RU2422510C2 - Method of producing purine ribonucleoside and ribonucleotides - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method implies using a Bacillus bacterium modified in such a manner that it contains an inactivated gene coding 3-hexulose-6-phosphatesynthase. The method involves cultivation of said bacterium in a nutrient medium. It is followed by recovery of said purine nucleoside from a culture fluid.

EFFECT: invention allows increasing production of purine nucleosides, such as inosine, xanthosine, guanosine or adenosine.

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Description

Technical field

The present invention relates to the microbiological industry, specifically to a method for the production of purine ribonucleosides, which are of value as a raw material for the synthesis of purine nucleotides. The method uses a Bacillus bacterium, modified in such a way that the expression of a gene encoding 3-hexulose-6-phosphate synthase in a modified bacterium is weakened.

Description of the Related Art

There are known methods for the enzymatic production of purine nucleosides using adenine auxotrophic strains. In addition, resistance to various compounds, such as purine analogues and sulfaguanidine, can be imparted to such strains. Examples of such strains include various strains belonging to the genus Bacillus (Japanese Patent Publication Nos. 38-23039 (1963), 54-17033 (1979), 55-2956 (1980) and 55-45199 (1980), Japanese Patent Application Laid-Open No. 56-162998 (1981), Japanese Patent Publication Nos. 57-14160 (1982) and 57-41915 (1982) and Japanese Patent Laid-open No. 59-42895 (1984), to the genus Brevibacterium (Japanese Patent Publication Nos. 51-5075 (1976) and 58-17592 (1972), and Agric. Biol. Chem., 42, 399 (1978)) to the genus Escherichia (international application PCT WO 9903988) and the like.

The traditional preparation of such mutant strains involves mutagenic treatment of microorganisms, such as UV irradiation and treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), selection of the desired strain using an appropriate selective medium. On the other hand, obtaining such mutant strains using genetic engineering methods is also practiced for strains belonging to the genus Bacillus (Japanese Patent Application Laid-open Nos. 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), patent U.S. 7,326,546 (2008)) and to the genus Brevibacterium (Japanese Patent Application Laid-Open No. 63-248394 (1988)). The productivity of these strains - producers of inosine - can be further improved by increasing the ability to secrete inosine (patent applications of the Russian Federation 2002101666, 2002104463 and 2002104464).

The ribulose monophosphate pathway (RuMP) is one of the metabolic pathways for the synthesis of compounds containing carbon-carbon bonds from monocarbon compounds, and this pathway has been found in many methane and methanolutilizing bacteria known as methylotrophs. Typical enzymes of this pathway are 3-hexulose-6-phosphate synthase (3-hexulose-6-phosphate synthase - HPS) and 6-phospho-3-hexuloisomerase (6-phospho-3-hexuloisomerase - PHI), which are not thought to exist outside methylotrophs. However, the putative product of the yckG gene, YckG of Bacillus subtilis, has a primary structure similar to that for HPS from methylotrophs. The sequence similarity of the yckF gene product — YckF and PHI from methylotrophs was also examined and it was found that the yckG and yckF genes from B. subtilis express proteins with the enzymatic activities of HPS and PHI, respectively. Both of these activities were induced in B. subtilis with formaldehyde, which demonstrates dependence on the yckH gene, but were not induced by methanol, formate, and methylamine. The destruction of both genes caused a moderate dependence on formaldehyde, suggesting that these enzymes may function in B. subtilis as a detoxification system for formaldehyde. The active yckG (for HPS) -yckF (for PHI) gene structure (currently called hxlA-hxlB) was found in non-methylotroph B. subtilis, which initially retained the RuMP pathway (Yasueda, H. et al., J. Bacteriology, 181 (23), p. 7154-7160 (1999)).

However, there are currently no reports of inactivation of a gene encoding 3-hexulose-6-phosphate synthase to improve the production of purine ribonucleosides.

Description of the invention

The aim of the present invention is to improve the microorganism intended for the production of purine nucleosides by fermentation, and to provide a method for producing nucleotides using the obtained strain.

This goal was achieved through the discovery of the fact that the weakening of the expression of the gene encoding hexulose-6-phosphate synthase can lead to an increase in the production of purine nucleosides such as inosine, xanthosine, guanosine or adenosine.

The present invention provides a bacterium belonging to the genus Bacillus, specifically Bacillus subtilis and Bacillus amyloliquefaciens, having an increased ability to produce purine nucleosides.

An object of the present invention is to provide a bacterium belonging to the genus Bacillus capable of producing purine nucleosides, characterized in that said bacterium is modified in such a way that expression of a gene encoding 3-hexulose-6-phosphate synthase in said bacterium is attenuated.

It is also an object of the present invention to provide a bacterium as described above, characterized in that said expression is attenuated by inactivation of said gene.

It is also an object of the present invention to provide a bacterium as described above, characterized in that the hxlA gene is a gene encoding 3-hexulose-6-phosphate synthase.

It is also an object of the present invention to provide a bacterium as described above, characterized in that said bacterium is B. subtilis.

It is also an object of the present invention to provide a bacterium as described above, characterized in that said bacterium is B. amyloliquefaciens.

It is also an object of the present invention to provide a bacterium as described above, characterized in that said purine nucleoside is selected from the group consisting of inosine, xanthosine, guanosine and adenosine.

It is also an object of the present invention to provide a method for producing a purine nucleoside, comprising culturing the above-described bacteria in a culture medium, leading to the secretion of said purine nucleoside into the culture medium, and isolating said purine nucleotide from the culture fluid.

It is also an object of the present invention to provide a method for producing a purine nucleoside as described above, characterized in that said purine nucleoside is selected from the group consisting of inosine, xanthosine, guanosine and adenosine.

It is also an object of the present invention to provide a method for producing a purine nucleoside, comprising culturing the above-described bacteria in a culture medium, leading to the secretion of said purine nucleoside into the culture medium, isolation of said purine nucleoside from the culture fluid, phosphorylation of said purine nucleoside, and isolation of purine nucleotide.

It is also an object of the present invention to provide a method for producing a purine nucleotide as described above, characterized in that the purine nucleotide is selected from the group consisting of 5′-inosinic acid, xanthosine 5′-phosphate, 5′-guanilic acid and 5′-adenylic acid.

The present invention will be described in more detail below.

BEST MODE FOR CARRYING OUT THE INVENTION

1. The bacterium according to the present invention

The bacterium according to the present invention is capable of producing a purine nucleoside and is modified in such a way that the expression of a gene encoding 3-hexulose-6-phosphate synthase in this bacterium is weakened. The bacterium belongs to the genus Bacillus.

Examples of microorganisms belonging to the genus Bacillus that can be used in the present invention include Bacillus subtilis subsp. subtilis strain 168 (B. subtilis 168) or Bacillus amyloliquefaciens (B. amyloliquefaciens). Bacteria B. amyloliquefaciens are fairly heterogeneous species. A number of B. amyloliquefaciens strains are known: SB, T, P, W, F, N, K, and H (Welker N.E., Campbell L.L., Unrelatedness of Bacillus amyloliquefaciens and Bacillus subtilis. J. BacterioL, 94: 1124-1130, 1967). Recently, Bacillus strains were isolated from plants, which can be considered as a separate ecotype of B. amyloliquefaciens (Reva et al., Taxonomic characterization and plant colonizing abilities of some bacteria related to Bacillus amyloliquefaciens and Bacillus subtilis. FEMS Microbiol. Ecol., 48: 249-259 , 2004). The nucleotide sequence of one of the strains, B. amyloliquefaciens FZB42 (Chen et al .. Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42. Nat. Biotechnol., 25: 1007-1014, 2007), was determined.

Examples of bacteria belonging to the genus Bacillus also include the following species: Bacillus licheniformis, Bacillus pumilis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, Bacillus stearothermophilus.

In addition to the properties already mentioned, the bacteria of the present invention may possess other characteristic properties, such as the need for various nutrients, antibiotic resistance, antibiotic sensitivity, which is not beyond the scope of the present invention.

The phrase "purine nucleoside" as used herein includes inosine, xanthosine, guanosine and adenosine, preferably inosine.

The phrase "ability to produce purine nucleoside" used here means the ability to synthesize and accumulate purine nucleoside in a nutrient medium. The phrase “a bacterium is capable of producing a purine nucleoside” means that a bacterium belonging to the genus Bacillus is capable of synthesizing and accumulating in the medium purines, such as purine nucleosides, in an amount greater than the wild-type strain or unmodified B. subtilis strain such as B subtilis 168. Preferably, this phrase means that the microorganism is capable of synthesizing and accumulating in a nutrient medium not less than 10 mg / l, more preferably not less than 50 mg / l inosine, xanthosine, guanosine or / and adenosine.

The term "activity of 3-hexulose-6-phosphate synthase (HPS)" means an activity that catalyzes the formation of D-arabino-3-hexulose-6-phosphate by Mg ²⁺ -dependent aldol condensation of formaldehyde with ribulose-5-phosphate. HPS activity can be measured as described by Yasueda, H. et al., (J. Bacteriology, 181 (23), p. 754-7160 (1999)).

The gene encoding HPS from Bacillus subtilis, more specifically the hxlA gene (synonymous with yckG) from Bacillus subtilis subsp. subtilis of strain 168 is known (nucleotides complementary to nucleotides from 374725 to 375357 in sequence with accession number NC_000964 in the GenBank database). The B. subtilis hxlA gene is located on the chromosome between the hxlB and hxlR genes. The nucleotide sequence of the B. subtilis hxlA gene and the amino acid sequence encoded by the hxlA gene are shown in the Sequence Listing Numbers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2), respectively.

A gene encoding HPS from Bacillus amyloliquefaciens, more specifically the hxlA gene of Bacillus amyloliquefaciens of strain FZB42, is known (nucleotides complementary to nucleotides 340797 to 341432 in sequence with accession number NC_009725 in the GenBank database). The hxlA gene from B. amyloliquefaciens is located on the chromosome between the hxlB and hxlR genes. The nucleotide sequence of the hxlA gene from B. amyloliquefaciens and the amino acid sequence encoded by the hxlA gene are shown in the Sequence Listing Numbers 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4), respectively.

Typically, the gene must be cloned into a vector capable of functioning in a bacterium belonging to the genus Bacillus before being introduced into the bacterium. For these purposes, you can use shuttle vectors: pHY300PLK, pMWMX1, pLF22, pKS1. The replacement of the wild-type gene with a mutant gene occurs due to homologous recombination.

Since representatives of different strains of the genus Bacillus may have some differences in the nucleotide sequences, the concept of the inactivated hxlA gene is not limited to the genes whose sequences are listed in the Sequence Listing under the numbers SEQ ID NO: 1 and SEQ ID NO: 3, but may also include genes homologous SEQ ID NO: 1 and SEQ ID NO: 3 encoding a variant of the HPS protein. The term “HPS protein variant” as used in the present invention means a protein with changes in the sequence, be it deletion, insertion, addition or substitution of amino acids, in which the activity of the HPS protein is maintained. The number of changes in a protein variant depends on the position or type of amino acid residue in the tertiary structure of the protein. It can be from 1 to 30, preferably from 1 to 15, more preferably from 1 to 5 in SEQ ID NO: 2 and SEQ ID No: 4. These variations in variants may occur in areas not critical to protein function. These changes are possible because some amino acids have a high homology to each other, therefore, such changes do not affect the tertiary structure or activity. Therefore, a variant of the protein encoded by the hxlA gene can be represented by proteins with a homology of at least 80%, preferably at least 90%, and most preferably at least 95%, with respect to the complete amino acid sequences shown in the sequence listing under numbers SEQ ID NO: 2 and SEQ ID NO: 4, provided that the HPS protein remains active until the hxlA gene is inactivated.

Homology between two amino acid sequences can be determined using known methods, for example, the BLAST 2.0 computer program, which counts three parameters: amino acid number, identity, and similarity.

In addition, the hxlA gene may be an option that hybridizes under stringent conditions with the nucleotide sequence shown in the Sequence Listing under the numbers SEQ ID NO: 1 or SEQ ID NO: 3, or with a probe that can be synthesized based on the specified nucleotide sequence, provided that prior to inactivation, it encodes a functional HPS protein. "Stringent conditions" include those conditions under which specific hybrids, for example hybrids with a homology of at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% and most preferably at least 95%, are formed, and non-specific hybrids, for example hybrids with less homology than indicated above, are not formed. A practical example of harsh conditions is a single wash, preferably two or three times, at a salt concentration of 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, at 60 ° C. The duration of washing depends on the type of membrane used for blotting and, as a rule, is as recommended by the manufacturer. For example, the recommended washing time for Hybond ™ N + nylon membrane (Amersham) under stringent conditions is 15 minutes. Double and triple washing is preferred. The length of the probe can be selected depending on the hybridization conditions, in this particular case, it can be about 100-1000 bp.

The expression of the hxlA gene can be attenuated by introducing mutations into the genes on the chromosome that the intracellular activity of the protein encoded by the gene is reduced compared to that in the unmodified strain. Such a gene mutation may be the replacement of one or more bases for amino acid substitution in the encoded protein (the Missense mutation), the introduction of a stop codon (the "nonsense" mutation), the deletion of one or more bases for reading frame shift, insertion of the resistance gene antibiotic or division of a gene or part thereof (J. Biol. Chem., 1997, 272 (13): 8611-8617, J. Antimicrobial Chemotherapy, 2000, 46: 793-96). The expression of the chaC gene or chaBC operon can also be attenuated by modifying the expression of regulatory sequences, such as a promoter, Shine-Dalgamo (SD) sequence, etc.

For example, the following methods can be used to introduce mutations by gene recombination. A mutant gene is constructed that encodes a mutant protein with reduced activity, and the bacterium for its modification is transformed with a DNA fragment containing the mutant gene. Then the native gene on the chromosome is replaced by homologous recombination with the mutant gene, and the resulting strain is selected. Such gene substitution using homologous recombination can be carried out using a linear DNA method known as “Red-dependent integration” or “Red-system integration” (Datsenko, KA, Wanner, BL, Proc.Natl.Acad.Sci.USA , 97, 12, 6640-6645 (2000), PCT application WO 2005/010175), or by a method using a plasmid whose replication is temperature sensitive (US Patent 6,303,383 or Japanese Patent Application JP 05-007491A). Further, the introduction of a site-specific mutation by replacing a gene using the aforementioned homologous recombination can also be carried out using a plasmid with reduced replication ability in the host cell.

Gene expression can also be attenuated by inserting a transposon or IS factor into the coding region of the gene (US Pat. No. 5,175,107), or by conventional methods such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine) .

Gene inactivation can also be carried out by traditional methods such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-specific mutagenesis, gene destruction using homologous recombination and / or mutagenesis due to insertion deletions.

The bacterium of the present invention can be obtained by attenuating the expression of a gene encoding HPS in a bacterium already capable of producing a purine nucleoside. On the other hand, the bacterium of the present invention can be obtained by imparting to a bacterium in which the expression of a gene encoding HPS is already impaired the ability to produce a purine nucleoside.

An example of a parent strain belonging to the genus Bacillus that can be used in the present invention is a B. subtilis producer of inosine producer B. subtilis KMBS375 (KMBS375: Ppur * -Δatt ΔpurA ΔpurR ΔpupG ΔdeoD guaB24; see Reference Example). Other parent strains belonging to the genus Bacillus that can be used in the present invention include B. subtilis strain AJ12707 (FERM P-12951) (Japanese patent application JP6113876A2), B. subtilis strain AJ3772 (FERM P-2555) (Japanese patent application JP62014794A2), Bacillus pumilus NA-1102 (FERM BP-289), Bacillus subtilis NA-6011 (FERM BP-291), Bacillus subtilis G1136A (ATCC No. 19222) (US Patent 3,575,809), reidentified as Bacillus amyloliquefaciens AJ1 On October 2005, at VKPM as Bacillus amyloliquefaciens G1136A (VKPM B-8994), on October 13, 2006, the strain was internationally deposited, Bacillus subtilis NA-6012 (FERM BP-292) (US patent 4,701,413), B pumilis Gottheil No. 3218 (ATCC No. 21005) (US patent 3,616,206), B. amyloliquefaciens AS115-7 strain (VKPM B-6134) (RF patent 2003678) and the like. Can also be used strain B. subtilis KMBS16. This strain is a derivative of the known B. subtilis 168 trpC2 strain with mutations introduced into the purr gene encoding the purine repressor (purR :: spc), the purA gene encoding the succinyl AMP synthase (purA :: erm) and the deoD gene encoding purine nucleoside phosphorylase (deoD :: kan) (RF patent application 2002103333, US patent 7,326,546, 2008).

The bacterium of the present invention can be further improved by enhancing the expression of one or more genes involved in purine biosynthesis. Examples of such genes include the operon pur from B. subtilis (Ebbole DJ and Zaikin HJ Biol. Chem., 262: 8274-87 (1987), Bacillus subtilis and Its Closest Relatives, Editor in Chief: AL Sonenshein, ASM Press, Washington DC, 2002). A strain is described that produces B. subtilis inosine with a modified regulatory region of pur operon (US patent 7,326,546, 2008).

Methods for preparing plasmid DNA, DNA restriction and ligation, transformation, selection of nucleotides as a primer, etc. may be conventional methods known to a person skilled in the art. These methods are described in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning: A Laboratory Manual, Third Edition", Cold Spring Harbor Laboratory Press (2001), and the like.

2. The method of production of nucleosides

The method of the present invention includes a method for producing a nucleoside, such as inosine and / or guanosine, comprising the steps of culturing the bacteria of the present invention in a culture medium for producing and storing the nucleoside in the medium and recovering the nucleoside from the culture fluid.

In the present invention, the cultivation of bacteria, the selection of nucleosides from the culture fluid and its purification, etc. can be carried out in a manner similar to traditional fermentation methods in which a nucleoside is prepared using a microorganism. The nutrient medium for the production of purine nucleoside may be a conventional medium containing a carbon source, a nitrogen source, inorganic ions, organic components and other necessary components. As a carbon source, saccharides such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose and starch hydrolysates can be used; alcohols such as glycerin, mannitol and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid, succinic acid, and the like. Inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate can be used as a nitrogen source; organic nitrogen, such as soybean hydrolysates; ammonia gas; aqueous ammonia, etc. It is desirable that vitamins such as vitamin B ₁ , essential substances, for example, organic additives, such as nucleic acids, adenine and RNA, or yeast extract and the like. could be present in appropriate or even minimal amounts. In addition, if necessary, small amounts of calcium phosphate, magnesium sulfate, iron ions, manganese ions and the like can be added.

The cultivation is preferably carried out under aerobic conditions for 16-72 hours, maintain the temperature of the cultivation 30-45 ° C, pH 5-8. The pH can be adjusted using inorganic or organic acidic or alkaline substances, such as ammonia gas.

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 and purified using any combination of traditional methods, such as ion exchange chromatography and precipitation.

3. Method for the production of purine nucleotides

A method for producing purine nucleotides includes the steps of growing the bacteria of the present invention in a culture medium, leading to the secretion of the purine nucleoside by the bacterium into the culture medium, phosphorylation of the resulting purine nucleoside and isolation of the purine nucleotide. In addition, the method of 5'-inosinic acid includes the steps of growing the bacteria of the present invention in a culture medium, leading to the secretion of inosine into the culture medium, phosphorylation of inosine and the allocation of 5'-inosinic acid. In addition, a method for producing 5'-xanthyl acid includes the steps of growing the bacteria of the present invention in a culture medium, leading to the secretion of xanthosine in the culture medium, phosphorylation of inosine and the release of 5'-xanthyl acid. In addition, a method for producing 5′-guanilic acid includes the steps of growing the bacteria of the present invention in a culture medium, leading to the secretion of guanosine in the culture medium, phosphorylation of guanosine and the release of 5′-guanilic acid. And the method for producing 5'-guanilic acid includes the steps of growing the bacteria of the present invention in a culture medium, allowing the secretion of xanthosine into the culture medium, phosphorylation of xanthosine, amination of 5'-xanthyl acid and the isolation of 5'-guanilic acid.

In the present invention, the cultivation of bacteria, the isolation of inosine from the culture medium and purification, etc. can be carried out in a similar manner to traditional fermentation methods in which inosine is prepared using a microorganism. Furthermore, in the present invention, the steps of phosphorylation of inosine and the isolation of 5'-inosinic acid can be carried out by conventional fermentation methods in which a purine nucleotide, such as 5'-inosinic acid, is formed from a purine nucleoside, such as inosine.

Phosphorylation of a purine nucleoside can be enzymatic using various phosphatases, nucleoside kinases or nucleoside phosphotransferases, or chemically using phosphorylating agents such as POCl ₃ and the like. You can use phosphatase capable of catalyzing the C-5'-selective transfer of the phosphoryl group of pyrophosphate to nucleosides (Mihara et. Al. Phosphorylation of nucleosides by the mutated acid phosphatase from Morganella morganii. Appl. Environ. Microbiol., 66: 2811-2816 (2000 )), or acid phosphatase, which uses polyphosphoric acid (salts), phenylphosphoric acid (salts) or carbamylphosphoric acid (salts) as a donor of phosphoric acid (WO 9637603 A1), or the like. Also, phosphatase can be used as an example of phosphatase, which catalyzes the transfer of a phosphoryl group to C-2 ', 3' or 5 'nucleotides using p-nitrophenyl phosphate as a substrate (Mitsugi, K., et al. Agric. Biol. Chem., 28 , 586-600 (1964)), inorganic phosphate (Japanese Patent Laid-Open No. No. JP42-1186) or acetyl phosphate (Japanese Patent Laid-Open No. No. JP61-41555), or the like. As an example of nucleoside kinase, E. coli nucleoside kinase, guanosine / inosine kinase (Mori, H. et. Al. Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the produced gene product. J. Bacteriol. 177: 4921- can be used. 4926 (1995); 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 used. , New York) or the like. Chemical phosphorylation can be carried out using agents such as POCl ₃ (Yoshikawa, K. et. Al. Studies of phosphorylation. III. Selective phosphorylation of unprotected nucleosides. Bull. Chem. Soc. Jpn. 42: 3505-3508 (1969)) or like that.

Amination of 5'-xanthyl acid can be enzymatic using, for example, E. coli GMP synthetase (Fujio et. Al. High level of expression of XMP aminase in Escherichia coli and its application for the industrial production of 5'-guanylic acid. Biosci. Biotech. Biochem. 1997, 61: 840-845; EP0251489B1).

Brief Description of the Drawings

Figure 1 shows the structure of plasmid pKS1 with the designation of unique restriction sites.

BEST MODE FOR CARRYING OUT THE INVENTION

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

Example 1. Construction of a strain with an inactivated hxlA gene

To inactivate the hxlA gene on the B. subtilis KMBS375 chromosome (constructing the KMBS375 strain is described in the Reference example), the regions localized before and after the hxlA gene were cloned at two sites of pKS1 plasmid (Shatalin KY and Neyfakh AA, FEMS Microbiology Letters, 245: 315- 9 (2005)) (Fig. 1). A 886 bp fragment amplifying the region in front of the hxlA gene was amplified in PCR using primers P1 (SEQ ID NO: 5) and P2 (SEQ ID NO: 6) and B. subtilis KMBS375 chromosomal DNA as a template. Primers P1 and P2 contain recognition sites for endonucleases SacII and PstI, respectively. A 923 bp fragment amplifying the region after the hxlA gene was amplified in PCR using primers P3 (SEQ ID NO: 7) and P4 (SEQ ID NO: 8) and B. subtilis KMBS375 chromosomal DNA as a template. Primers P3 and P4 contain recognition sites for the endonucleases ClaI and KpnI, respectively. The purified PCR product obtained using primers P1 and P2 was treated with SacII and PstI endonucleases and then cloned at the corresponding sites in plasmid pKS1. The purified PCR product obtained using primers P3 and P4 was treated with ClaI and KpnI endonucleases and also cloned at the corresponding sites in plasmid pKS1. After cloning both fragments, a plasmid containing the Km ^R cassette and regions localized before and after the hxlA gene was treated with SacII and KpnI endonucleases and the resulting fragment (3194 bp) was subcloned into the non-replicative vector pBluescript II KS (Stratagene) from Bacillus. The resulting plasmid was used to transform B. subtilis KMBS375 and 12 Km ^R transformants were isolated. To confirm the inactivation of the hxlA gene as a result of integration of the Km ^R cassette, PCR test was performed using primers P1 and P4. The size of the DNA fragment obtained in PCR using primers P1 and P4 with the natural allele of the hxlA gene is 2476 bp; however, the presence of a 3194 bp DNA fragment confirms the integration of the Km ^R cassette into the hxlA gene region. In additional check PCR using primers P5 (SEQ ID NO: 9 - primer P5 contains 20 N. complementary to the region located at 1013 bp from the start of hxlA gene translation, before the region complementary to primer P1) and P6 ( SEQ ID NO: 10 - primer P6 contains 21 N. complementary to the region localized inside the Km ^R cassette), the presence of a fragment obtained in PCR of 1491 bp confirms the integration of the Km ^R cassette into the hxlA gene on the KMBS375 chromosome. One of the KMBS375 transformants containing the integrated Km ^R cassette was used in further work. This strain is named KMBS375Δhxl :: Km.

Example 2. The production of inosine strain B. subtilis KMBS375Δhxl :: Km

To assess the effect of hxlA gene inactivation on inosine production, strains of B. subtilis KMBS375 and KMBS375Δhxl :: Km were cultured at 34 ° C for 18 hours in L-broth, then 0.3 ml of culture was inoculated in 3 ml of fermentation medium in 20 × 200 mm tubes and cultured at 34 ° C for 72 hours on a rotary shaker.

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

Glucose 30.0 NH ₄ Cl 32.0 KH ₂ PO ₄ 1.0 MgSO ₄ · 7H ₂ O 0.4 FeSO ₄ · 7H ₂ O 0.01 MnSO ₄ · 5H ₂ O 0.01 Substitutional Nitrogen 1.35 DL-methionine 0.3 Adenine 0.1 Guanine 0.05 Antifoam (adecanol) 0.5 ml CaCO ₃ 50.0 pH 6.0 (brought by KOH)

After cultivation, the amount of inosine accumulated in the medium was determined by HPLC.

HPLC conditions:

Column: Luna C18 (2) 250 × 3 mm, 5u (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. Volume of administration: 5 μl. Detection: UV 250 nm.

Retention Time (min):

Xanthosine 13.7 Inosine 9.6 Hypoxanthine 5.2 Guanosine 11.4 Adenosine 28.2

Table 1 Strain OD ₅₆₂ Inosine, g / l Productivity, inosine, g / l / OD Exit, % KMBS375 13.6 4.72 ± 0.1 0.347 ± 0.006 15.73 ± 0.33 KMBS375Δhxl :: Km 12.8 5.13 ± 0.01 0.401 ± 0.001 17.09 ± 0.02

As can be seen from Table 1, the strain KMBS375Δhxl :: Km produces more inosine than the strain KMBS375.

Reference example. Construction of strain KMBS375

<Construction of the prototroph B. subtilis 168 Marburg>

B. subtilis 168 Marburg (ATCC6051) is a Trp auxotroph due to the presence of the trpC2 mutant allele (trpC; indole-3-glycerol phosphate synthase gene) on the chromosome (Albertini AM, and A. Galizzi. 1999. The sequence of the trp operon of Bacillus subtilis 168 (trpC2) revisited. Microbiology. 145: 3319-3320). The trpC2 allele, in which three “att” nucleotides are added at position 328 m from the start codon of trpC2, was amplified by PCR and introduced into B. subtilis 168 Marburg, as described below.

(1) Amplification of the allele trpC ⁺ allele by recombinant PCR

To amplify the 5'-end of the trpC ⁺ allele and the preceding region, PCR primers 34 (SEQ ID NO. 38) and 35 (SEQ ID NO. 39) were constructed based on information from the GenBank database (Accession No. NC_000964) and Albertini and Galizzi (Microbiology. 145: 3319-3320).

PCR (94 ° C, 30 seconds; 53 ° C, 1 minute; 72 ° C, 1 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was performed using the above primers and chromosomal DNA of B. subtilis 168 strain Marburg as a matrix for amplification of fragments containing the region of the 5'-end of the trpC ⁺ allele and the region preceding it.

To amplify the 3'-end of the trpC ⁺ allele and the subsequent region, PCR primers 36 (SEQ ID NO. 40) and 37 (SEQ ID NO. 41) were constructed based on information from the GenBank database (Accession No. NC_000964) and Albertini and Galizzi (Microbiology. 145: 3319-3320).

PCR (94 ° C, 30 seconds; 53 ° C, 1 minute; 72 ° C, 1 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was performed using the above primers and chromosomal DNA of B. subtilis 168 strain Marburg as a matrix for amplification of fragments containing the region of the 3'-end of the trpC ⁺ allele and the region following it.

To amplify the complete sequence of the trpC ⁺ allele by recombinant DNA, two DNA fragments amplified as described above were purified using a MicroSpin Column S-400 (Amersham Pharmacia Biotech), an appropriate amount of their mixture was used as a template in PCR (94 ° C, 30 seconds ; 55 ° C, 1 minute; 72 ° C, 2 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) using primers 34 (SEQ ID NO. 38) and 37 (SEQ ID NO. 41), thus, an amplified fragment (about 1.2 kb) of the trpC ⁺ allele was obtained.

(2) Construction of a Trot Prototroph from B. subtilis 168 Marburg strain

The target trpC ⁺ allele fragment was isolated from the gel after agarose gel electrophoresis. Competent cells of strain B. subtilis 168 Marburg, prepared as described by Dubnau and Davidoff-Abelson (Dubnau, D., and R. Davidoff-Abelson, J. Mol. Biol., 56: 209-221 (1971)), transformed with this fragment DNA and selected colonies capable of growing on plates with minimal agar medium. Chromosomal DNA was isolated from these colonies. DNA fragments were amplified by PCR using this chromosomal DNA as a template and primers 34 (SEQ ID NO. 38) and 37 (SEQ ID NO. 41), then sequencing was performed to confirm the insertion of three nucleotides into the trpC2 allele and the absence of other mutations, which could result from PCR. The recombinant thus obtained was not auxotrophic for Trp; this strain was named KMBS275.

<Construction of auxotrophic according to His B. subtilis 168 Marburg>

Deletion without violating the reading frame was performed in the hisC gene encoding histidinolphosphate aminotransferase (ΔhisC: deletion of 204 nucleotides 403-606) and introduced into B. subtilis 168 Marburg strain (trpC2), as described below.

(1) Amplification of ΔhisC by Recombinant PCR

To amplify the 5'-end of the ΔhisC allele and the preceding region, PCR primers 38 (SEQ ID NO. 42) and 39 (SEQ ID NO. 43) were constructed based on information from the GenBank database (Accession No. NC_000964). Primer 38 contains an EcoRI site at the 5 ′ end. The primer contains 39 joints formed during deletion without violating the reading frame.

PCR (94 ° C, 30 seconds; 55 ° C, 1 minute; 72 ° C, 1 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was performed using the above primers and chromosomal DNA of B. subtilis 168 strain Marburg as a matrix for amplification of fragments containing the region of the 5'-end of the ΔhisC allele and the region preceding it.

To amplify the 3'-end of the ΔhisC allele and the subsequent region, PCR primers 40 (SEQ ID NO. 44) and 41 (SEQ ID NO. 45) were constructed based on information from the GenBank database (Accession No. NC_000964). Primer 40 contains a junction formed by a deletion without violating the reading frame. Primer 41 contains a BamHI site at the 5 ′ end.

PCR (94 ° C, 30 seconds; 55 ° C, 1 minute; 72 ° C, 1 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was performed using the above primers and chromosomal DNA of B. subtilis 168 strain Marburg as a matrix for amplification of fragments containing the region of the 3'-end of the ΔhisC allele and the region following it.

To amplify the complete ΔhisC sequence by recombinant PCR, two DNA fragments amplified as described above were purified using a MicroSpin Column S-400 (Amersham Pharmacia Biotech), an appropriate amount of their mixture was used as template in PCR using primers 38 (SEQ ID NO. 42) and 41 (SEQ ID NO. 45), performed PCR (94 ° C, 30 seconds; 55 ° C, 1 minute; 72 ° C, 2 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) thus, an amplified fragment (about 1.5 kb) of ΔhisC was obtained.

(2) Cloning of the ΔhisC Gene

The amplified fragment was digested with restriction enzymes EcoRI and BamHI (37 ° C, overnight) and separated on an agarose gel. The target fragment was isolated from the gel and ligated with the integrative vector pJPM1 of B. subtilis chromosome (Mueller, JP, G. Bukusoglu, and AL Sonenshein. 1992. Transcriptional regulation of Bacillus subtilis glucose starvation-inducible genes: control of gsiA by the ComP-ComA signal transduction system. J. Bacteriol. 174: 4361-4373), which was pretreated with the same enzymes (37 ° C, 3 hours). A plasmid was selected into which the ΔhisC gene was correctly integrated; sequencing confirmed the absence of mutations that could result from PCR; the plasmid was named pKM186.

(3) Construction of His auxotroph from B. subtilis 168 Marburg strain

Competent B. subtilis 168 Marburg strain cells prepared as described above were transformed with pKM186 plasmid and colonies (recombinants resulting from a single crossing-over) capable of growing on LB agar plates containing 2.5 μg / ml chloramphenicol (Cm) were selected.

One of the obtained recombinants was inoculated in 10 ml of LB medium supplemented with 20 mg / L Gua (LB + Gua medium) and cultured for 2 days at 37 ° C. Colonies showing sensitivity to chloramphenicol were selected using LB + Gua agar medium with / without Cm. Chromosomal DNA was isolated from the obtained Cm-sensitive colonies. PCR was performed in the same manner as described above using primers 38 (SEQ ID NO. 42) and 41 (SEQ ID NO. 45). Strains were identified in which the hisC gene on the chromosome was replaced by the destroyed hisC gene (ΔhisC) as a result of double recombinant crossing-over. The resulting double recombinant strain was named KMBS276 (ΔhisC trpC2).

<Construction of strain B. subtilis KMBS375>

1. Construction of strain KMBS350 (ΔpurA ΔpurR ΔpupG)

The destroyed genes pupG, purR, and purA (guanosine / inosine phosphorylase, purine operon repressor, succinyl-AMP synthetase, respectively) were sequentially introduced into the recombinant strain KMBS276 (ΔhisC trpC2) - derived from the strain B. subtilis 168 Marburg strain and obtained from as indicated below.

(1) Introduction ΔpupG (deletion without violating the reading frame of 204 nucleotides 334-537) in strain KMBS276

(i) Amplification of ΔpupG by Recombinant PCR

To amplify the 5'-end of ΔpupG and the preceding region, PCR primers 42 (SEQ ID NO. 46) and 43 (SEQ ID NO. 47) were constructed based on information from the GenBank database (Accession No. NC_000964). Primer 42 contains a BamHI site at the 5 ′ end. Primer 43 contains a junction formed by a deletion without violating the reading frame.

PCR (94 ° C, 30 seconds; 55 ° C, 1 minute; 72 ° C, 1.5 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was performed using the above primers and strain B chromosome DNA. subtilis 168 Marburg as a matrix for amplification of fragments containing the region of the 5'-end ΔpupG and the region preceding it.

To amplify the 3'-end of ΔpupG and the subsequent region, PCR primers 44 (SEQ ID NO. 48) and 45 (SEQ ID NO. 49) were constructed based on information from the GenBank database (Accession No. NC_000964). Primer 44 contains a junction formed by a deletion without violating the reading frame. Primer 45 contains a BamHI site at the 5 ′ end.

PCR (94 ° C, 30 seconds; 50 ° C, 1 minute; 72 ° C, 1.5 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was performed using the above primers and strain B chromosome DNA. subtilis 168 Marburg as a matrix for amplification of fragments containing the region of the 3'-end of ΔpupG and the next region.

To amplify the complete ΔpupG sequence by recombinant PCR, two DNA fragments amplified as described above were purified using a MicroSpin Column S-400 (Amersham Pharmacia Biotech), an appropriate amount of their mixture was used as a template in PCR using primers 42 (SEQ ID NO. 46) and 45 (SEQ ID NO. 49), performed PCR (94 ° C, 30 seconds; 55 ° C, 1 minute; 72 ° C, 3 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) thus, an amplified fragment (about 2 kb) of ΔpupG was obtained.

(ii) Cloning of the ΔpupG gene

The amplified fragment was treated with BamHI restriction enzyme (37 ° C, overnight) and separated on an agarose gel. The target fragment was isolated from the gel and ligated with the integrative vector pJPM1 of the B. subtilis chromosome (J. Bacteriol. 174: 4361-4373) pretreated with the same enzyme (37 ° C, 3 hours) followed by treatment with calf intestine phosphatase (calf intestine phosphatase). A plasmid was selected into which the ΔpupG gene was correctly integrated; DNA sequencing confirmed the absence of other mutations that could result from PCR; the plasmid was named pKM199.

(iii) Introduction of ΔpupG to strain KMBS276

Competent cells of strain KMBS276, prepared as described above, were transformed with plasmid pKM199 and colonies (recombinants resulting from a single crossing-over) capable of growing on LB agar plates containing 2.5 μg / ml Cm were selected.

One of the obtained recombinants was inoculated in 10 ml of LB + Gua medium and cultured for 2 days at 37 ° C. Colonies that were sensitive to chloramphenicol were selected using LB + Gua agar medium with / without Cm. Chromosomal DNA was isolated from the cells of the obtained Cm-sensitive colonies. PCR was performed as described above using primers 42 (SEQ ID NO. 46) and 45 (SEQ ID NO. 49). Strains were identified in which the pupG gene on the chromosome was replaced by the destroyed pupG gene (ΔpupG) by double recombinant crossing-over. The resulting double recombinant strain was named KMBS334 (ΔpupC ΔhisC trpC2).

(2) Introduction of ΔpurR (deletion without reading frame shift 795 nucleotides 31-825) in strain KMBS334

(i) Amplification of ΔpurR by Recombinant PCR

To amplify the 5'-end of ΔpurR and the preceding region, PCR primers 46 (SEQ ID NO. 50) and 47 (SEQ ID NO. 51) were constructed based on information from the GenBank database (Accession No. NC_000964). Primer 46 contains the BamHI site at the 5'-end. Primer 47 contains a junction formed by a deletion without violating the reading frame.

PCR (94 ° C, 30 seconds; 55 ° C, 1 minute; 72 ° C, 1.5 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was performed using the above primers and strain B chromosome DNA. subtilis 168 Marburg as a matrix for amplification of fragments containing the region of the 5'-end of ΔpurR and the region preceding it.

To amplify the 3'-end of ΔpurR and the region that follows, PCR primers 48 (SEQ ID NO. 52) and 49 (SEQ ID NO. 53) were constructed based on information from the GenBank database (Accession No. NC_000964). Primer 48 contains a junction formed by deletion without violating the reading frame. Primer 49 contains a BamHI site at the 5 ′ end.

PCR (94 ° C, 30 seconds; 50 ° C, 1 minute; 72 ° C, 1.5 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was performed using the above primers and strain B chromosome DNA. subtilis 168 Marburg as a matrix for amplification of fragments containing the region of the 3 ′ end of ΔpurR and the region following it.

To amplify the complete ΔpurR sequence by recombinant PCR, two DNA fragments amplified as described above were purified using a MicroSpin Column S-400 (Amersham Pharmacia Biotech), an appropriate amount of their mixture was used as a template in PCR using primers 46 (SEQ ID NO. 50) and 49 (SEQ ID NO. 53), performed PCR (94 ° C, 30 seconds; 55 ° C, 1 minute; 72 ° C, 3 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) thus, an amplified fragment (about 2.1 kb) of ΔpurR was obtained.

(ii) Cloning of the ΔpurR gene

The amplified fragment was treated with BamHI restriction enzyme (37 ° C, overnight) and separated on an agarose gel. The target fragment was isolated from the gel and ligated with the integrative vector pJPM1 of the B. subtilis chromosome (J. Bacteriol. 174: 4361-4373), pretreated with the same enzyme (37 ° C, 3 hours), followed by treatment with calf intestine phosphatase. A plasmid was selected into which the ΔpurR gene was correctly integrated; DNA sequencing confirmed the absence of other mutations that could result from PCR; the plasmid was named pKM200.

(iii) Introduction ΔpurR in strain KMBS334

Competent cells of strain KMBS334, prepared as described above, were transformed with plasmid pKM200 and colonies (recombinants resulting from a single crossing-over) capable of growing on plates with LB agar containing 2.5 μg / ml Cm were selected.

One of the obtained recombinants was inoculated in 10 ml of LB + Gua medium and cultured for 2 days at 37 ° C. Colonies that were sensitive to chloramphenicol were selected using LB + Gua agar medium with / without Cm. Chromosomal DNA was isolated from the cells of the obtained Cm-sensitive colonies. PCR was performed as described above using primers 46 (SEQ ID NO. 50) and 49 (SEQ ID NO). Strains were identified in which the purR gene on the chromosome was replaced by the destroyed purR gene (ΔpurR) by double recombinant crossing-over. The resulting double recombinant strain was named KMBS337 (ΔpurR ΔpupG ΔhisC trpC2).

(i) Amplification of ΔpurA by Recombinant PCR

To amplify the 5'-end of ΔpurA and the preceding region, PCR primers 50 (SEQ ID NO. 54) and 51 (SEQ ID NO. 55) were constructed based on information from the GenBank database (Accession No. NC_000964). Primer 50 contains a BamHI site at the 5 ′ end. Primer 51 contains a junction formed by deletion without violating the reading frame.

PCR (94 ° C, 30 seconds; 55 ° C, 1 minute; 72 ° C, 1 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was performed using the above primers and chromosomal DNA of B. subtilis 168 strain Marburg as a matrix for amplification of fragments containing the region of the 5'-end of ΔpurA and the region preceding it.

To amplify the 3'-end of ΔpurA and the region following it, PCR primers 52 (SEQ ID NO. 56) and 53 (SEQ ID NO. 57) were constructed based on information from the GenBank database (Accession No. NC_000964). Primer 52 contains a junction formed during division without violating the reading frame. Primer 53 contains a BamHI site at the 5 ′ end.

PCR (94 ° C, 30 seconds; 50 ° C, 1 minute; 72 ° C, 1 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was performed using the above primers and chromosomal DNA of B. subtilis 168 strain Marburg as a matrix for amplification of fragments containing the region of the 3 ′ end of ΔpurA and the region following it.

To amplify the entire ΔpurA sequence by recombinant PCR, two DNA fragments amplified as described above were purified using a MicroSpin Column S-400 (Amersham Pharmacia Biotech), an appropriate amount of their mixture was used as a template in PCR using primers 50 (SEQ ID NO. 54) and 53 (SEQ ID NO. 57), performed PCR (94 ° C, 30 seconds; 55 ° C, 1 minute; 72 ° C, 2 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) thus, an amplified fragment (about 1.4 kb) of ΔpurA was obtained.

(ii) Cloning of the ΔpurA gene

The amplified fragment was treated with BamHI restriction enzyme (37 ° C, overnight) and separated on an agarose gel. The target fragment was isolated from the gel and ligated with the integrative vector pJPM1 of the B. subtilis chromosome (J. Bacteriol. 174: 4361-4373), pretreated with the same enzyme (37 ° C, 3 hours), followed by treatment with calf intestine phosphatase. A plasmid was selected into which the ΔpurA gene was correctly integrated; DNA sequencing confirmed the absence of other mutations that could result from PCR; the plasmid was named pKM208.

(iii) Introduction of ΔpurA to strain KMBS337

Competent cells of strain KMBS337 prepared as described above were transformed with plasmid pKM208 and colonies (recombinants resulting from a single crossing-over) capable of growing on LB agar plates containing 2.5 μg / ml Cm were selected.

One of the obtained recombinants was inoculated in 10 ml of LB + Gua medium and cultured for 2 days at 37 ° C. Colonies that were sensitive to chloramphenicol were selected using LB + Gua agar medium with / without Cm. Chromosomal DNA was isolated from the cells of the obtained Cm-sensitive colonies. PCR was performed as described above using primers 50 (SEQ ID NO. 54) and 53 (SEQ ID NO. 57). Strains were identified in which the purA gene on the chromosome was replaced by the destroyed purA gene (ΔpurA) by double recombinant crossing-over. The resulting double recombinant strain was named KMBS349 (ΔpurA ΔpurR ΔpupG ΔhisC trpC2).

(3). Introducing trpC ⁺ and hisC ⁺ in KMBS349

Competent cells of strain KMBS349, prepared as described above, transformed the chromosomal DNA of strain KMBS275 (see <Construction of the B. subtilis 168 Marburg prototroph>) and selected colonies capable of growing on plates with minimal agar medium containing 20 mg / l adenine (Ade) .

Adenotin auxotrophic strains were selected from the obtained recombinants using a minimal agar medium with / without Ade. Then, using gel PCR (the PCR conditions corresponding to the pupG, purR, and purA genes were described above), strains were identified in which three genes on the chromosome were replaced by disrupted variants of these genes (ΔpupG, ΔpurR and ΔpurAS). The resulting strain was named KMBS350 (ΔpurA ΔpurR ΔpupG).

2. Construction of strain KMBS356 (ΔpurA ΔpurR ΔpupG guaB24)

A guanine auxotrophy mutation while maintaining a residual expression level of guaB24 (the same as guaB (A1) described in US2006275874A1) in the guaB gene encoding IMP dehydrogenase was introduced into the recombinant strain KMBS349 (ΔpurA up trPrRp subtilis 168 Marburg, and a prototroph was obtained from the resulting strain, as set forth below.

(one). Construction of the strain with the destroyed gene guaB from KMBS349

The competent cells of strain KMBS349 (ΔpurA ΔpurR ΔpupG ΔhisC trpC2), prepared as described above, were transformed with the chromosomal DNA of strain KMBS193 (guaB :: kan trpC2; US2006275874A1) and colonies were selected that were grown on plates with 2.5 μl medium (B μl medium) Kan).

Of the recombinants obtained, Kan resistant strains were identified, Gua auxotrophic. The resulting strain was named KMBS351 (guaB :: kan ΔpurA ΔpurR ΔpupG ΔhisC trpC2).

(2). Construction of a strain with a mutant (while maintaining a residual expression level) gene guaB from strain KMBS349

The competent cells of strain KMBS351 (guaB :: kan ΔpurA ΔpurR ΔpupG ΔhisC trpC2), prepared as described above, were transformed with the chromosomal DNA of strain YMBS9 (guaB24 trpC2; the competent cells of strain KMBS193 were transformed with the obtained by PCR method gu subtilis 168 Marburg containing the mutation guaB24 on the chromosome, yielding YMBS9) and colonies capable of growing on plates with agarized minimal agar medium containing 20 mg / L Ade, His and Trp were selected.

The strains auxotrophic for Ade and His were selected from the obtained recombinants using a minimal agar medium with / without Ade + His. Then, using gel PCR (the PCR conditions corresponding to the pupG and purr genes were described above) and DNA sequencing, strains were identified in which two genes on the chromosome were replaced by the corresponding variants of the destroyed genes (ΔpupG and ΔpurR) and guaB :: kan was replaced by guaB24 correctly. The resulting strain was named KMBS353 (guaB24 ΔpurA ΔpurR ΔpupG ΔhisC trpC2).

(3). Introduction of trpC ⁺ and hisC ⁺ in KMBS353

Competent cells of strain KMBS353 prepared as described above transformed the chromosomal DNA of strain KMBS350 (see 1. Construction of KMBS350 (ΔpurA ΔpurR ΔpupG)) and colonies capable of growing on a minimal agar medium containing 20 mg / L Ade were selected.

Then, using DNA sequencing of the guaB gene in the obtained recombinants, strains were identified in which the wild-type guaB gene on the chromosome was replaced with the mutation guaB24 allele, while maintaining a residual expression level. The resulting strain was named KMBS356 (guaB24 ΔpurA ΔpurR ΔpupG).

3. Construction of strain KMBS375 (Ppur * -Δatt ΔdeoD ΔpurA ΔpurR ΔpupG)

To the recombinant strain KMBS337 (ΔpurR ΔpupG ΔhisC trpC2; see 1. (2) Introduction of ΔpurR (deletion without reading frame shift 795 nucleotides 31-825) in KMBS334), derived from the strain B. subtilis 168 Marburg, the modified purine opon promoter was sequentially introduced (Ppur) and deletion attenuator sequence (Ppur * -Δatt; the same as Ppur1-Δatt described in US 2006275874 A1), the destroyed purA gene, adenine auxotrophy mutation with a residual guaB24 expression level (same as guaB (A1) described in US 2006275874 A1) in the guaB gene encoding IMP dehydrogenase, and the destroyed version of the deoD gene encoding go purinnucleoside phosphorylase, as set forth below.

(one). Construction of strain with destroyed Ppur from KMBS337

Competent cells of strain KMBS337 (ΔpurR ΔpupG ΔhisC trpC2) prepared as described above were transformed with the chromosomal DNA of strain KMBS198 (Ppur :: cat trpC2; US 2006275874 A1) and colonies capable of growing on LB + Gua + Cm medium were selected.

Strains auxotrophic for Ade and His were selected from the obtained recombinants using minimal agar medium with / without Ade + His. Then, using the PCR method in gel (the PCR conditions corresponding to the pupG and purr genes were described above), strains were identified in which two genes on the chromosome were replaced by the corresponding variants of the destroyed genes (ΔpupG and ΔpurR). The resulting strain was named KMBS340 (Ppur :: cat ΔpurR ΔpupG ΔhisC trpC2).

(2). Replacing Ppur :: cat with Ppur * -Δatt

Competent cells of strain KMBS340 (Ppur :: cat ΔpurR ΔpupG ΔhisC trpC2), prepared as described above, transformed the chromosomal DNA of strain KMBS261 (Ppur1-Δatt purR :: spc ΔpupG * trpC2; US 2006275874 A1 and selected to grow agar medium containing 20 mg / L Trp and His.

The strains auxotrophic for Ade and His were selected from the obtained recombinants using a minimal agar medium with / without Ade + His. Then, using gel PCR (the PCR conditions corresponding to the pupG and purR genes were described above) and DNA sequencing, strains were identified in which two genes on the chromosome were replaced by the corresponding variants of the destroyed genes (ΔpupG and ΔpurR) and Ppur * -Δatt was correctly replaced by Ppur :: cat. The resulting strain was named KMBS352 (Ppur * -Δatt ΔpurR ΔpupG ΔhisC trpC2).

(3). Introduction ΔpurA (deletion without shift of reading frame 369 nucleotides 307-675) in KMBS352

Competent cells of strain KMBS352 (Ppur * -Δatt ΔpurR ΔpupG ΔhisC trpC2), prepared as described above, transformed the chromosomal DNA of strain KMBS350 (ΔpurA ΔpurR ΔpupG; see 1. Construction of KMBS350 (Δpur Apur )pur) and minimal agar medium containing 20 mg / L His and Ade.

Strains with Ade auxotrophy acquired as a result of "congression" were selected among the obtained recombinants using minimal agar medium with / without Ade + His. It was found that one of these strains contains hisC ⁺ instead of ΔhisC. Then, using the gel PCR method (the PCR conditions corresponding to the purA gene were described above) and DNA sequencing, strains were identified in which the purA gene on the chromosome was replaced by a destroyed variant of this gene (ΔpurA) and Ppur * -Δatt was not replaced Ppur is wild type. The resulting strain was named KMBS359 (Ppur * -Δatt ΔpurA ΔpurR ΔpupG).

(four). Construction of the strain with the destroyed gene guaB from KMBS359

The competent cells of the strain KMBS359 (Ppur * -Δatt ΔpurA ΔpurR ΔpupG), prepared as described above, transformed the chromosomal DNA of the strain KMBS351 (guaB :: kan ΔpurA ΔpurR ΔpupG ΔhisC trpC2; see section 2 (c) KMBS349) and colonies capable of growing on plates with LB + Gua medium supplemented with 2.5 μg / ml Kan were selected.

Among the recombinants obtained, Kan resistant strains, auxotrophic for Gua, were selected. Then, using the PCR method (PCR conditions for Ppur were described in the application US 2006275874 A1), strains were identified in which Ppur * -Δatt was not replaced by wild-type Ppur. The resulting strain was named KMBS364 (guaB :: kan Ppur * -Δatt ΔpurA ΔpurR ΔpupG).

(5). Construction of a strain with a mutation with a residual expression level in the guaB gene from KMBS364

Competent cells of strain KMBS364 (guaB :: kan Ppur * -Δatt ΔpurA ΔpurR ΔpupG), prepared as described above, transformed the chromosomal DNA of strain KMBS356 (guaB24 ΔpurA ΔpurR ΔpupG; see 2. (3) Introduction of trpC ⁺ CBS ⁺ and ⁺ CBS ⁺ ) and colonies capable of growing on plates with minimal medium containing 20 mg / L Ade were selected.

Then, using DNA sequencing, strains were identified in which the guaB :: kan allele was correctly replaced by guaB24, and Ppur * -Δatt was not replaced by wild-type Ppur. The resulting strain was named KMBS365 (guaB24 Ppur * -Δatt ΔpurA ΔpurR ΔpupG).

(6). Introduction ΔdeoD (deletion without shift of the reading frame of 189 nucleotides 262-450) in KMBS365

(i) Amplification of ΔdeoD by Recombinant PCR

To amplify the 5'-end of ΔdeoD and the preceding region, PCR primers 54 (SEQ ID NO. 58) and 55 (SEQ ID NO. 59) were constructed based on information from the GenBank database (Accession No. NC_000964). Primer 54 contains the BamHI site at the 5'-end. Primer 55 contains a junction formed by deletion without violating the reading frame.

PCR (94 ° C, 30 seconds; 55 ° C, 1 minute; 72 ° C, 1.5 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was performed using the above primers and strain B chromosome DNA. subtilis 168 Marburg as a matrix for amplification of fragments containing the region of the 5'-end of ΔdeoD and its preceding region.

To amplify the 3'-end of ΔdeoD and the subsequent region, PCR primers 56 (SEQ ID NO. 60) and 57 (SEQ ID NO. 61) were constructed based on information from the GenBank database (Accession No. NC_000964). Primer 56 contains a junction formed by deletion without violating the reading frame. Primer 57 contains a BamHI site at the 5 ′ end.

PCR (94 ° C, 30 seconds; 50 ° C, 1 minute; 72 ° C, 1 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was performed using the above primers and chromosomal DNA of B. subtilis 168 strain Marburg as a matrix for amplification of fragments containing the region of the 3'-end of ΔdeoD and the region following it.

To amplify the complete ΔdeoD sequence by recombinant PCR, two DNA fragments amplified as described above were purified using a MicroSpin Column S-400 (Amersham Pharmacia Biotech), an appropriate amount of their mixture was used as template in PCR using primers 54 (SEQ ID NO .58) and 57 (SEQ ID NO. 61), performed PCR (94 ° C, 30 seconds; 50 ° C, 1 minute; 72 ° C, 3 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer) ), thus, an amplified fragment (about 2.0 kb) of AdeoD was obtained.

(ii) Cloning of the AdeoD Gene

The amplified fragment was treated with BamHI restriction enzyme (37 ° C, overnight) and separated on an agarose gel. The target fragment was isolated from the gel and ligated with the integrative vector pJPMl of the B. subtilis chromosome (J. Bacteriol. 174: 4361-4373) pretreated with the same enzyme (37 ° C, 3 hours), followed by treatment with calf intestine phosphatase. A plasmid was selected into which the AdeoD gene was correctly integrated; DNA sequencing confirmed the absence of other mutations that could result from PCR; the plasmid was named pKM201.

(ii) Introduction ΔdeoD in strain KMBS365

Competent cells of the KMBS365 strain prepared as described above were transformed with plasmid pKM201 and colonies (recombinants resulting from a single crossover) capable of growing on LB agar plates containing 2.5 μg / ml Cm were selected.

One of the obtained recombinants was inoculated in 10 ml of LB + Gua medium and cultured for 2 days at 37 ° C. Colonies that were sensitive to chloramphenicol were selected using LB + Gua agar medium with / without Cm. Chromosomal DNA was isolated from the cells of the obtained Cm-sensitive colonies. PCR was performed as described above using primers 54 (SEQ ID NO. 58) and 57 (SEQ ID NO. 61). The strains were identified in which the deoD gene on the chromosome is replaced by the destroyed deoD gene (ΔdeoD) by double recombinant crossing-over. The resulting double recombinant strain was named KMBS375 (ΔdeoD guaB24 Ppur * -Δatt ΔpurA ΔpurR ApupG).

Example 3. Construction of a strain of Bacillus amyloliquefaciens with inactivated hxlA gene. The production of inosine and gaunosin strain Bacillus amyloliquefaciens AJ 1991

To inactivate the hxlAB operon on the chromosome of the Bacillus amyloliquefaciens AJ 1991 strain, a nucleoside producer, the region upstream of the hxlA gene was amplified by PCR using hxlupBcu ⁺ primers (SEQ ID NO: 35) and hxlupPst- (SEQ ID NO: .2, A) and the genomic DNA of strain B. amyloliquefaciens IAM1523 (University of Tokyo, Japan) as a matrix. The resulting fragment was treated with restriction enzymes Bcul and PstI and cloned into the plasmid pNZTl (Zakataeva NP et al. A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains. Appi Microbiol Biotechnol (2010) 85: 1201-1209) pretreated with the same restriction enzymes to obtain plasmid pNZTl-hxlup. Then, the site located after the hxIA gene was amplified using hxldwPst + primers (SEQ ID NO: 37) and hxldwSal- (SEQ ID NO: 38) and the genomic DNA of B. amyloliquefaciens strain IAM1523. The resulting fragment was treated with restriction enzymes SalI and PstI, and cloned into the plasmid pNZTl-hxlup, previously treated with the same restriction enzymes, to obtain the plasmid pNZTl-hxlupdw (Figure 2, B). The correctness of the obtained construct was confirmed by analysis of the nucleotide sequence.

Plasmid pNZTl-hxlupdw was integrated into the B. amyloliquefaciens AJ 1991 strain, the producer of inosine and gaunosin, deposited on October 3, 2005 with VKPM as Bacillus amyloliquefaciens G1136A (VKPM B-8994). The replacement of the native hxlAB operon with the deletion variant ΔhdAB was carried out using the two-stage recombination method (Zakataeva NP et al. A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains. Appl Microbiol Biotechnol (2010) 85: 1201- 1209).

To assess the effect of hxlA gene inactivation on nucleoside production, strain AJ1 991 and its derivative AJ1991ΔhxlAB were cultured at 34 ° C for 18 hours in L-broth, then 0.3 ml of culture was inoculated in 3 ml of fermentation medium in 20 × 200 mm tubes and cultured at 34 ° C for 72 hours on a rotary shaker.

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

Glucose 60.0 NH ₄ Cl 15.0 KH ₂ PO ₄ 1.0 MgSO ₄ · 7H ₂ O 0.4 FeSO ₄ · 7H ₂ O 0.01 MnSO ₄ · 4H ₂ O 0.01 Mameno (total nitrogen) 0.8 Adenine 0.3 CaCO ₃ 25 pH 6.4 (adjusted using KOH)

Sterilization: 116 ° C, 30 min. Glucose and CaCO _{3 were} sterilized separately. Glucose: 110 ° C, 30 min; CaCO ₃ : 116 ° C, 30 min.

The amount of nucleosides accumulated in the medium was determined by HPLC as described above (see Example 2). The results of three independent experiments are shown in Table 2.

table 2 Strain OD ₅₆₂ Inosine, g / l Guanosine, g / l Aj 1991 13.4 4.10 ± 0.07 1.90 ± 0.02 AJ1991ΔhxlAB 12.7 4.43 ± 0.05 1.95 ± 0.03

As can be seen from Table 2, the strain AJ1991ΔhxlAB produces more inosine and guanosine than the parent strain AJ1991.

Although the invention has been described in detail with reference to the best mode of carrying out the invention, it will be apparent to those skilled in the art that various changes can be made and equivalent replacements made, and such changes and replacements are not outside the scope of the present invention.

Each of the above documents has a link, and all cited documents are part of the description of the present invention.

Claims (9)

1. A bacterium belonging to the genus Bacillus, with the ability to produce purine nucleosides, characterized in that said bacterium is modified so that a gene encoding 3-hexulose-6-phosphate synthase (HPS) is inactivated in it. 2. The bacterium according to claim 1, characterized in that said gene is the hx1A gene. 3. The bacterium according to claim 1, characterized in that said bacterium is the bacterium B.subtilis. 4. The bacterium according to claim 1, characterized in that said bacterium is the bacterium B.amyloliquefaciens. 5. The bacterium according to claim 1, characterized in that said purine nucleoside is selected from the group consisting of inosine, xanthosine, guanosine and adenosine. 6. A method of producing a purine nucleoside, comprising cultivating the bacterium according to any one of claims 1 to 5 in a nutrient medium, leading to the accumulation of said purine nucleoside in a nutrient medium, and isolating said purine nucleoside from the culture fluid. 7. The method for producing purine nucleoside according to claim 6, characterized in that said purine nucleoside is selected from the group consisting of inosine, xanthosine, guanosine and adenosine. 8. A method for producing a purine nucleotide, comprising culturing the bacterium according to any one of claims 1 to 5 in a nutrient medium, leading to the accumulation of said purine nucleoside in a culture medium, isolation of said purine nucleoside from the culture fluid, phosphorylation of said purine nucleoside and isolation of purine nucleotide. 9. A method for producing a purine nucleotide according to claim 8, characterized in that the purine nucleotide is selected from the group consisting of 5'-inosinic acid, xanthosine-5'-phosphate, 5'-guanilic acid and 5'-adenylic acid.

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