to the genus Escherichia and to the genus Bacillus and with increased activity of protein RhtA encoded rhtA gene (ybiF), has produced more inosine or xanthosine than the parent strains (patent RF №2002101666).

Description of the invention

The aim of the present invention is to increase the productivity of inosine strains - producers of inosine and the provision of a method of producing inosine and 5’-Yasinovka acid using these strains.

This goal was achieved by establishing the fact that the gene yijE, presumably coding for the store membrane protein not involved in the biosynthesis pathway of purine nucleosides, confers resistance to the analogue of the purine bases, 8-asadinho, when natural allele of the specified gene introduced into the cells of strain on mnogostadiinoi plasmid. Protein YijE encoded by the genome yijE, not involved in the biosynthesis pathway of purine nucleosides and belongs to the family of exporters of antibiotics and metabolites (Jack et al. Eur. J. Biochem., 268, 3620-3639, 2001). Moreover, gene yijE can increase the production of nucleosides in the case when additional copies are introduced into the cells of the respective producers inosine belonging to the genus Escherichia. Thus was accomplished the present invention.

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

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

Further, the present image is eenie provides a method of obtaining a purine nucleoside fermentation including the stage of growth of the above microorganism in a nutrient medium to produce and accumulate purine nucleoside in a nutrient medium, and excretion of purine nucleoside from the culture fluid.

Further, the present invention provides a method of obtaining 5’-Yasinovka acid, comprising the stage of growth of bacteria according to the present invention in a nutrient medium, phosphorylation received and accumulated inosine and allocation received and accumulated 5’-Yasinovka acid.

The present invention includes the following.

The invention 1. The bacterium belonging to the genus Escherichia having the ability to produce inosine in which the activity of the protein, as described in paragraphs (A) or (B)in the cell mentioned bacteria raised:

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

(C) a protein that presents the amino acid sequence comprising deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence listed in sequence number 2, and which has activity, giving bacteria more resistant to 8-asadinho (hereinafter proteins, as described in paragraphs (a) and (b), referred to as “the tree according to the present invention”).

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

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

The invention 4. A method of obtaining a purine nucleoside, comprising the stage of growth of bacteria in accordance with any one of Inventions 1 to 3 in a nutrient medium and a selection from the culture fluid obtained and accumulated in it inosine.

The invention 5. The method in accordance with the Invention 4, in which the bacterium has enhanced expression of genes of the biosynthesis of purine nucleosides.

The invention 6. The method of obtaining 5’-Yasinovka acid, comprising the stage of growth of bacteria in accordance with any one of Inventions 1 through 3 in a nutrient medium, phosphorylation received and accumulated inosine and allocation received and accumulated 5’-Yasinovka acid.

The invention 7. The method in accordance with the Invention 6, in which the bacterium is modified to increase the expression of genes of the biosynthesis of purine nucleosides.

The present invention is more thoroughly discussed 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 having the ability to produce inosine in which the activity of the protein, as described in paragraphs (A) or (B)in the cell mentioned bacteria is increased: (A) a protein that presents the amino acid sequence listed in sequence number 2; (C) a protein that presents the amino acid sequence comprising deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence listed in sequence number 2, and which has activity, giving bacteria resistance to 8-asadinho.

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

Used here, the term “capacity for the production of inosine” means the ability to production and accumulation of inosine in a nutrient medium. The term “capable of production of inosine” means that the micro is organism to the genus Escherichia that has the capacity to production and accumulation in the medium of inosine in number, greater than the natural strain of E. coli, such as E. coli strains W3110 and MG1655, and preferably means that the microorganism is able to produce and accumulate in the medium, the amount of not less than 10 mg/l, more preferably not less than 50 mg/l of inosine.

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

To proteins according to the present invention include proteins, are described in the following paragraphs (a) And (b):

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

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

A protein that presents the amino acid sequence listed in sequence number 2, is a protein YijE.

Protein YijE not involved in the biosynthesis pathway is Ugrinovich nucleosides and belongs to the family of exporters of antibiotics and metabolites (Jack et al. Eur. J. Biochem., 268, 3620-3639, 2001). Protein YijE is a highly hydrophobic protein, consisting of 312 amino acids, containing the projected 10 transmembrane segments, and have an unknown function. Protein YijE is encoded by gene yijE. Gene yijE (nucleotides with 4133655 on 4134593 in sequence with inventory number NC_000913, gi:16131781 in GenBank) is located in the E. coli chromosome between genes katG yijF. Gene yijE 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 32, preferably from 2 to 16 and more preferably from 2 to 5 for the protein (A).

The term “resistance to 8-asadinho” refers to the ability of bacteria to grow on minimal medium containing 8-azadani in concentration at which the wild-type strain or a parent strain cannot grow, or the ability of bacteria to grow on a nutrient medium containing 8-azadani, with greater rate than the wild-type strain or a parent strain. The above-mentioned concentration of 8-asadena is usually from 50 to 5000 μg/ml, preferably from 100 to 1000 μg/ml

Methods of increasing the activity of the protein according to the present invention, in particular the methods of increasing the number of molecules of a specified protein in the cell, include methods to change the program sequence, regulating the expression of DNA that encodes a protein according to the present invention, and methods of increasing the number of copies of the gene, but are not limited to.

A change in the sequence that regulates the expression of DNA that encodes a protein according to the present invention can be achieved by placing the DNA that encodes a protein according to the present invention, under the control of a strong promoter. As strong promoters are known, for example, lac promoter, trp promoter, trc promoter, plthe promoter of phage lambda. On the other hand, the promoter can be enhanced, for example, by introducing mutations in the indicated promoter to increase the level of transcription of a gene located after the promoter. Further, it is known that substitution of several nucleotides in the area between the binding site of the ribosome (RBS) and the start-codon, and in particular in the sequence immediately before the start codon, has a significant impact on transliruemie mRNA. For example, it was found 20-fold change of expression level depending on the nature of three nucleotides preceding the start-codon (Gold et al, Annu. Rev. Environ., 35, 365-403, 1981; Hui et al, EMBO J., 3, 623-629, 1984).

Moreover, an “enhancer” can be added to increase the transcription level of the specified gene. Introduction DNA containing either the gene or the promoter in the chromosomal DNA op is Sano, for example, in patent application laid Japan No. 1-215280 (1989).

Alternatively, the number of copies of a gene can be increased by introduction of a gene in mnogoopytny vector with the formation of recombinant DNA, followed by the introduction of such recombinant DNA in a microorganism. Examples of vectors used for the introduction of recombinant DNA, are plasmid vectors such as pMW118, pBR322, pUC19, pBluescript KS+pACYC177, pACYC184, pAYC32, pMW119, pET22b and the like, phage vectors, such as 11059, 1BF101, M13mp9, phage Mu (lined patent application of Japan No. 2-109985) and the like, and the transposon (Berg, D.E. and Berg, C.M., Bio/Technol, 1,417 (1983)), such as Mu, Tn10, Tn5, and the like. In addition, amplification of gene expression can be achieved by integration of the gene into a bacterial chromosome by the method of homologous recombination or the like.

Methods of using a strong promoter or enhancer may be combined with methods to increase the number of copies of the gene.

Methods of obtaining chromosomal DNA hybridization, polymerase chain reaction (PCR), to obtain plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a nucleating and similar methods may be conventional methods, well known to the person skilled in the art. These methods are described in the book of Sambrook, J. and Russell D., "Molecular Cloning A Laboratory Manual, Third Edition", Cold Spring HarborLaboratory Press (2001) and other similar publications.

For removing microorganism belonging to the genus Escherichia, and with increased expression of the gene encoding the protein according to the present invention, the necessary parts of genes can be obtained by PCR on the basis of already available information about the genes of E. coli. For example, gene yijE, it is believed that encodes the Transporter, may be cloned from chromosomal DNA of E. coli strains To 12 W3110 and E. coli MG1655 using the method of PCR. Chromosomal DNA used for this purpose, can also be obtained from any other strain of E. coli.

To proteins according to the present invention include mutants and variants of the protein YijE that may exist due to natural diversity, provided that these mutants and variants demonstrate the functional properties of the protein YijE, at least resistance to 8-asadinho. DNA encoding these mutants and variants can be obtained by DNA extraction, which hybridized gene yijE (SEQ ID NO:1) or part of a specified gene in stringent conditions and which encodes a protein that increases the production of purine nucleotides. The term “stringent conditions”referred to here means the conditions under which the formation of the so-called specific hybrids, and non-specific - not formed. For example, to the hard conditions include conditions under which hybridize DNA, becausee a high degree of homology, for example, DNA having a homology of at least 70% relative to each other. Alternatively, an example of the stringent conditions are conditions that match the conditions of washing by hybridization to Southern, for example 60°, 1× SSC, 0.1% SDS, preferably 0.1× SSC, 0.1% SDS. As a probe for DNA, the coding options and hybridization with the gene yijE, can also be used a part of the nucleotide sequence at number 1. The probe of this kind can be obtained from PCR using as a nucleating oligonucleotides derived from the nucleotide sequence under number 1, and the DNA fragment containing the nucleotide sequence at number 1 as the matrix. In the case when the probe is a DNA fragment with a length of about 300 base pairs, conditions of washing in hybridization correspond to, for example, 50°, 2× SSC and 0.1% SDS.

The bacterium according to the present invention can be obtained by introducing the above-mentioned DNA in a bacterium already having the ability to production of purine nucleosides. On the other hand, the bacterium according to the present invention can be obtained by giving the bacteria that already contains the DNA, the ability to production of purine nucleosides.

As the parent strain producing inosine, which Akti the activity of proteins according to the present invention will be raised, can be used E. coli strain AJ13732 (FADRaddeddyicPpgixapA(pMWKQ)) (WO 9903988). The specified strain is derived from a known strain W3110 containing the mutations introduced in the purF gene encoding PRPP amidotransferase, purR gene encoding the repressor of the biosynthesis of purines, deoD gene encoding phosphorylase purine nucleosides, gene Riga, encoding succinyl AMR synthase, add gene encoding adelaideans, edd gene encoding 6-phosphoglyceraldehyde, pgi gene encoding phosphoglucomutase, gene Hara encoding xanthocephalus (purF-, purA-, deoD-, purR-, add-, edd-, pgi-, xapA-), as well as containing plasmid pMWKQ is derived from the vector pMW218, in which genes are purFKQ encoding PRPP amidotransferase, insensitive to guanosin monophosphate (GMP) (WO 9903988).

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

The bacterium according to the present invention can be further improved by increasing the expression of one or more genes involved in the biosynthesis of purines. Examples of such genes are genes pur regulon from E. coli (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F.C.Neidhardt, ASM Press, Washington D.C., 1996). Described E. coli strain producing inosine containing mutant purF gene encoding PRPP amidotransferase, free from inhibition of GMP and AMR-type feedback, inactivated purR gene encoding the repressor of the biosynthesis of purines (WO 9903988).

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

2. The method of obtaining purine nucleosides.

The methods according to the present invention is a method of producing inosine, including the stage of growth of bacteria according to the present invention in a nutrient medium for the purpose of production and accumulation of inosine in a nutrient medium, and the selection of inosine from Kul is turalei liquid.

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

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

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

3. The method of obtaining 5’-Yasinovka acid.

The methods according to the present invention also includes a method of obtaining 5’-Yasinovka acid, comprising the stage of growth of bacteria according to the present invention in a nutrient medium, phosphorylation received and accumulated inosine and allocation received and accumulated 5’-and osinowo acid.

According to the present invention, the cultivation, isolation and purification of inosine from cultural or similar fluid may be carried out in a manner similar to traditional methods of fermentation, in which inosine is produced using a microorganism. Further, according to the present invention phosphorylation received and accumulated inosine, as well as the allocation received and accumulated 5’-Yasinovka acid can be carried out by a method similar to the traditional methods, in which the purine nucleotides such as 5’-insinua acid, derived from a purine nucleoside, such as inosine.

Phosphorylation of purine nucleoside can be carried out enzymatically with the use of various phosphatases, nucleotidases and nucleotidyltransferase or chemically using fosfauriliruetsa agents, such as l3or similar. Can be used phosphatase capable of selective catalysis of the transfer of the phosphoryl group of pyrophosphate in the 5’-position of the nucleoside (Mihara et. al, Phosphorylation of nucleosides by the mutated acid phosphatase from Morganella morganii. Appl. Environ. Environ. 2000, 66:2811-2816), or acid phosphatase, using polyphosphoric acid (salt), phenylphosphino acid (salt) or carbamylphosphate acid (salt) as a donor of phosphoric acid (WO 9637603 A1), or the like. Also the quality is as example of the phosphatase may be given phosphatase, capable of catalytic transfer of the phosphoryl group in the 2’, 3’, 5’-position of the nucleoside using as the substrate p-nitrophenylphosphate (Mitsugi, K., et al, Agric. Biol. Chem. 1964, 28, 586-600), inorganic phosphate (JP42-1186), or acetyl phosphate (JP61-41555), or similar. As an example nucleosidase can be given guanosin-insinking from E. coli (Mori et. al. Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product. J. Bacteriol. 1995. 177:4921-4926; WO 9108286), or similar, as an example nucleotidyltransferase can be given nucleotidyltransferase described Hammer-Jespersen, K. (Nucleoside catabolism, p.203-258. In A Munch-Petesen (ed.), Metabolism of nucleotides, nucleosides, and nucleobases in microorganism. 1980, Academic Press, New York), or similar. Chemical phosphorylation of nucleosides can be accomplished using fosforiliruyusciye agent, such as l3(Yoshikawa et. al. Studies of phosphorylation. III. Selective phosphorylation of unprotected nucleosides. Bull. Chem. Soc. Jpn. 1969, 42:3505-3508), or similar.

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

Captions to drawings

Figure 1. Scheme chromosomal DNA insert contained in phasmida pAZA9, which made the cells resistant to 8-asadinho. Figure 2 shows the structure of plasmid pYIJE1. Figure 3 show the structure of the plasmid pYIJE3.

The best way of carrying out the invention

Example 1. Cloning of genes that give resistance to purine analogues grounds of .coil in phasmida mini-Mu.

Genes from E.coli, which causes resistance to purine analogues grounds were originally cloned in vivo using phasmida mini-Mu d5005 (Groisman, E.A., et al., J. Bacteriol., 168, 357-364 (1986)). Strain MG1655, lysogeny on phage uts62, was used as donor. Freshly prepared lysates were used to infect Lisovenko on phage MuSts derivative of this strain. The obtained cells were cultivated in minimal medium M9 with glucose containing kanamycin (40 μg/ml) and 8-azadani (200 mg/ml). Were selected colonies that appeared after 48 to 72 hours of cultivation, from the colonies was isolated plasmid DNA and used to transform strain MG1655 standard methods. Transformants were selected on plates with agar L-broth containing kanamycin as described above. From transformants resistant to 200 μg/ml 8-asadena, was isolated plasmid DNA. The nucleotide sequence of the ends of chromosomal DNA inserts was determined using a nucleating listed in the List of sequences 3 and 4 (SEQ ID NO:3 and 4), respectively, for the left and right sections of the accession of phage Mu. The complete nucleotide sequence of GE is Ohm strain E. coli K-12 defined (Science, 277, 1453-1474, 1997). Therefore, these data on nucleotide sequences were compared with information from GenBank, and were therefore identified the cloned fragments.

It turned out that were cloned several types of inserts belonging to different parts of the chromosome. One of them, AZA-9, which is the chromosomal insertion (2237 KBP) in phasmida pAZA9 shown in figure 1.

Example 2. Identification of the gene yijE, giving resistance to 8-asadinho. Cloning of the gene yijE in vector pAYCTER3.

Fragment AZA-9 contained at least 3 genes. In order to identify the gene that gives resistance to 8-asadinho, each gene was subcloned in mnogoopytny vector pUC21 and tested for the ability to give stability to the specified reagent. More specifically, the gene yijE, containing approximately 200 BP before the intended start-codon, was carved out of a piece of AZA-9 using enzymes Bsp119I and PauI and inserted into the vector pUC21, pretreated with enzymes Bsu15I and luI. Thus was obtained a plasmid pYIJE1 containing gene yijE (Figure 2). In addition, gene yijE was periglomerular of plasmid pUC21 sites BglII and SalI vector pAYCTER3 derived from pAYC32 constructed by introducing polylinker of plasmids pUC19 and strong terminator gene rrb. Thus was obtained a plasmid pYIJE2 (Figure 3). Vector pAYCTER3 I have is very stable srednekovoi vector, constructed based on the plasmid RSF1010 (Christoserdov A. Y., Tsygankov Y. D, Plasmid, 1986, v.16, pp.161-167) by introducing plasmid pAYC32 of polylinker of plasmids pUC19 and strong terminator gene rrb. Plasmids pYIJE1 and pYIJE2 and vectors pUC21 and pAYCTER3 were introduced into E. coli strain TG1. So were the strains TG1(pYIJEl), TG1(pYIJE2), TG1(pUC21) and TG1 (pAYCTER3). It was then determined the ability of these strains to grow in the presence of 8-asadena on cups with minimal agar medium M9 with glucose containing graded concentrations of inhibitor. Cups were seeded with cells (105up to 106night of culture grown on minimal medium containing 100 mg/ml ampicillin in the case of strains with plasmids. The degree of growth was evaluated after incubation for 44 hours at 37° C. the Results are presented in Table 1.

Table 1
StrainGrowth on minimal medium
not containing addedcontaining 8-azadani, ug/ml
1003005001000
TG1+----
TG1 (pUC21)+----
TG1 (pYIJEl)++++±
TG1 (pAYCTER3)+----
TG1 (pYIJE2)+++±-
Explanation. +: good growth; ±: poor growth; -: no growth.

As can be seen from Table 1, gene amplification ij significantly increased the resistance of cells to 8-asadinho. In addition, the level of resistance depends on the number of copies of the plasmid.

Gene yijE (SEQ ID NO:1) encodes a protein consisting of 312 amino acid residues and having a calculated molecular mass of 34.1 kDa. Sequence analysis of the protein YijE known method (Kyte and Doolittle, J. Mol. Biol., 157, 105-132 (1982), Tusnady and Simon, J. Mol. Biol., 283, 489-506 (1998)) showed that it is highly hydrophobic protein containing the projected 10 transmembrane segments. Protein YijE belongs to the family of exporters of antibiotics and metabolites (Jack et al. Eur. J. Biochem., 268, 3620-3639, 2001). On this basis, we can predict that the gene yijE encodes a protein involved in the transport of cells of some metabolites. The fact that increased expression of a specified gene makes the cells resistant to 8-asadinho indicates that a possible substrates are PR is spodnie purines.

Example 3. The effect of amplification of the gene ij products inosine strain of E. coli-producing inosine.

Strain AJ13732(pMWKQ) producing inosine was transformed vector pUC21 and plasmid pYIJE1. So were the strains AJ13732(pMWKQ, pUC21) and AJ13732(pMWKQ, pYIJE1). Each of these strains were 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 obtained culture was transferred to 3 ml of culture medium for fermentation, containing 100 mg/l ampicillin and 75 mg/l kanamycin, in vitro 20× 200 mm and were incubated at 37° C for 72 hours on a rotary shaker.

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

Glucose 40.0

(NH4)2SO416.0

KN2RHO41.0

MgSO4×7H2O 1.0

FeSO4×7H2O 0.01

MnSO4×5H2O 0.01

Yeast extract 8.0

Caso330.0

Glucose and manganese sulfate were sterilized separately. Caso3sterilized by heating at 180° C for 2 hours. the pH was maintained in the area of 7.0. Antibiotics were added to the medium after sterilization.

After growing the number of inosine accumulated in the medium was determined by HPLC. A sample of the culture fluid (500 μl) was centrifuged at 1500 rpm for 5 min, the supernatant was diluted with water 4 times and p is analiziroval using HPLC.

Conditions for analysis by HPLC:

Column: Luna C18(2) 250× 3 mm, 5 u (Phenomenex, USA). Buffer: 2% v/v2H5HE; 0.8% v/v triethylamine, 0.5% v/v acetic acid (glacial), pH 4.5. Temperature: 30° C. flow Rate: 0.3 ml/min Volume of sample: 5 µl. UV detector: 250 nm.

Retention time (min):

Xanthosine 13.7

Inosine 9.6

Gipoksantin 5.2

Guanosin 11.4

Adenosine 28.2

The results are presented in Table 2.

Table 2
StrainOD540Inosine, g/l
AJ13732(pMWKQ)4.35.9
J13732 (pMWKQ, pUC21)4.35.6
J13732 (pMWKQ, pYIJEl)4.56.5

As can be seen from Table 2, gene amplification yij increased production of inosine strain J13732 (pMWKQ).

1. A method of producing inosine, including the stage of growth of bacteria Escherichia coli in a nutrient medium and a selection from the culture fluid obtained and accumulated in it inosine, wherein using the bacterium Escherichia coli, the ability to produce inosine which is increased by increasing the activity of the proteins selected from the gr is PI (A) protein, which presents the amino acid sequence listed in sequence 2 (SEQ ID NO:2); (C) a protein that presents the amino acid sequence comprising deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence listed in sequence 2 (SEQ ID NO:2), and which has activity, giving bacteria more resistant to 8-asadinho.

2. The method according to claim 1, characterized in that the activity of proteins described in paragraphs (A) or (B)is increased by transformation of bacteria with DNA that encodes a protein, as described in paragraphs (A) or (B), or by changing the regulation of expression of the indicated DNA in the chromosome of said bacterium.

3. The method according to claim 2, characterized in that the transformation is done using mnogoopytnogo vector.

4. The method according to claim 1, characterized in that the indicated bacteria the expression of genes of the biosynthesis of inosine increased.

5. The method according to claim 1, characterized in that as a producer of inosine using a strain of Escherichia coli AJ13732 (pMWKQ, pYIJE1).

6. The method of obtaining 5`-Yasinovka acid, comprising the stage of growth of bacteria Escherichia coli in a nutrient medium, phosphorylation received and accumulated inosine and allocation obtained 5`-Yasinovka acid, otlichuy is the, using the bacterium Escherichia coli, the ability to produce inosine which is increased by increasing the activity of the proteins selected from the group of (A) a protein that presents the amino acid sequence listed in sequence 2 (SEQ ID NO:2); (C) a protein that presents the amino acid sequence comprising deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence listed in sequence 2 (SEQ ID NO:2), and which has activity, giving bacteria more resistant to 8-asadinho.

7. The method according to claim 6, characterized in that the indicated bacteria the expression of genes of the biosynthesis of inosine increased.

8. The strain Escherichia coli AJ13732 (pMWKQ, pYIJE1) producing inosine.



 

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FIELD: biotechnology, microbiology, dairy industry.

SUBSTANCE: the strain of microorganism Lactobacillus lactis VKPM B-8354 is prepared without using mutagens and genetic methods and shows resistance against broad spectrum of lactophages. The strain ferments effectively milk from different trading sorts, with broad range of fatness and different methods of thermal treatment. Individual specific properties of the strain allows its applying as a monostrain ferment. Curd obtained with applying the strain L. lactis VKPM B-8354 shows good organoleptic qualities, nonacid taste and homogenous consistence. The strain is suitable especially for plants with small volume of manufacture but with varied assortment.

EFFECT: valuable properties of strain.

3 tbl, 2 dwg, 5 ex

FIELD: biotechnology, microbiology, agriculture.

SUBSTANCE: the strain Lactobacillus plantarum 578/25 is obtained by method of step-by-step selection and selected by its ability to produce significant amount of crude protein and to accumulate the biomass. The strain is deposited in the VGNKI collection at number VGNKI-03.04.09.-DEP. Invention provides eliminating the pollution of environment in producing the protein fodder, to elevate the protein specific yield, to reduce energy consumptions in preparing protein fodder, to simplify and to accelerate the process of its preparing, to simplify apparatus equipment, to utilize waste in manufacturing using the natural raw.

EFFECT: valuable properties of strain.

2 tbl, 10 ex

FIELD: biotechnology, agriculture, microbiology.

SUBSTANCE: invention relates to a new isolated strain of Lactobacillus acidophilus 1660/08 as a producer of the protein fodder. The strain Lactobacillus acidophilus 1660/08 is obtained by the selection method and selected by its ability to form significant amount of crude protein and to accumulate the biomass. The strain is deposited in the VGNKI collection at number VGNKI-03.04.10.-DEP. Invention provides eliminating the environment pollution in producing the protein fodder, to enhance the specific protein yield, to reduce energy consumptions in preparing protein fodder, to simplify and to accelerate the process in its preparing, to simplify equipment fitting out and to utilize waste in manufactures using natural raw.

EFFECT: valuable properties of strain.

2 tbl, 10 ex

FIELD: biotechnology, microbiology, agriculture.

SUBSTANCE: the strain Lactobacillus buchneri 600 is isolated from barley grains at milkwax stage of ripeness. The strain Lactobacillus buchneri 600 is registered in 05. 08. 2002 year in the All-Russian State collection of microorganism strains used in veterinary science and animal husbandry at number Lactobacillus buchneri VGNKI 02.08.04-DEP and deposited in the collection 000 "Biotrof". Invention provides the more effective multiplication of the strain in maize ensilaging green mass and preserving flattened grains with enhanced formation of lactic acid, inhibition of putrid microflora that allows preparing fodder from vegetable raw with enhanced quality. Invention can be used in fodder production for ensilage maize green mass and preserving flattened grains.

EFFECT: valuable properties of strain.

3 tbl, 2 ex

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

The invention relates to the microbiological industry

The invention relates to the field of Microbiology and can be used as a means for protection of plants from diseases and pests

The invention relates to agricultural biotechnology, in particular to the production and use of biological means of combating plant pathogens
The invention relates to the microbiological industry (biotechnology)

The invention relates to agriculture and can be used in the manufacture of various types of granulated mineral fertilizers (ammonium nitrate, required for plant growth, urea, ammofoska and others) for crop production

The invention relates to biotechnology and can be used in medicine to detect the genetic material of the virus Crimean-Congo hemorrhagic fever (CCHF) in samples

The invention relates to biotechnology, biology, medicine and veterinary

The invention relates to biotechnology, namely the genetic engineering of animals, and can be used in veterinary Microbiology to identify the causative agent of microbacteria ruminants Fusobacterium necrophorum and differentiating it from atypical forms of Fusobacterium pseudonecrophorum and other microflora

The invention relates to biotechnology, in particular genetic engineering, and can be used for DNA identification of human herpes virus type 6 (HHV-6)

The invention relates to the field of molecular biology and can be used for the detection of human immunodeficiency virus type 1 (HIV-1)

The invention relates to the microbiological industry

The invention relates to biotechnology and is a way to obtain purine nucleosides such as inosine and xanthosine, as well as a method of obtaining a purine nucleotides, such as inosine-5'-phosphate, xanthosine-5'-phosphate and guanosine-5'-phosphate, as producers use strains of bacteria, as belonging to the genus Escherichia and the genus Bacillus, in which the production of purine nucleosides these bacteria increased by increasing the activity of the proteins encoded by the genome rhtA (ybiF)
The invention relates to biotechnology, namely, construction of Escherichia coli - producer cholera toxin, and can be used to create a diagnostic test systems, allowing to detect toxigenic clones of Vibrio cholerae and Escherichia coli, as well as for the design of live vaccine strains against diarrhoeal diseases caused by pathogenic strains of V. cholerae and E.
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