RU2403286C2 - Mutant phosphoribosyl pyrophosphate synthetase, dna coding said synthetase, bacterium coding said dna, method of producing purine nucleosides and method of producing purine nucelotides - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and is a method of producing purine nucleosides using a bacterium belonging to the Bacillus genus, containing a fragment of DNA which codes mutant bacterial phosphoribosyl pyrophosphate synthetase, in which the L-amino acid residue which corresponds to position 120, 135 or 194 in the wild type phosphoribosyl pyrophosphate synthetase sequence from Bacillus subtilis is substituted with a residue of another L-amino acid, and sensitivity of the said enzyme to inhibiting purine nucleotides on the type of feedback is low.

EFFECT: invention enables to obtain highly effective purine nucleosides.

13 cl, 2 dwg, 2 tbl, 5 ex

Description

Technical field

The present invention relates to biotechnology, specifically to a method for the production of purine ribonucleosides, such as inosine and guanosine, which are of value as a raw material for the synthesis of 5'-inosinic acid and 5-guanilic acid, respectively. More specifically, the present invention relates to a novel feedback-resistant inhibition enzyme involved in purine biosynthesis. More specifically, the present invention relates to a novel feedback resistant inhibition mutant phosphoribosyl pyrophosphate synthetase (PRPP synthetase - phosphoribosylpyrophosphate synthetase) from bacteria of the genus Bacillus. The invention also relates to a method for producing purine ribonucleosides and purine ribonucleotides by fermentation using bacterial strains containing a feedback inhibition resistant enzyme encoded by the mutant prs gene.

Description of the Related Art

There are known methods for the enzymatic production of purine nucleosides using adenine auxotrophic strains or strains that are further conferred resistance to various compounds, such as purine analogues and sulfaguanidine, which strains belong to the genus Bacillus (published Japanese patents Nos. 38-23039 (1963), 54-17033 (1979), 55-2956 (1980), and 55-45199 (1980), Japanese Patent Application Laid-Open No. 56-162998 (1981), Published Japanese Patents Nos. 57-14160 (1982) and 57-41915 (1982) and Japanese Patent Application Laid-Open No. 59-42895 (1984)) or to the genus Brevibacterium (published Japanese Patent Nos. 51-5075 (1976) and 58-17592 (1972), and Agric. Biol. Chem., 42, 399 (1978), or to the genus Escherichia (PCT International Application 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-M-nitrosoguanidine), and 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 US 7326546 (2008)) and to the genus Brevibacterium (Japanese Patent Application Laid-Open No. 63-248394 (1988)). The productivity of these inosine producing strains can be further improved by increasing the ability to secrete inosine (RF patent applications 2002101666, 2002104463 and 2002104464).

5-Phosphoribosyl-α-1-pyrophosphate (hereinafter - “PRPP”) and glutamine are the initial substrates for purine biosynthesis. PRPP synthetase [EC 2.7.6.1], which catalyzes the reaction of ribose-5-phosphate with ATP to form PRPP and AMP, binds the pentose phosphate pathway with the biosynthesis of purine and pyrimidine nucleotides, with the biosynthesis of histidine and tryptophan and pyridine nucleotide coenzymes ( F. In Metabolism of Nucleotides, Nucleosides and Nucleobases in Microorganisms (ed. Munch-Petersen A.), 1-25. Academic Press, London; 1983).

PRPP synthetase is subject to feedback inhibition by purines, pyrimidines and amino acids. Many purine nucleotides inhibit the activity of PRPP synthetase in competition with adenosine 5'-triphosphate (hereinafter, “ATP”). However, the only strong nucleotide inhibitor is adenosine-5'-diphosphate (ADP-adenosine-5'-diphosphate); it competes with ATP and is an allosteric inhibitor that binds to a site other than the active site (Hove-Jensen, B. et al., J. Biol. Chem. 261: 6765-6771 (1986); Amvig K. et al. , Eur. J. Biochem. 192: 195-200 (1990)).

Human hyperuricemia and gout are known to be due to increased activity and resistance to purine nucleotides of PRPP synthetase (Becker MA et al. Arthritis Rheum, 18: 6 Suppl: 687-94 (1975); Zoref E. et al., J. Clin. Invest., 56: 1093-9 (1975); Becker MA et al., J. Clin. Invest., 96: 2133-41 (1995)). Uric acid synthesis in individuals with increased activity of PRPP synthetase is the result of increased production of PRPP and subsequent acceleration of de novo purine nucleotide synthesis. It was shown that the increased activity of PRPP synthetase is the result of several mutations in the corresponding gene, which is a specific replacement of amino acid residues in the human enzyme: asparagine to histidine at position 51, asparagine to serine at position 113, leucine to isoleucine at position 128, alanine to valine at position 189. Such a mutant PRPP synthetase is resistant to purine nucleotides that inhibit the normal enzyme by a mechanism that is not competitive with ATP (Roessler. BJ et al. J. Biol. Chem., v.268, No. 35, 26476-26481 ( 1993); Becker, MA et al., J. C lin. Invest., 96: 2133-41 (1995)).

A process for the production of purine nucleosides through the fermentation of a microorganism belonging to the genus Escherichia and having the ability to produce purine nucleoside with the mutation prs D128A (US patent application 20070161090 (2007)) is disclosed. In addition, a method for producing L-histidine using a mutant PRPP synthetase is described, characterized in that the L amino acid residue corresponding to position 115 in wild-type Escherichia coli PRPP synthetase is replaced by another amino acid residue (US Patent Application 20050176033, (2005)). In addition, it was shown that a strain of Ashbya gossypii with increased expression of a mutant PRPP synthetase with a H196Q mutation resistant to ADP inhibition synthesizes more riboflavin than a strain with increased expression of wild-type PRPP synthetase (US patent 6878536 (2005)). However, there are currently no reports describing a mutant PRPP synthetase from a bacterium of the Bacillus genus that is resistant to feedback inhibition by purine nucleotides and the use of such a mutant PRPP synthetase to improve nucleoside production using a microorganism producing a purine nucleoside belonging to the genus Bacillus in particular Bacillus subtilis and Bacillus amyloliquefaciens. Moreover, the sequence of the prs Bacillus amyloliquefaciens K gene has never been published before.

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 the production of purine nucleosides using the obtained strains.

This goal was achieved by constructing a new mutant PRPP synthetase from Bacillus. Given the high degree of conservatism of the protein PRPP synthetase (Eriksen T. et al .. Nature Struct. Biol, v.7, No. 4, pp.303-308 (2000)), mutant PRPP synthetases from B. subtilis and B were constructed. amyloliquefaciens with mutations corresponding to known mutations in the human enzyme. It was shown that only some mutations when introduced into the gene encoding PRPP synthetase lead to an increase in the production of purine nucleosides in producer strains of purine nucleosides. Thus, the present invention has been made.

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.

The aim of the present invention is the provision of a mutant bacterial phosphoribosyl pyrophosphate synthetase (PRPP synthetase), characterized in that the L-amino acid residue corresponding to position 120, or 135, or 194 in the sequence of wild-type PRPP synthetase Bacillus subtilis, is replaced by a residue of another L-amino acid, and feedback sensitivity of the enzyme to inhibition by purine nucleotides is reduced.

It is also an object of the present invention to provide a mutant PRPP synthetase as described above, characterized in that the asparagine residue at position 120 in the sequence of wild-type PRPP synthetase is substituted with a serine residue.

It is also an object of the present invention to provide a mutant PRPP synthetase as described above, characterized in that the leucine residue at position 135 in the wild-type PRPP synthetase sequence is replaced with an isoleucine residue.

It is also an object of the present invention to provide a mutant PRPP synthetase as described above, characterized in that the alanine residue at position 194 in the sequence of wild-type PRPP synthetase is substituted with a Valine residue.

It is also an object of the present invention to provide the mutant PRPP synthetase described above, characterized in that the wild-type PRPP synthetase is a PRPP synthetase from Bacillus subtilis or Bacillus amyloliquefaciens.

Another objective of the present invention is the provision of the above mutant PRPP synthetase, including deletion, substitution, insertion or addition of one or more amino acids in one or in many positions other than position 120, or 135, or 194, characterized in that the inhibition of the enzyme purine feedback nucleotides are reduced.

It is also an object of the present invention to provide DNA encoding the mutant PRPP synthetase described above.

It is also an object of the present invention to provide a bacterium containing the above-described DNA and capable of producing purine nucleosides.

It is also an object of the present invention to provide said bacterium, characterized in that said bacterium is Bacillus subtilis.

It is also an object of the present invention to provide said bacterium, characterized in that said bacterium is Bacillus amyloliquefaciens.

Another objective of the present invention is the provision of a method for the production of purine nucleoside, comprising culturing the above bacteria in a nutrient medium, leading to the secretion of the specified purine nucleoside in the specified nutrient medium, and the allocation of the specified purine nucleoside from the culture fluid.

It is also an object of the present invention to provide the method described above, characterized in that the purine nucleoside is inosine, xanthosine, guanosine or adenosine.

It is also an object of the present invention to provide a method for producing a purine nucleotide, 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 the method described above, wherein the purine nucleotide is 5'-inosinic acid, xanthosine-5'-phosphate, 5'-guanilic acid or 5'-adenyl acid.

PRPP synthetase with the substitution of the L-amino acid residue corresponding to position 120, or 135, or 194 in the sequence of wild-type Bacillus PRPP synthetase may be referred to as "mutant PRPP synthetase", DNA encoding a mutant PRPP synthetase may be referred to as "mutant prs gene "or" mutant PRPP synthetase gene ", and PRPP synthetase without substitution may be referred to as" wild-type PRPP synthetase ". The term "mutant PRPP synthetase" means that enzyme activity due to mutations leading to the replacement of amino acid residues at position 120, or 135, or 194, is inhibited by purine nucleotides, in particular ADP, to a lesser extent than the enzyme of the wild-type strain.

The present invention will now be described in detail.

1. Mutant PRPP synthetase and mutant prs gene according to the present invention.

It is known that the increased activity of human PRPP synthetase is the result of several mutations of the corresponding gene, leading to a specific replacement of amino acid residues in the mature enzyme: asparagine to histidine at position 51, asparagine to series at position 113, leucine to isoleucine at position 128, alanine to valine to position 189. Such a mutant PRPP synthetase is resistant to purine nucleotides that inhibit the normal enzyme by a mechanism that is not competitive with ATP (Becker MA et al., J. Clin. Invest., 96: 2133-41 (1995)). Given the high degree of conservatism of the prs gene (Eriksen, T. et al. Nature Struct. Biol, v. 7, No. 4, pp. 303-308 (2000)), mutant Bacillus PRPP synthetases with mutations D58H, N120S, L135I or A194V, the corresponding mutations N51H, N113S, L128I or A189V in a human enzyme. Such mutations are unknown for Bacillus PRPP synthetases. The term "PRPP synthetase from Bacillus" means a PRPP synthetase of bacteria belonging to the genus Bacillus. Preferred species are B. subtilis or B. amyloliquefaciens.

Mutant PRPP synthetase was obtained taking into account known sequences by introducing mutations into the wild-type prs gene using conventional methods. As the wild-type prs gene, the prs gene from B. subtilis (nucleotides 57743 to 58696 in sequence with accession number NC_000964 in the GenBank database; SEQ ID NO: 1) or the prs gene from B. amyloliquefaciens K (SEQ ID NO : 3). The B. subtilis pr gene is located between the ORF gcaD and the ctc ORF on the chromosome of B. subtilis strain 168. Therefore, the prs gene can be obtained by PCR (White, TJ et al. Trends Genet. 5, 185 (1989)) using primers constructed based on the nucleotide sequence of a gene. The gene encoding the B. amyloliquefaciens K PRPP synthetase (SEQ ID NO: 1) can be obtained in a similar manner.

Mutant PRPP synthetase may include division, substitution, insertion or addition of one or more amino acids at one or a plurality of positions other than position 120, or 135, or 194, provided that the activity of the PRPP synthetase is not impaired. The term "PRPP synthetase activity" means the activity of catalysis of the reaction for the formation of 5-phosphoribosyl-α-1-pyrophosphate (PRPP) from ribose-5-phosphate and ATP to form AMP. The activity of PRPP synthetase in extracts and the degree of inhibition of the ADP enzyme can be measured using a partially modified method of K. F. Jensen et al. (Analytical Biochemistry, 98, 254-263 (1979)). Specifically, [α- ³² P] ATP can be used as a substrate and the AMP formed in the [ ³² P] reaction should be determined.

The number of "several" amino acids varies depending on the position or type of amino acid residues in the tertiary structure of the protein. This is due to the following reasons. Namely, some amino acids have a high degree of homology with each other and therefore such changes do not greatly affect the tertiary structure of the protein. Therefore, the mutant PRPP synthetase of the present invention can be an enzyme with a homology of at least 30-50%, preferably 50-70%, relative to the complete amino acid sequence of the main PRPP synthetase having PRPP synthetase activity.

In the present invention, “L-amino acid residue corresponding to position 120, or 135, or 194” means an amino acid residue corresponding to position 120, or 135, or 194 in the amino acid sequence of SEQ ID NO: 2.

To determine the L-amino acid residue corresponding to mutations N51H, N113S, L128I or A189V of human PRPP synthetase, it is necessary to align the amino acid sequence of the human PRPP synthetase with the amino acid sequence of interest to the PRPP synthetase from a bacterium of the genus Bacillus, and determine the bacterial of interest PRPP synthetase L-amino acid residues corresponding to numbers 51, 113, 128 or 189 in the amino acid sequence of human PRPP synthetase.

Substitution, division, insertion or addition of one or more amino acid residues must be conservative mutations in order to maintain the activity of the enzyme. Conservative substitutions are typical conservative mutations. Examples of conservative substitutions include replacing Ala with Ser or Thr, replacing Arg with Gln, His or Lys, replacing Asn with Glu, Gin, Lys, His or Asp, replacing Asp with Asn, Glu or Gin, replacing Cys with Ser or Ala, replacing Gln with Asn, Glu, Lys, His, Asp or Arg, replacing Glu with Asn, Gln, Lys or Asp, replacing Gly with Pro, replacing His with Asn, Lys, Gln, Arg or Tur, replacing Ile with Leu, Met, Val or Phe, replace Leu with Ile, Met, Val or Phe, replace Lys with Asn, Glu, Gln, His or Arg, replace Met with Ile, Leu, Val or Phe, replace Phe with Trp, Tyr, Met, Ile or Leu, replacing Ser with Thr or Ala, replacing Thr with Ser or Ala, replacing Trp with Phe or Tyr, replacing Tyr with His, Phe or Trp, and replacing Val with Met, Ile or Leu.

DNA encoding essentially the same protein as the mutant PRPP synthetases described above is obtained, for example, by modifying the nucleotide sequence, for example, by the method of site-directed mutagenesis, so that one or more amino acid residues at a particular site suggest a deletion, replacement, insertion or addition. DNA modified as described above is obtained by conventional mutagenesis techniques known in the art. Mutagenic treatment includes a method for processing DNA containing the mutant prs gene in vitro, for example, hydroxylamine, and a method for treating a microorganism, for example, bacteria with the mutant prs gene belonging to the genus Bacillus, ultraviolet radiation or a mutagen such as N-methyl-N ' N-nitro-N-nitrosoguanidine (NTG) and nitrous acid, commonly used for such processing. Substitution, deletion, insertion or addition of a nucleotide as described above also includes naturally occurring mutations (mutant or variant) due to individual differences or differences between species or genera of bacteria containing PRPP synthetase.

DNA encoding essentially the same protein as mutant PRPP synthetase can be obtained by isolating DNA that hybridizes under stringent conditions with DNA having the sequence of the known prs gene, or with its part as a probe, and encoding a protein with PRPP synthetase activity from mutagenized cells with mutant PRPP synthetase.

"Stringent conditions" include those conditions under which specific hybrids are formed and non-specific hybrids are not formed. It is difficult to precisely determine these conditions using numerical values. However, stringent conditions include, for example, conditions under which DNA with a high degree of homology, for example, DNA with a homology of at least 50%, hybridizes with each other, and DNA with a lower homology does not hybridize with each other.

Calculation methods such as BLAST, FASTA, and CrustalW can be used to evaluate the homology of proteins or DNA.

BLAST (Basic Local Alignment Search Tool) - heuristic search algorithm used by the programs blastp, blastn, blastx, megablast, tblastn and tblastx; these programs attribute values to indicators using statistical methods Karlin, Samuel and Stephen F. Altschul ("Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes". Proc. Natl. Acad. Sci. USA, 1990, 87: 2264-68; "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc. Natl. Acad. Sci. USA, 1993, 90: 5873-7). FASTA method described by W.R. Pearson (Rapid and Sensitive Sequence Comparison with FASTP and FASTA, Methods in Enzymology, 1990, 183: 63-98). The Clustal W method is described by Thompson J.D., Higgins D.G. and Gibson T.J. ("CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice", Nucleic Acids Res. 1994, 22: 4673-4680).

On the other hand, an example of stringent conditions is the conditions under which DNA hybridizes with each other at a salt concentration that meets the usual washing conditions for Southern hybridization, i.e., 60 ° C, 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS. Part of the sequence SEQ ID NO: 1 can also be used as a probe for DNA encoding the variants and hybridizing with the prs gene. Such a probe can be prepared by PCR using oligonucleotides constructed on the basis of the nucleotide sequence of SEQ ID NO: 1 as primers and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 1, as a matrix. When a DNA fragment of about 300 bp is used as a probe, washing conditions for hybridization include, for example, 50 ° C, 2 × SSC and 0.1% SDS. The duration of washing depends on the type of membrane used for blotting, and is generally recommended by the manufacturer. For example, the recommended duration of washing a Hybond ™ N + nylon membrane (Amersham) under stringent conditions is 15 minutes.

A gene hybridizing under stringent conditions as described above includes genes with a stop codon formed within the coding region and coding for an inactive protein due to a mutation in the active center. However, this difficulty can easily be eliminated by ligating the gene with a commercial expression vector and examining the expressed protein for PRPP synthetase activity.

2. The bacterium of the present invention

The bacterium of the present invention is a purine nucleoside producing bacterium containing DNA encoding the mutant PRPP synthetase of the present invention. More specifically, the bacterium of the present invention contains DNA with a mutant prs gene expressed on a chromosome or plasmid in the bacterium, and has an increased ability to produce purine nucleosides.

The bacterium of the present invention is a bacterium belonging 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). 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 have 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 purines, such as purine nucleosides, in an amount greater than that of a wild-type B. subtilis strain such as B. subtil is 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 of inosine, xanthosine, guanosine or / and adenosine.

Transformation of a bacterium with DNA encoding a protein means introducing DNA into a bacterial cell, for example, by conventional methods for introducing a mutation of the present invention into a gene encoding a PRPP synthetase. Typically, for this introduction, it is necessary to clone a gene in a vector capable of functioning in a bacterium belonging to the genus Bacillus. For these purposes, shuttle vectors can be used: pH Y300PLK, pMWMXl, pLF22, pKSl. The replacement of the wild-type gene with a mutant gene occurs due to homologous recombination.

Methods for the preparation of chromosomal DNA, hybridization, PCR, preparation of plasmid DNA, restriction and ligation of DNA, transformation, selection of oligonucleotides as a primer, etc. may be conventional methods known to a person skilled in the art. These methods are described, for example, 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.

The gene encoding the protein of the Bacillus amyloliquefaciens K PRPP synthetase, the prsBA gene, is sequenced by the authors of the present invention (SEQ ID NO: 3). The sequence of the gene encoding the protein PRPP synthetase from Bacillus subtilis, the prs gene of Bacillus subtilis subsp.subtilis strain 168, is known (nucleotides 57743 to 58696 in the sequence with accession number NC_000964 in the GenBank database; (SEQ ID NO: 1)). The prs gene of Bacillus subtilis is located on the chromosome between the gcaD and ctc genes in the region of 7 °. Therefore, these prs genes can be obtained by PCR (polymerase chain reaction; refer to White, T.J. et al. Trends Genet., 5, 185 (1989)) using primers designed based on the nucleotide sequence of the gene. Genes encoding the protein of the PRPP synthetase of other microorganisms can be obtained in a similar manner (see Example 3).

Examples of the prs gene of Bacillus subtilis also include DNA encoding a protein of PRPP synthetase with the amino acid sequence shown in SEQ ID NO: 2 and / or a protein with an amino acid sequence including a deletion, substitution, insertion or addition of one or more amino acids in the amino acid sequence, presented in SEQ ID NO: 2.

Examples of the prs gene of Bacillus amyloliquefaciens K also include DNA encoding a protein PRPP synthetase with the amino acid sequence shown in SEQ ID NO: 4, and / or a protein with an amino acid sequence including a deletion, substitution, insertion or addition of one or more amino acids in the amino acid sequence presented in SEQ ID NO: 4.

The bacterium of the present invention can be obtained by introducing the aforementioned DNA into a bacterium initially capable of producing a purine nucleoside. On the other hand, the bacterium of the present invention can be obtained by imparting the ability to produce purine nucleoside bacteria containing the aforementioned DNA.

An example of a parent strain belonging to the genus Bacillus that can be used in the present invention is a producer strain of inosine B, subtilis KMBS375. (KMBS375: Ppur * -Δatt ΔpurA ΔpurR ΔpupG ΔdeoD guaB24; see the Reference example). Other parent strains belonging to the genus Bacillus that can be used in the present invention include B. subtilis strain AJ 12707 (FERM P-12951) (Japanese patent application JP 6113876A2), B. subtilis strain AJ3772 (FERM P-2555) (patent Japanese application JP 62014794A2), Bacillus pumilus NA-1102 (FERM BP-289), Bacillus subtilis NA-6011 (FERM BP-291), Bacillus subtilis G1136A (ATCC No. 19222) (US Pat. No. 3,575,809), reidentified as Bacillus amyloliquefaciens 1991, deposited on October 3, 2005 at the 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 4701413), B . pumilis Gottheil No.3218 (ATCC No. 21005) (US Pat. No. 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 the purine nucleoside phosphorylase (deoD :: kan) (RF patent application 2002103333, US patent 7326546, 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 onepohpur from B. subtilis (Ebbole DJ and Zalkin HJ Biol. Chem., 262: 8274-87 (1987), Bacillus subtilis and Its Closest Relatives, Editor in Chief: AL Sonenshein, ASM Press, Washington DC, 2002 ) A producer strain of B. subtilis inosine is described with a modified regulatory region of the rig operon (US patent 7326546, 2008).

3. 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 cultivation temperature of 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.

4. Method for the production of purine nucleotides

The method of the present invention includes a method for producing purine nucleotides, comprising the steps of growing a bacterium of the present invention in a culture medium, which leads 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 the present invention includes a method for producing 5'-inosinic acid, comprising 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 release of 5'-inosinic acid. In addition, the method of the present invention includes a method for producing 5'-xanthyl acid, comprising the steps of growing the bacteria of the present invention in a culture medium, resulting in the secretion of xanthosine in the culture medium, phosphorylation of inosine and the isolation of 5'-xanthyl acid. In addition, the method of the present invention includes a method of producing 5'-guanilic acid, comprising the steps of growing the bacteria of the present invention in a culture medium, leading to the secretion of guanosine into the culture medium, phosphorylation of guanosine and the release of 5'-guanilic acid. The method of the present invention also includes a method for producing 5'-guanilic acid, comprising the steps of growing the bacteria of the present invention in a culture medium, allowing the secretion of xanthosine in 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 purine nucleoside can be enzymatic using various phosphatases, nucleoside kinases or nucleoside phosphotransferases, or chemical using phosphorylating agents such as POCl ₃ and the like. You can use phosphatase that can catalyze 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 using as phosphoric acid donor polyphosphoric acid (salts), phenylphosphoric acid (salts) or carbamylphosphoric acid (salts) (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-nitrophenylphosphate as a substrate (Mitsugi, K., et al., Agric. Biol. Chem., 28, 586-600 (1964)), inorganic phosphate (Japanese Laid-open Patent Application No. JP 42-1186) or acetyl phosphate (Japanese Laid-open Patent Application No. JP 61-41555), 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 sort gene product. J. Bacteriol. 177: 4921-4926 can be used. (1995); WO 9108286) or the like. As an example of nucleoside phosphotransferase, the nucleoside phosphotransferase described by Hammer-Jespersen, K. (Nucleoside catabolism, p.203-258. In A Munch-Petesen (ed.). Metabolism of nucleotides, nucleosides, and nucleobases in microorganism. 1980, Academic Press, can be 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 GMF 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; EP 0251489 B1).

Brief Description of Drawings

The Figure 1 shows the sequence alignment of PRPP synthetase: Hum, human isoenzyme I; Bs, Bacillus subtilis; Ba _K , Bacillus amyloliquefaciens, strain K; Ba _FZB42 , Bacillus amyloliquefaciens FZB42 _FZB42 . Amino acids in the human enzyme, which are replaced by other amino acids to give the enzyme resistance to inhibition of ADP and GDP, are shown in bold. The corresponding amino acids of the Bacillus enzyme are also shown in bold. Figure 2 depicts the organization of the plasmid pKSl-PRSBS.

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. Cloning of the wild-type prs gene from B. subtilis and construction of mutant genes.

The complete nucleotide sequence of B. subtilis 168 strain has been determined (F. Kunst, N. Ogasawara, I. Moszer, et al., The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390: 249-256 (1997)). Primers 1 (SEQ ID NO. 5) and 2 (SEQ ID NO. 6) were synthesized based on the published nucleotide sequence to amplify the prs gene. Using these primers and B. subtilis 168 genomic DNA as a template, the B. subtilis prs gene (prsBS) was amplified with adjacent regions. The resulting fragment is 1.8 kbp. treated with restriction enzymes SalI and Ecl136I and cloned in a heat-sensitive low-copy shuttle vector pKS treated with SalI and SmaI (Shatalin KY and Neyfakh AA, FEMS Microbiology Letters, 245: 315-9 (2005)) to obtain plasmid pKSl-PRSBS. The prsBS gene contained on this plasmid was then mutagenized using the QuikChange site directed mutagenesis method (Stratagene). For this, primers 3 (SEQ ID NO.7) and 4 (SEQ ID NO.8) were used to introduce the N120S mutation and primers 5 (SEQ ID NO.9) and 6 (SEQ ID NO.9) and 6 (SEQ ID NO.10) were used to introduce the A194V mutation and plasmid pKSl-PRSBS as a matrix. As a result, plasmids pKSl-PRSBS 120 and pKSl-PRSBS 194 containing prs _BS N120S or prs _BS A194V, respectively, were obtained and confirmed the presence of mutations of interest by sequencing.

Example 2. The effect of mutations prs _BS N120S or prs _BS A194V on the production of inosine by the strain of the producer of inosine B. subtilis

Plasmids pKSl-PRSBS 120 and pKSl-PRSBS 194 containing prs _BS N120S or prs _BS A194V were introduced by transformation into the B. subtilis KMBS375 inosine producer strain, after which the prsBS strain was replaced by prs _BS N120S or prs _BS A194V. The mutant gene was integrated into the KMBS375 chromosome using the method described for gene inactivation in B. anthracis (Shatalin and Neyfakh, FEMS Microbiology Letters, 2005) with modifications that made it possible to introduce mutations of interest (including point mutations) into the chromosome without selection. This method includes the following steps.

1) Introduction of the obtained plasmid by transformation into the Bacillus strain, selection of clones with the plasmid at 30 ° C on LA (peptone 10 g / l, yeast extract 5 g / l, NaCl 3 g / l, agar 18 g / l) with 10 μg / ml erythromycin.

2) Growing the plasmid-transformed strain on LB medium (without erythromycin) with aeration at 37 ° C overnight and sieving the culture on LB agar with 10 μg / ml erythromycin. About 100% of erythromycin-resistant cells of such an overnight culture contain a plasmid integrated into the chromosome as a result of a single crossing-over. Therefore, the obtained clones contain two alleles of the gene of interest in the chromosome: the wild-type gene and the mutant one.

3) Growing individual clones containing an integrated plasmid on LB medium without erythromycin with aeration for 48 hours at 30 ° C. The resulting culture was plated on LA plates (without erythromycin) and grown at 34 ° C to obtain individual colonies.

4) Testing of individual clones for resistance to erythromycin; usually 0.5-50% of the colonies are sensitive to erythromycin. As a result of cultivation, a second crossingover and plasmid excision occurred, which led to the fact that either the wild-type or mutant gene remained on the chromosome.

5) Testing erythromycin-sensitive clones for the required mutation using the PCR method in gel. In this method, PCR is carried out - analysis of colonies with 2 pairs of primers. 5'-end primer - common to any pair. 3'-terminal primers are distinguished by 3'-terminal nucleotides. One 3'-terminal primer (using which the maximum product with DNA will be obtained from the colonies formed by cells with the mutant gene as a template) contains a mutant terminal nucleotide, and the other 3'-terminal primer (using which the maximum product with DNA from colonies formed by wild-type cells as a template) contains a wild-type terminal nucleotide. In addition, the penultimate nucleotide of both 3'-terminal primers was specially modified to further hinder primer annealing to a non-specific matrix and thus to better distinguish between wild-type and mutant colonies.

To select strains containing prs _BS N120S or prs _BS A194V mutations, 5'-end primer 7 (SEQ ID NO.11), 3'-end primer 8 (SEQ ID NO.12) and 5'- were used for gel PCR. end primer 7 (SEQ ID NO. 11), 3'-end primer 9 (SEQ ID NO.13) for identification of prs _BS 120S, and 5'-end primer 7 of SEQ ID NO.11), 3'-end primer 10 (EQ ID NO.14) and the 5'-end primer 7 (SEQ ID NO.11), the 3'-end primer 11 (SEQ ID NO.15) to identify prs _BS A 194V.

Thus, derivatives of KMBS375 containing prs _BS N120S mutations (KMBS375 prs _N120S ) or prs _BS A194V (KMBS375 prs _A194V ) were _obtained . After confirming the accuracy of substitution by sequencing, the obtained strains KMBS375 prs _N120S , KMBS375 prs _A194V and the control strain KMBS375 were evaluated in vitro fermentation. For this, each of the strains was cultured at 34 ° С for 18 hours in L-broth and 0.3 ml of the obtained culture was inoculated in 3 ml of fermentation medium in 20 × 200 mm tubes and cultured on a rotary shaker at 34 ° С for 72 hours.

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 (ADESANO1) 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 C 18 (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 ₆₀₀ H × R, g / l Exit, % GR, g / l Exit, % KMBS375 9.0 6.7 ± 0.4 22.2 ± 1.4 0.25 ± 0.04 0.83 ± 0.12 KMBS375 prs _N120S 9.7 7.9 ± 0.4 26.3 ± 1.2 0.29 ± 0.05 0.97 ± 0.18 KMBS375 prs _A194V 9.6 7.3 ± 0.6 24.3 ± 2.1 0.30 ± 0.01 1.00 ± 0.02

As can be seen from Table 1, the use of a mutant prs gene encoding a PRPP synthetase resistant to feedback inhibition by purine nucleotides improved the inosine productivity of B. subtilis strain KMBS375.

Example 3. Cloning of the wild-type prs gene B. amyloliquefaciens K and the construction of mutant genes.

To clone a DNA fragment containing the prs gene of strain B. amyloliquefaciens K (prs _BA ), Primer 12 (SEQ ID NO.16) and primer 13 (SEQ ID NO.17) were constructed using sequences in the structural parts of the B. subtilis gsaD and ctc genes located on both sides of the prs gene, following the assumption that these chromosome regions of B. subtilis are homologous to those of B. amyloliquefaciens. Using these primers and genomic DNA of B. amyloliquefaciens IAM1523 (University of Tokyo, Japan), a PCR product of the expected size was obtained as a template. The fragment was sequenced and found that it contains a gene with very high homology to the prsBS gene and to the prs gene of plants of the bacterium Bacillus amyloliquefaciens FZB42, the genome of which was recently sequenced (Fig. 1).

Based on the obtained sequence of the prs region from B. amyloliquefaciens K, primer 14 (SEQ ID NO.18) and primer 15 (SEQ ID NO.19) were constructed to subclone the prs gene with flanking regions sufficient for further homologous recombination.

Using these primers and genomic DNA of B. amyloliquefaciens IAM1523 as the template for the preparation, a PCR product that was digested with restriction enzymes SalI and PvuII and cloned into the pKSl vector treated with restriction enzymes SalI and SmaI to form plasmid pKSl-PRSBA. Then, site-directed mutagenesis of the prs _Ba gene contained on this plasmid was carried out as described in Example 1, using plasmid pKSl-PRSBA as template, primer 16 (SEQ ID NO.20) and primer 17 (SEQ ID NO.21); primer 18 (SEQ ID NO.22) and primer 19 (SEQ ID NO.23); primer 20 (SEQ ID NO.24) and primer 21 (SEQ ID NO.25); primer 22 (SEQ ID NO.26) and primer 23 (SEQ ID NO.27), respectively, for the introduction of mutations D58H, N120S, L135I or A194V, corresponding mutations N51H, N113S, L128I or A189V human PRPP synthetase.

Thus, plasmids pKS1-PRSBA58, pKS1-PKSBA120, pKS1-PRSBA135 and pKS1-PRSBA194 containing the genes prs _BA D58H, prs _BA N120S, prs _BA L135I and prs _BA A194V, respectively, were obtained.

Example 4. The effect of mutations prs _BA D58H, prs _BA N120S, prs _BA L135I or prs _BA A194V on the production of inosine and guanosine by the producer strain of inosine and guanosine B. amyloliquefaciens

The resulting plasmids pKSl-PRSBA58, pKSl-PRSBA120, pKSl-PRSBA135 and pKSl-PRSBA194 were transformed into B. subtilis 168 to obtain B. subtilis 168 bearing pKSl-PRSBA58, pKSl-PRSBA120, pKSl-PRSBA135 or pKSl-PRSBA194. Then, the E40 phage grown on each of the obtained strains was used for transduction of plasmids pKSl-PRSBA58, pKSl-PRSBA120, pKSl-PRSBA135 and pKSl-PRSBA194 into the producer strain of inosine and guanosine B. amyloliquefaciens AJ 1991. Thus, B. amyloliquefaciens AJ derivatives were constructed. AJ 1991 bearing pKSl-PRSBA58, pKS1-PRSBA120, pKSl-PRSBA135 or pKSl-PRSBA194.

The integration of mutant genes into the AJ1991 chromosome was carried out as described in Example 2. For the selection of strains containing the prs _BA D58H, prs _BA N120S, prs _BA L135I or prs _BA A194V mutations, the 5'-terminal primer 24 (SEQ ID NO.28) was used. The 3'-end primer 25 (SEQ ID NO.29) and the 3'-end primer 26 (SEQ ID NO.30) to identify the prs _BA D58H mutation; The 5'-end primer 27 (SEQ ID NO.31), the 3'-end primer 28 (SEQ ID NO.32) and the 3'-end primer 29 (SEQ ID NO.33) to identify prs _BA N120S mutation; The 5'-end primer 27 (SEQ ID NO.31), the 3'-end primer 30 (SEQ ID NO.34) and the 3'-end primer 31 (SEQ ID NO.35) to identify the prs _BA L135I mutation; 5'-end primer 27 (SEQ ID NO.31), 3'-end primer 32 (SEQ ID NO.36) and 3'-end primer 33 (SEQ ID NO.37) to identify prs _BA A194V mutation. Integration was also confirmed by sequencing. Thus, the strains AJ1991 prs _BA D58H, AJ1991 prs _BA N120S, AJ1991 prs _BA L135I and AJ1991 prs _BA A194V were constructed.

The strains AJ1991 prs _BA D58H, AJ1991 prs _BA N120S, AJ1991 prs _BA L135I, AJ1991 prs _BA A194V and the control strain AJ1991 were cultured at 34 ° C for 18 hours in L-broth and 0.3 ml of the resulting cultures were inoculated in 3 ml of fermentation medium in 3 ml of fermentation medium 20 × 200 mm tubes and cultured on a rotary shaker at 30 ° C for 72 hours. The results are presented in Table 2.

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

Glucose 80.0 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

table 2 Strain OD ₆₀₀ Inosine, g / l Guanosine, g / l Aj1991 13.5 3.47 ± 0.18 2.05 ± 0.04 AJ1991 prs _BA D58H 14.3 2.65 ± 0.25 1.70 ± 0.03 AJ1991 prs _BA N120S 14.7 3.98 ± 0.09 2.40 ± 0.02 AJ1991 prs _BA L135I 10.0 4.45 ± 0.06 2.73 ± 0.04 AJ1991 prs _BA A194V 13.2 3.97 ± 0.18 2.45 ± 0.21

As can be seen from Table 2, among the mutant prs _BA genes encoding a PRPP synthetase resistant to inhibition by purine nucleotides by feedback, the gene containing the D58H mutation impaired the inosine and guanosine productivity of B. amyloliquefaciens strain, and the genes containing the N120S mutation , L135I or A194V, improved nucleoside productivity. On the other hand, the results show that there is no strict correlation between the resistance of PRPP synthetase to inhibition by purine nucleotides by feedback type and the production of purine nucleosides. If the mutant PRPP synthetase is resistant to feedback inhibition by purine nucleotides, this does not necessarily lead to an increase in the production of purine nucleosides by a bacterium containing such PRPP synthetase. This is an explicit indication of the inventive step of the present invention.

Reference example. Design 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 were added at position 328 m from the start codon of trpC2, was amplified by PCR and introduced into B. subtilis 168 Marburg as follows.

(1) Amplification of the 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 region following it, 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 trpC ⁺ allele sequence by recombinant PCR, two DNA fragments amplified as described above were purified using 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 B. subtilis 168 Marburg strain cells prepared as described by Dubnau and Davidoff-Abelson (Dubnau, D. and R. Davidoff-Abelson, J. Mol. Biol., 56: 209-221 (1971)) were transformed with this DNA fragment 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 His auxotrophic strain of B. subtilis 168 Marburg>

The open reading frame was deleted in the hisC gene encoding histidinolphosphate aminotransferase (ΔhisC: division of 204 nucleotides 403-606) and introduced into the strain B. subtilis 168 Marburg (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 fusions formed by a deletion in an open 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 fusion formed by a deletion in an open reading frame. Primer 41 contains a BamH1 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 AhisC 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 pJPMl of the 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 auxotrophic strain 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, and obtained from protrof, as indicated below.

(1) Introduction ΔpupG (deletions 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 fusion formed by a deletion in an open 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 chromosomal DNA of strain B. 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 region following it, 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 fusion formed by division in an open 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 chromosomal 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 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 (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.

(in) Introduction of ΔpyrC 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 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 cells obtained from 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 (ΔpuC ΔhisC trpC2).

(2) Introduction ΔpurR (deletions in the open reading frame of 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 fusion formed by a deletion in an open 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 chromosomal DNA of strain B. 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 following it, 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 fusion formed by a deletion in an open 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 chromosomal 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 BamHl 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 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 (Δ purR) 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 BamRl site at the 5'-end. Primer 51 contains a fusion formed by a deletion in an open 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 fusion formed by a deletion in an open 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 3 ° end region 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 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 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 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 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 (Δ purA) by double recombinant crossing-over. The resulting double recombinant strain was named KMBS349 (ΔpurA ΔpurR ΔpupG ΔhisC trpC2).

(four). 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 prototroph B. subtilis 168 Marburg>) 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 pur A 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 ΔpurA). The resulting strain was named KMBS350 (ΔpurA ΔpurR ΔpupG).

2. Construction of strain CMV $ 356 ((ΔpurA ΔpurR ΔpupG guaB24)

A guanine auxotrophy mutation while maintaining a residual expression level of guaB24 (the same as guaB (A1) described in US 2006275874 A1) in the guaB gene encoding IMP dehydrogenase was introduced into the recombinant strain KMBS349 (ΔpurA ΔpurR hpgRp B. subtilis 168 Marburg, and a prototroph was prepared from the resulting strain as follows.

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

Competent cells of strain KMBS349 (ΔpurA ΔpurR ΔpurG ΔhisC trpC2) prepared as described above were transformed with the chromosomal DNA of strain KMBS193 (guaB :: kan trpC2; US 2006275874 A1) and colonies were selected that were able to grow on plates with ml L 2.5B medium kanamycin (Kan).

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

(2). Construction of a strain with a mutant (retaining a residual expression level, type "leaky") gene guaB from strain KMBS349

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; competent cells of strain KMBS193 were transformed by PCR guided strain from the B region DNA fragment 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 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 a deletion attenuator sequence (Ppur * -Δatt; same as Ppurl-Δatt described in US 2006275874 A1), the disrupted 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 the strain with destroyed Rrig from strain KMBS337

Competent cells of strain KMBS337 (ΔpurR ΔpurC 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 gel PCR method (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 Δpur :: 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 selectively grown 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 correct replaced by Ppur :: cat. The resulting strain was named KMBS352 (Ppur * -Δatt ΔpurR ΔpupG ΔhisC trpC2).

(3). Introduction of ΔpurA (open reading frame deletion of 369 nucleotides 307 to 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 (ΔpurA ΔpurR ΔpupG)), and colonies capable of growing on a minimal agar medium containing 20 mg / L His and Ade were selected.

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 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

Competent cells of strain KMBS359 (Ppur * -Δatt ΔpurA ΔpurR ΔpupG) prepared as described above were transformed with the chromosomal DNA of strain KMBS351 (guaB :: kan ΔpurA ΔpurR ΔpupG ΔhisC trpC2; see 2. ) 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

The competent cells of the KMBS364 strain (guaB :: kan Ppur * -Δatt ΔpurA ΔpurR ΔpurC2), prepared as described above, transformed the chromosomal DNA of the strain KMBS356 (guaB24 ΔpurA ΔpurR ΔpupG; see 2. (3) Introduction of ираtr3 + C3tB + C3r colonies capable of growing on plates with minimal medium containing 20 mg / l Ade.

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 fusion formed by a deletion in an open 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 chromosomal DNA of strain B. 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 region that follows, 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 fusion formed by a deletion in an open reading frame. Primer 57 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 conducted using the above-described primers and a template of the chromosomal DNA of B. subtilis 168 Marburg strain to amplify fragments containing the 3'-end region and the downstream region of the ΔdeoD.

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 a 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 ΔdeoD was obtained.

(ii) Cloning of the ΔdeoD 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 ΔdeoD gene was correctly integrated; DNA sequencing confirmed the absence of other mutations that could result from PCR; the plasmid was named pKM201.

(iii) Introduction ΔdeoD in strain KMBS365

Competent cells of strain KMBS365, prepared as described above, were transformed with plasmid pKM201 and colonies (recombinants resulting from a single crossover) 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 cells obtained from Cm-sensitive colonies. PCR was performed as described above using primers 54 (SEQ ID NO.58) and 57 (SEQ ID NO.61). Strains were identified in which the deoD gene on the chromosome was replaced by the destroyed deoD gene (Δ deoD) by double recombinant crossing-over. The resulting double recombinant strain was named KMBS375 (ΔdeoD guaB24 Ppur * -Δatt ΔpurA ΔpurR ΔpupG).

Although the invention has been described in detail with reference to the Best Mode for 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 beyond 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 (13)

1. Mutant bacterial phosphoribosyl pyrophosphate synthetase (PRPP synthetase), characterized in that the L-amino acid residue corresponding to position 120, 135 or 194 in the sequence of wild-type PRPP synthetase from Bacillus subtilis is replaced by another L-amino acid residue and the sensitivity of this enzyme to inhibition purine nucleotides as feedback reduced. 2. The mutant PRPP synthetase according to claim 1, characterized in that the asparagine residue corresponding to position 120 in the sequence of wild-type PRPP synthetase from Bacillus subtilis is replaced by a serine residue. 3. The mutant PRPP synthetase according to claim 1, characterized in that the leucine residue corresponding to position 135 in the sequence of wild-type PRPP synthetase from Bacillus subtilis is replaced by an isoleucine residue. 4. The mutant PRPP synthetase according to claim 1, characterized in that the alanine residue corresponding to position 194 in the sequence of wild-type PRPP synthetase from Bacillus subtilis is replaced by a valine residue. 5. The mutant PRPP synthetase according to claim 1, characterized in that the mutant PRPP synthetase is an enzyme from Bacillus subtilis or Bacillus amyloliquefaciens. 6. A DNA fragment encoding the mutant PRPP synthetase according to any one of claims 1 to 5. 7. A bacterium belonging to the genus Bacillus, containing the DNA fragment according to claim 6 and having the ability to produce purine nucleosides. 8. The bacterium according to claim 7, characterized in that said bacterium is the bacterium Bacillus subtilis. 9. The bacterium according to claim 7, characterized in that said bacterium is the bacterium Bacillus amyloliquefaciens. 10. A method for the production of purine nucleoside, comprising cultivating the bacterium according to any one of claims 7 to 9 in a nutrient medium, leading to the secretion of said purine nucleoside into said nutrient medium, and isolation of said purine nucleoside from the culture fluid. 11. The method of production according to claim 10, characterized in that the purine nucleoside is inosine, xanthosine, guanosine or adenosine. 12. A method for producing a purine nucleotide, comprising cultivating a bacterium according to any one of claims 7 to 9 in a nutrient medium, which results in the secretion of said purine nucleoside into said culture medium, isolation of said purine nucleoside from the culture fluid, phosphorylation of said purine nucleoside and isolation of said purine nucleotide . 13. The method for producing a purine nucleotide according to claim 12, wherein the purine nucleotide is 5'-inosinic acid, xanthosine-5'-phosphate, 5'-guanilic acid or 5'-adenyl acid.

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