# Patent

## Title
Method for producing purine nucleosides and nucleotides by fermentation using bacterium belonging to the genus Bacillus or Escherichia

## Abstract

Methods for producing purine nucleosides, and purine nucleotides, such as inosine and 5′-inosinic acid are provided which include using a bacterium belonging to the genus Bacillus or to the genus Escherichia wherein the purine nucleoside productivity of said bacterium is enhanced by increasing an activity of the YdhL protein. Also disclosed is the amino acid sequence of the YdhL protein from Bacillus amyloliquefaciens and the gene encoding it.

## Background
This application is a continuation under 35 U.S.C. §120 of PCT/JP2005/006833, filed Mar. 31, 2005, and claims priority under 35 U.S.C. §119 to Russian Patent Application No. 2004109599, filed Mar. 31, 2004, and Russian Patent Application No. 2005104627, filed Feb. 22, 2005, all of which are incorporated by reference. The Sequence Listing on Compact Disk filed herewith is also hereby incorporated by reference in its entirety (File Name: US-166 Seq List; File Size: 20 KB; Date Created Sep. 29, 2006).


FIELD OF THE INVENTION
The present invention relates to a method for producing purine nucleosides, which are important as a raw material in the syntheses of 5′-inosinic acid and 5′-guanylic acid. The present invention also relates to novel microorganisms used in the production method. The present invention also relates to novel DNA and protein species which confer to a bacterium expressing the novel DNA resistance to purine base analogues and purine nucleosides.
BRIEF DESCRIPTION OF THE RELATED ART
Conventionally, nucleosides such as inosine have been industrially produced via fermentation methods which utilize adenine auxotrophic strains or strains which have been imparted with drug resistance to various drugs, such as purine analogues and sulfaguanidine. Examples of such strains include strains belonging to the genus Bacillus (Japanese Patent Publication 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 Application Laid-Open No. 59-42895 (1984)), or the genus Brevibacterium (Japanese Patent Publication Nos. 51-5075 (1976) and 58-17592 (1972), and Agric. Biol. Chem., 42, 399 (1978)), or the genus Escherichia (WO9903988) and the like.
Acquisition of such mutant strains typically involves subjecting microorganisms to a mutagenesis treatment such as UV irradiation or nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine) treatment, and selecting the desired strain via a suitable selection medium. On the other hand, the breeding of such mutant strains using genetic engineering techniques has also been 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)), or the genus Brevibacterium (Japanese Patent Application Laid-Open No. 63-248394 (1988)), or the genus Escherichia (WO9903988).
The productivity of these inosine-producing strains could be further improved by increasing the inosine-secretion ability. Currently, it is generally accepted that the penetration of metabolites across the cytoplasmic cell membrane is usually mediated by specific efflux transporter proteins (Pao, S. S. et al., Microbiol. Mol. Biol. Rev., 62 (1), 1-34, (1998); Paulsen, I. T. et al., J. Mol. Biol., 277, 573-592 (1998); Saier, M. H. et al., J. Mol. Microbiol. Biotechnol., 257-279 (1999)). The present inventors have previously shown that inosine- or xanthosine-producing strains belonging to the genus Escherichia or Bacillus which have an enhanced activity of the RhtA protein encoded by the rhtA (ybif) gene, an enhanced activity of the YijE protein encoded by the yijE gene, and an enhanced activity of the YdeD protein encoded by the ydeD gene produced more inosine or xanthosine than their respective parental strains (Japanese Patent Application Laid-Open No. 2003-219876A, Russian patent Nos. 2244003C and 2244004C, respectively). Recently, YdhL from B. subtilis was found to lower the sensitivity to the purine analogue 2-fluoroadenine. Besides, indirect evidence indicates that YdhL decreases the amount of the internal pool of hypoxanthine (Johansen et al, J. Bacteriol., 185, 5200-5209, 2003). However, the effect of YdhL overexpression on nucleoside secretion and/or nucleoside production has not been shown.
Moreover, YdhL from Bacillus amyloliquefaciens has never been reported, nor has the effect of YdhL overexpression on nucleoside secretion and/or nucleoside production ever been shown.
SUMMARY OF THE INVENTION
An object of the present invention is to enhance production of purine nucleosides by purine nucleoside-producing strains as well as provide methods for producing inosine and 5′-inosinic acid using these strains.
This object was achieved by identifying the ydhL gene of B. subtilis. This gene encodes a putative membrane protein which confers resistance to purine base analogues, such as 8-azaadenine, 6-methylpurine, 2,6-diaminopurine, 6-mercaptopurine, 8-azaguanine and purine nucleosides, such as inosine and guanosine, when the wild-type allele of the gene placed on a multicopy vector was introduced into an E. coli strain. Also, the previously unknown ydhL gene from Bacillus amyloliquefaciens was isolated and its nucleotide sequence was determined. Furthermore, it was determined that the ydhL gene from Bacillus subtilis or Bacillus amyloliquefaciens can enhance purine nucleoside production when additional copies of the gene are introduced into the cells of the respective inosine-producing strain belonging to the genus Bacillus or genus Escherichia. Thus the present invention has been completed.
Thus, the present invention provides a microorganism belonging to the genus Bacillus or genus Escherichia which has an ability to produce purine nucleosides.
It is an object of the present invention to provide YdhL protein from Bacillus amyloliquefaciens selected from the group consisting of:

    
    
        (A) a protein which comprises the amino acid sequence shown in SEQ ID NO: 2; and
        (B) a protein which comprises an amino acid sequence of SEQ ID NO: 2, and which includes a deletion, substitution, insertion or addition of one or several amino acids, and which has an activity of making a bacterium belonging to the genus Bacillus or to the genus Escherichia resistant to purine base analogues and/or purine nucleosides such as inosine, and guanosine, when said activity of said protein is enhanced in said bacterium.
    
    


It is a further object of the present invention to provide a DNA coding for the protein described above.
It is a further object of the present invention to provide the DNA as described above, wherein said DNA is selected from the group consisting of:

    
    
        (a) a DNA which comprises the nucleotide sequence of SEQ ID No. 1, and
        (b) a DNA which is hybridizable with the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and encodes a protein having an activity of making a bacterium belonging to the genus Bacillus or to the genus Escherichia resistant to purine base analogues and/or purine nucleosides when activity of said protein is enhanced in said bacterium.
    
    


It is a further object of the present invention to provide the DNA as described above, wherein the stringent conditions comprise washing with 1×SSC, 0.1% SDS, at 60° C.
It is a further object of the present invention to provide a bacterium, which belongs to the genus Bacillus or to the genus Escherichia, wherein said bacterium has a purine nucleoside-producing ability, and said bacterium has enhanced activity of a YdhL protein.
It is a further object of the present invention to provide the bacterium as described above, wherein the activity of said YdhL protein has been enhanced by increasing a copy number of a ydhL gene, or by modifying an expression regulatory sequence of the ydhL gene.
It is a further object of the present invention to provide the bacterium as described above, wherein said ydhL gene is selected from the group consisting of:

    
    
        (a) a DNA comprising a nucleotide sequence of SEQ ID NO: 1; and
        (b) a DNA which is able to hybridize under stringent conditions with a nucleotide sequence of SEQ ID NO: 1 or a probe that can be prepared from the nucleotide sequence of SEQ ID NO: 1, and which encodes a protein having an activity of making a bacterium belonging to the genus Bacillus or to the genus Escherichia resistant to purine base analogues and/or purine nucleosides when activity of said protein is enhanced in said bacterium.
    
    


It is a further object of the present invention to provide the bacterium as described above, wherein the stringent conditions comprise washing with 1×SSC, 0.1% SDS, at 60° C.
It is a further object of the present invention to provide the bacterium as described above, wherein said ydhL gene is selected from the group consisting of:

    
    
        (a) a DNA comprising a nucleotide sequence of SEQ ID NO: 3; and
        (b) a DNA hybridizable under stringent conditions with a nucleotide sequence of SEQ ID NO: 3 or a probe that can be prepared from the nucleotide sequence of SEQ ID NO: 3, and which encodes a protein having an activity of making a bacterium belonging to the genus Bacillus or to the genus Escherichia resistant to purine base analogues and/or purine nucleosides when activity of said protein is enhanced in said bacterium.
    
    


It is a further object of the present invention to provide the bacterium as described above, wherein the stringent conditions comprise washing with 1×SSC, 0.1% SDS, at 60° C.
It is a further object of the present invention to provide a bacterium belonging to the genus Bacillus or to the genus Escherichia which bacterium has a purine nucleoside-producing ability, and said bacterium has an enhanced activity of a protein selected from the group consisting of

    
    
        (A) a protein encoded by a ydhL gene from Bacillus amyloliquefaciens, 
        (B) a protein encoded by a ydhL gene from Bacillus subtilis, and
        (C) a protein encoded by an ortholog of either of said ydhL genes in (A) or (B).
    
    


It is a further object of the present invention to provide the bacterium described above, wherein the purine nucleoside is inosine, xanthosine, or guanosine.
It is a further object of the present invention to provide the bacterium described above, wherein the activity of the protein is enhanced by transformation of the bacterium with a DNA coding for the protein, or by alteration of an expression regulatory sequence of said DNA on the chromosome of the bacterium.
It is a further object of the present invention to provide the bacterium described above, wherein the transformation is performed with a low copy number vector.
It is a further object of the present invention to provide a method for producing a purine nucleoside comprising cultivating the bacterium described above in a culture medium, allowing said purine nucleoside to be secreted into said culture medium, and collecting said purine nucleoside from the culture medium
It is a further object of the present invention to provide the method described above, wherein the purine nucleoside is inosine, xanthosine, or guanosine.
It is a further object of the present invention to provide the method as described above, wherein the bacterium has been modified to have an enhanced expression of purine nucleoside biosynthesis genes.
It is a further object of the present invention to provide a method for producing purine nucleotides comprising cultivating the bacterium as described above in a culture medium, phosphorylating the purine nucleoside to generate purine nucleotides, allowing the purine nucleotide to be secreted into said culture medium, and collecting purine nucleotide.
It is a further object of the present invention to provide the method as described above, wherein the purine nucleotide is 5′-inosinic acid, 5′-xanthylic acid, or 5′-guanylic acid.
It is a further object of the present invention to provide the method described above, wherein the bacterium has been modified to have an enhanced expression of purine nucleoside biosynthesis genes.
It is a further object of the present invention to provide a method for producing 5′-guanylic acid comprising cultivating a bacterium as described above in a culture medium, phosphorylating xanthosine to generate 5′-xanthylic acid, aminating 5′-xanthylic acid to generate 5′-guanylic acid, and collecting 5′-guanylic acid.
The present invention is described in detail below.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an alignment of the B. amyloliquefaciens YdhL protein (YDHL_BA; SEQ ID NO: 2) coded by a DNA fragment within the pYDHL4 plasmid, and the B. subtilis YdhL protein (YDHL BS; SEQ ID NO: 3) coded by a DNA fragment within the pYDHL1 plasmid. The symbol “*” means a common amino acid between YDHL_BA and YDHL_BS. The symbol “:” means a similarity of amino acid between YDHL_BA and YDHL_BS.
FIG. 2 shows the structure of the pMWMX1 plasmid.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. The Proteins of the Present Invention
The proteins of the present invention encompass a protein which has the amino acid sequence shown in SEQ ID NO: 2 or 4, and a protein which has an amino acid sequence which includes deletions, substitutions, insertions or additions of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 or 4, and which has an activity of making a bacterium belonging to the genus Bacillus or to the genus Escherichia resistant to purine base analogues, or purine nucleosides such as inosine or guanosine.
The protein which has the amino acid sequence shown in SEQ ID NO: 2 is YdhL protein from Bacillus amyloliquefaciens. The amino acid sequence of the protein and nucleotide sequence of ydhL gene have not been previously disclosed.
The YdhL protein from Bacillus subtilis is not involved in the biosynthetic pathway of purine nucleotides and belongs to the Major Facilitator Superfamily (Pao, S. S., et al, Microbiol. Mol. Biol. Rev. 62 (1): 1-32, (1998)) and to the arabinose efflux permease cluster of the orthologous group (COG) 2814 (Tatusov, R. L. et al, Nucleic Acids Res., 29: 22-28 (2001)). E. coli proteins belonging to this family, AraJ and YdeA, are also involved in arabinose export (Bost, S. et al, J. Bacteriol., 181: 2185-2191 (1999)). The genome of Bacillus subtilis contains the following 5 genes which encode homologs of the ydhL gene: ybcL, yceJ, yfhI, ytbD, and ywfA. The YdhL protein from B. subtilis is a highly hydrophobic protein composed of 388 amino acids, and contains 12 putative transmembrane segments having unknown function. The YdhL protein is encoded by the ydhL gene. The ydhL gene of Bacillus subtilis subsp. subtilis strain 168 (numbers from 624675 to 625838 in GenBank, accession number CAB12399.2; GI:32468705) is located on chromosome between the ydhK and ydhM genes at 53.50°.
The number of “several” amino acids which can be altered in the proteins of the present invention differs depending on the position and/or the type of amino acid residue in the three dimensional structure of the protein. It may be 1 to 40, preferably 1 to 20, and more preferably 1 to 5 for the protein as shown in SEQ ID No. 2 or 4. This is due to the fact that some amino acids are highly homologous to one another, so the three dimensional structure or activity is not affected by such a change. Therefore, the protein which has an amino acid sequence which includes deletions, substitutions, insertions or additions of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 or 4 may be one which has homology of not less than 30 to 50%, preferably 50 to 70%, and most preferably 70 to 90% with respect to the entire amino acid sequence of YdhL protein, and which also retains the activity of the protein.
A protein having a homology of not less than 90%, preferably not less than 95%, more preferably not less than 98%, to the amino acid sequence shown in SEQ ID No. 2 or 4 and which retains the activity of the YdhL protein is encompassed by the present invention and is the most preferred.
To evaluate the degree of homology, several well-known calculation methods, such as BLAST search, FASTA search, and CrustalW, can be used.
BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed by the programs blastp, blastn, blastx, megablast, tblastn, and tblastx; these programs assign significance to their findings using the statistical methods of 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, 87:2264-68 (1990); “Applications and statistics for multiple high-scoring segments in molecular sequences”. Proc. Natl. Acad. Sci. USA, 90:5873-7 (1993)). The FASTA search method is described by W. R. Pearson (“Rapid and Sensitive Sequence Comparison with FASTP and FASTA”, Methods in Enzymology, 183:63-98 (1990)). The ClustalW 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. 22:4673-4680 (1994)).
Changes to the proteins of the present invention such as those described above are typically conservative changes so as to maintain the activity of the protein. Substitution changes include those in which at least one residue in the amino acid sequence has been removed and a different residue is inserted in its place. Examples of amino acids which may be substituted for an original amino acid in the above protein and which are regarded as conservative substitutions include ala substituted with ser or thr; arg substituted with gln, his, or lys; asn substituted with glu, gln, lys, his, asp; asp substituted with asn, glu, or gln; cys substituted with ser or ala; gln substituted with asn, glu, lys, his, asp, or arg; glu substituted with asn, gln, lys, or asp; gly substituted with pro; his substituted with asn, lys, gln, arg, tyr; ile substituted with leu, met, val, phe; leu substituted with ile, met, val, phe; lys substituted with asn, glu, gln, his, arg; met substituted with ile, leu, val, phe; phe substituted with trp, tyr, met, ile, or leu; ser substituted with thr, ala; thr substituted with ser or ala; trp substituted with phe, tyr; tyr substituted with his, phe, or trp; and val substituted with met, ile, leu.
The “activity” in reference to the proteins of the present invention means one which makes a bacterium harboring the protein resistant to purine base analogues, such as 8-azaadenine, 2,6-diaminopurine, 6-mercaptopurine, 6-thioguanine, and/or resistant to purine nucleosides, such as inosine and guanosine.
The phrase “resistant to purine base analogues and/or purine nucleosides” means a characteristic of a bacterium to grow on minimal medium containing purine base analogues, such as 8-azaadenine, 6-methylpurine, 2,6-diaminopurine, 6-mercaptopurine, 8-azaguanine, or purine nucleosides such as inosine or guanosine, in concentration, whereby the wild-type or parental strain cannot grow under similar conditions, or a characteristic of a bacterium to grow on the medium containing the purine base analogues or purine nucleosides faster than the wild-type or parental strain of the bacterium. The above-mentioned concentration of the purine base analogues is 300 μg/ml for 8-azaadenine; 400 μg/ml for 2,6-diaminopurine; 400 μg/ml for 6-mercaptopurine; and for the purine base nucleosides, it is 300 μg/ml, preferably 1000 μg/ml, for inosine, and 10 μg/ml for guanosine.
II. The Bacterium of the Present Invention
The bacterium of the present invention is a bacterium wherein purine nucleoside production by the bacterium is enhanced by increasing an activity of the protein. More specifically, the bacterium of present invention is a bacterium wherein purine nucleoside production by the bacterium is enhanced by increasing an activity of the protein of the present invention in the bacterium. Even more specifically, the bacterium of the present invention harbors the DNA of the ydhL gene or its ortholog on the chromosome or a plasmid in the bacterium. The DNA of the ydhL gene or its ortholog is overexpressed, and as a result, the bacterium has an ability to produce greater amounts of purine nucleosides.
Enhancement of the ydhL gene expression can be confirmed by measuring the amount of RNA which is produced by ydhL gene expression in the bacterium of the present invention by northern hybridization or RT-PCR (Molecular cloning: Cold spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001), and comparing it to that of a wild-type or non-modified strain. The expression of the ydhL gene in the microorganism of the present invention is enhanced more than that of a wild-type or non-modified strain, and preferably not less than 1.5-fold, more preferably not less than 2-fold, and most preferably not less than 3-fold when compared to expression of the ydhL gene in a wild-type or non-modified strain.
The bacterium of the present invention is a bacterium belonging to either the genus Bacillus or Escherichia. An example of the microorganism belonging to the genus Escherichia which can be used in the present invention is Escherichia coli (E. coli). Examples of the microorganism belonging to the genus Bacillus which can be used in the present invention include Bacillus subtilis subsp. subtilis str. 168 (B. subtilis 168) or Bacillus amyloliquefaciens (B. amyloliquefaciens).
Examples of bacteria belonging to genus Bacillus also include the following:
Bacillus lichenifonnis 
Bacillus pumilis 
Bacillus megaterium 
Bacillus brevis 
Bacillus polymixa 
Bacillus stearothermophilus. 
Bacteria belonging to genus of Escherichia reported in Neidhardt et al. (Neidhardt, F. C. et al., Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1), such as Escherichia coli, can be utilized. Examples of wild-type strains of Escherichia coli include, but are not limited to, the K12 strain and derivatives thereof, Escherichia coli MG1655 strain (ATCC No. 47076), and W3110 strain (ATCC No. 27325). These strains are available from the American Type Culture Collection (ATCC, Address: P.O. Box 1549, Manassas, Va. 20108, United States of America).
In addition to the properties already mentioned, the bacteria of the present invention may have other specific properties such as various nutrient requirements, drug resistance, drug sensitivity, and drug dependence without departing from the scope of the present invention.
The phrase “purine nucleoside” as used herein includes inosine, xanthosine, guanosine, and adenosine, preferably inosine.
The phrase “purine-nucleoside producing ability” as used herein means an ability to produce and cause accumulation of a purine nucleoside in a medium. The phrase “having purine-nucleoside producing ability” means that the microorganism belonging to the genus Escherichia or Bacillus is able to produce and cause accumulation of purine nucleosides in a medium in an amount larger than a wild-type strain of E. coli, such as E. coli W3110 and MG1655, or a wild-type strain of B. subtilis such as B. subtilis 168. Preferably this phrase means that the microorganism is able to produce and cause accumulation in a medium in an amount of not less than 10 mg/l, more preferably not less than 50 mg/l of inosine, xanthosine, guano sine or/and adeno sine.
As used herein, when an activity of a protein of the present invention is enhanced in a bacterium, this means that the number of the protein molecules in the bacterium is increased, or that the activity per protein molecule itself is increased.
The term “orthologous gene” or an “ortholog” means a gene from one species which corresponds to a gene in another species that is related via a common ancestral species (a homologous gene), but which has evolved to become different from the gene of the other species. Orthologous genes may or may not have the same function. If complete genomes of organisms are sequenced, the following technique could be used to search for orthologs. A BLAST search is performed using a gene from one organism to find the best homolog in the other organism. Then the second backward BLAST search is performed using the best homolog from the other organism found in the first round of BLAST search to find the best homolog or ortholog in the first organism. If the gene used in the first round and the gene found in the second round coincide, the gene from first organism and the best homolog in the other organism are orthologs. In the process of the BLAST search, the homology of proteins coded by the genes of interest is determined.
For the purpose of the present invention, proteins encoded by orthologous gene are homologous proteins having the same activity. Searching for such orthologs is performed by determining the sequence of genes and proteins, and activity of proteins (see, for example, FIG. 1 and Example 2).
The gene coding for the YdhL protein of Bacillus amyloliquefaciens, the ydhL gene, has been sequenced (SEQ ID NO: 1). The gene coding for YdhL protein from Bacillus subtilis, ydhL gene of Bacillus subtilis subsp. subtilis str. 168, has been elucidated (numbers from 13042 to 14208 in EMBL accession number Z99107) (SEQ ID NO: 3). The ydhL gene from Bacillus subtilis is located on the chromosome between ydhK and ydhM genes at 53.50°. Therefore, said ydhL genes can be obtained by PCR (polymerase chain reaction; refer to White, T. J. et al., Trends Genet., 5, 185 (1989)) utilizing primers prepared based on the nucleotide sequence of the gene. Genes coding for YdhL protein from other microorganisms can be obtained in a similar manner (see Example 2).
The ydhL gene derived from Bacillus amyloliquefaciens can be also exemplified by a DNA which encodes the proteins of the present invention, such as a protein which has the amino acid sequence shown in SEQ ID NO: 2, and/or a protein which has the amino acid sequence of SEQ ID NO: 2, but which includes the deletion, substitution, insertion or addition of one or several amino acids, and which has an activity of making a bacterium belonging to the genus Bacillus or to the genus Escherichia resistant to purine base analogues and/or purine nucleosides such as inosine and guanosine, when the activity of the protein is enhanced in the bacterium. An example of the DNA is SEQ ID NO: 1.
The ydhL gene derived from Bacillus subtilis encompasses a DNA which encodes the proteins of the present invention including a protein which has the amino acid sequence shown in SEQ ID NO: 4, and/or a protein which has the amino acid sequence of SEQ ID NO. 4, but includes the deletion, substitution, insertion or addition of one or several amino acids, and which has an activity of making a bacterium belonging to the genus Bacillus or to the genus Escherichia resistant to purine base analogues and purine nucleosides such as inosine and guanosine, when the activity of the protein is enhanced in the bacterium. An example of the DNA is SEQ ID NO: 3.
The DNAs which encode substantially the same proteins as the YdhL proteins described above may be obtained, for example, by modifying the nucleotide sequence of the DNA encoding the YdhL proteins, for example, by means of site-directed mutagenesis so that one or more amino acid residues at a specified site are deleted, substituted, inserted, or added. DNA modified as described above may be obtained by conventionally known mutation treatments. Such treatments include hydroxylamine treatment of the DNA encoding proteins of present invention, or treatment of the bacterium containing the DNA with UV irradiation or a reagent such as N-methyl-N′-nitro-N-nitrosoguanidine or nitrous acid.
A DNA encoding substantially the same protein as the YdhL protein derived from Bacillus amyloliquefaciens can be obtained by expressing the DNA having mutations as described above in an appropriate cell, and investigating the activity of any expressed product. A DNA encoding substantially the same protein as the YdhL protein can also be obtained by isolating a DNA from mutant DNA encoding the YdhL protein or from a mutant-containing cell, wherein the DNA is hybridizable with a probe having a nucleotide sequence which contains, for example, the nucleotide sequence shown as SEQ ID NO: 1, under stringent conditions, and encodes a protein having the activity of the YdhL protein. The “stringent conditions” referred to herein are conditions under which so-called specific hybrids are formed, and non-specific hybrids are not formed. It is difficult to clearly express this condition by using any numerical value. However, for example, the stringent conditions include conditions under which DNAs which are highly homologous, for example, DNAs having homology of not less than 50%, preferably not less than 70%, more preferably not less than 90%, still more preferably not less than 95% are able to hybridize with each other, but DNAs having homology lower than the above are not able to hybridize with each other. Alternatively, the stringent conditions may be exemplified by conditions under which DNA is able to hybridize at a salt concentration equivalent to typical washing conditions in Southern hybridization, i.e., approximately 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS, at 60° C. Duration of washing depends on the type of membrane used for blotting and, as a rule, is recommended by manufacturer. For example, recommended duration of washing the HybondTM N+nylon membrane (Amersham) under stringent conditions is 15 minutes. Preferably, washing may be performed 2 to 3 times.
A partial sequence of the nucleotide sequence of SEQ ID NO: 1 can also be used as a probe. Probes may be prepared by PCR using primers based on the nucleotide sequence of SEQ ID NO: 1, and a template which contains a DNA having the nucleotide sequence of SEQ ID NO: 1. When a DNA fragment having a length of about 300 bp is used as the probe, the hybridization conditions for washing might be, for example, 50° C., 2×SSC and 0.1% SDS.
All of the above is applicable for the YdhL protein and ydhL gene derived from Bacillus subtilis. 
Techniques for enhancing the activity of the protein of present invention, especially techniques for increasing the number of protein molecules in a bacterial cell, include altering the expression regulatory sequence of a DNA coding for the protein of the present invention, and increasing the copy number of the gene, but are not limited thereto.
Altering the expression regulatory sequence of a DNA coding for the protein of present invention can be achieved by locating the DNA coding for the protein of present invention under the control of a strong promoter. For example, lac promoter, trp promoter, trc promoter, PL promoter of lambda phage are all known strong promoters for E. coli. Especially, veg promoter, spac promoter, xylE promoter are known as strong promoters for Bacillus. Alternatively, a promoter's activity can be enhanced by, for example, introducing a mutation into the promoter to increase the transcription level of a gene located downstream of the promoter (WO00/18935). Furthermore, the mRNA translatability can be enhanced by introducing a mutation into a spacer region between the ribosome binding site (RBS) and the start codon. For example, a 20-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon (Gold et al., Annu. Rev. Microbiol., 35, 365-403, 1981; Hui et al., EMBO J., 3, 623-629, 1984).
Furthermore, to increase the transcription level of the gene, an enhancer may be newly introduced. The introduction of DNA containing either a gene or promoter into chromosomal DNA is described in, for example, Japanese Patent Application Laid-Open No. 1-215280 (1989).
Alternatively, the copy number of the gene may be increased by inserting the gene into a multi-copy vector to form a recombinant DNA, followed by introduction of the recombinant DNA into a microorganism. Examples of vectors autonomously replicable in Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pACYC184, (pHSG and pACYC are available from Takara Bio), RSF1010, pBR322, pMW219 (pMW is available from Nippon Gene), and so forth. Examples of vectors which are autonomously replicable in Bacillus bacteria include pUB10, pC194, pE194. Preferably, low-copy vectors are used. Examples of low-copy vectors include but are not limited to pSC101, pMW118, pMW119, and the like. The term “low-copy vector” is used for vectors which result in a copy number of up to 5 copies per cell. Methods of transformation include any known methods in the art. For example, a method of treating recipient cells with calcium chloride so as to increase permeability of the cells to DNA has been reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)) and may be used in the present invention.
Enhancement of gene expression may also be achieved by introducing multiple copies of the gene into the bacterial chromosome via, for example, homologous recombination, Mu integration, or the like. For example, one act of Mu integration permits introduction into the bacterial chromosome of up to 3 copies of the gene.
The techniques described above of using a strong promoter or enhancer can be combined with the techniques based on increasing the copy number or expression of the gene.
Ordinary methods known in the art for preparation of chromosomal DNA, hybridization, PCR, preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer and the like may be used in the present invention. These methods are described in Sambrook, J., and Russell D., “Molecular Cloning A Laboratory Manual, Third Edition”, Cold Spring Harbor Laboratory Press (2001) and the like.
To breed a microorganism belonging to the genus Bacillus which results in increased expression of the gene or genes coding for the protein or proteins of the present invention, necessary regions of the gene or genes may be obtained by PCR (polymerase chain reaction), primarily based on available information about the B. subtilis genes. For example, ydhL gene, which seems to be a gene coding for a transporter, can be cloned from the chromosomal DNA of B. subtilis strains using PCR. The chromosomal DNA used for this may be derived from any other strain of B. subtilis. 
The protein of present invention includes mutants and variants of the YdhL protein which exist due to natural diversity, and homologous proteins of YdhL which are present in different bacteria, provided these mutant, variant, or homologous proteins demonstrate the functional property of the YdhL protein, that is, imparting resistance to purine base analogues and purine nucleosides such as inosine, or guanosine.
The bacterium of the present invention can be obtained by introducing the aforementioned DNAs coding for the proteins of present invention into a bacterium inherently having an ability to produce purine nucleosides such as inosine. Alternatively, the bacterium of present invention can be obtained by imparting an ability to produce purine nucleosides such as inosine to a bacterium already harboring the DNAs.
An example of the parent strain belonging to the genus Bacillus that may be used in the present invention is the B. subtilis strain KMBS16-1. This strain is a derivative of the inosine-producing strain B. subtilis KMBS16 in which a purA::erm mutation was changed to a purA::cat mutation. In turn, B. subtilis KMBS16 is a derivative of the known B. subtilis trpC2, which has mutations introduced into the purA gene coding for succinyl-AMP synthase (purA::erm), the purR gene coding for a purine repressor (purR::spc), and the deoD gene coding for purine nucleoside phosphorylase (deoD::kan) (US 2004-0166575A). Other parent strains belonging to the genus Bacillus that may 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), 6-ethoxypurine-resistant strain AJ13937 (FERM BP-18665) (US2004-0166575), or the like
An example of an inosine-producing strain belonging to the genus Escherichia is FADRaddedd(pMWKQ). This strain is a derivative of the known strain W3110, which has mutations introduced into the purF gene coding for PRPP amidotransferase, the purR gene coding for a purine repressor, the deoD gene coding for purine nucleoside phosphorylase, the purA gene coding for succinyl-AMP synthase, the add gene coding for adenosine deaminase, the edd gene coding for 6-phosphogluconate dehydrase (WO9903988), and harbors the pMWKQ plasmid, which contains the purFKQ gene coding for the PRPP amidotransferase and is insensitive to GMP (WO9903988).
Moreover, the examples also include releasing regulation of an enzyme involved in inosine biosynthesis, specifically, a method of canceling feedback inhibition of such an enzyme (WO99/03988). Examples of the means for canceling regulation of such an enzyme as mentioned above involved in the inosine biosynthesis include, for example, deletion of the purine repressor (U.S. Pat. No. 6,284,495). Examples of the method of deleting the purine repressor include disrupting a gene coding for the purine repressor (purR, GenBank Accession No. Z99104).
Moreover, the inosine-producing ability can also be increased by blocking a reaction branching off from the inosine biosynthesis pathway, which results in another metabolic product (WO99/03988). Examples of a reaction branching off from the inosine biosynthesis pathway and resulting in another metabolic product include reactions catalyzed by, for example, succinyl-adenosine monophosphate (AMP) synthase, inosine-guanosine kinase, 6-phosphogluconate dehydrase, phosphoglucoisomerase, and so forth. The succinyl-adenosine monophosphate (AMP) synthase is encoded by purA (GenBank Accession No. Z99104).
Furthermore, the inosine-producing ability can also be enhanced by weakening incorporation of inosine into cells. The incorporation of inosine into cells can be weakened by blocking a reaction involved in the incorporation of inosine into cells. Examples of the aforementioned reaction involved in the incorporation of the inosine into cells include a reaction catalyzed by, for example, nucleoside permease.
To increase the activity per protein molecule in the protein of the present invention, it is also possible to introduce a mutation into the structural gene of the protein to enhance the activity of the protein. In order to introduce a mutation into the gene, site-specific mutagenesis (Kramer, W. and Frits, H. J., Methods in Enzymology, 154, 350 (1987)), recombinant PCR (PCR Technology, Stockton Press (1989)), chemical synthesis of a specific portion of the DNA, hydroxylamine treatment of the gene of interest, treatment of microbial strains having the gene of interest by UV irradiation or a chemical agent such as nitrosoguanidine or nitrous acid, and the like can be used. A microorganism in which the activity of the protein is enhanced can be selected as a strain growing in a minimal medium containing 8-azaadenine, 6-methylpurine, 2,6-diaminopurine, 6-mercaptopurine, 8-azaguanine, or inosine in concentrations shown in Table 1 (see below).
The bacterium of the present invention may be further improved by enhancing the expression of one or more genes involved in purine biosynthesis. Such genes include the pur regulon of E. coli (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D.C., 1996). The inosine-producing E. coli strain having a mutant purF gene encoding a PRPP amidotransferase not subject to feedback inhibition by ADP and GMP, and an inactivated purR gene encoding the repressor in the purine nucleotide biosynthesis system has been described. (WO9903988).
III. Method for Producing Purine Nucleosides
The method of the present invention includes a method for producing purine nucleosides including the steps of cultivating the bacterium of the present invention in a culture medium, allowing the purine nucleosides to be produced and secreted by the bacterium, and collecting the purine nucleosides from the culture medium. Furthermore, the method of the present invention includes a method for producing inosine including the steps of cultivating the bacterium of the present invention in a culture medium, allowing inosine to be produced and secreted by the bacterium, and collecting inosine from the culture medium. Furthermore, the method of the present invention includes a method for producing xanthosine including the steps of cultivating the bacterium of the present invention in a culture medium, allowing xanthosine to be produced and secreted by the bacterium, and collecting xanthosine from the culture medium. Furthermore, the method of the present invention includes a method for producing guanosine including the steps of cultivating the bacterium of the present invention in a culture medium, allowing guanosine to be produced and secreted by the bacterium, collecting guanosine from the culture medium.
In the present invention, the cultivation, collection, and purification of purine nucleoside from the medium and the like may be performed in a manner similar to conventional fermentation methods wherein a purine nucleoside is produced using a microorganism. Culture medium for purine nucleoside production may be a typical medium containing a carbon source, a nitrogen source, inorganic ions, and other organic components as required. As the carbon source, saccharides such as glucose, lactose,
sucrose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose and hydrolyzates of starches; alcohols such as glycerol, mannitol and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid and succinic acid and the like can be used.
As the nitrogen source, inorganic ammonium salts such as ammonium sulphate, ammonium chloride, and ammonium phosphate; organic nitrogen such as of soybean hydrolyzates; ammonia gas; aqueous ammonia and the like can be used. It is desirable that vitamins such as vitamin B1, required substances, for example, nucleic acids such as adenine and RNA, or yeast extract and the like are contained in appropriate amounts as trace organic nutrients. Other than these, small amounts of calcium phosphate, magnesium sulphate, iron ions, manganese ions and the like may be added, if necessary.
Cultivation is preferably performed under aerobic conditions for 16 to 72 hours, and the culture temperature during the cultivation is controlled within 30 to 45° C. and the pH between 5 and 8. The pH can be adjusted by using an inorganic or organic acidic or alkaline substance as well as ammonia gas.
After cultivation, solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and then the target purine nucleoside can be recovered from the fermentation liquor by any of a combination of conventional techniques, such as ion exchange resins and precipitation.
4. Method for Producing Purine Nucleotides
The method of the present invention includes a method for producing purine nucleotides including the steps of cultivating the bacterium of the present invention in a culture medium, phosphorylating the desired purine nucleoside to generate a purine nucleotide corresponding to the purine nucleoside, optionally allowing secretion of the purine nucleotide into the culture medium by the bacterium, and collecting the purine nucleotide. Furthermore, the method of the present invention includes a method for producing 5′-inosinic acid including the steps of cultivating the bacterium of the present invention in a culture medium, phosphorylating inosine to generate 5′-inosinic acid, optionally allowing secretion of 5′-inosinic acid into the culture by the bacterium, and collecting 5′-inosinic acid. Furthermore, the method of the present invention includes a method for producing 5′-xanthylic acid including the steps of cultivating the bacterium of the present invention in a culture medium, phosphorylating xanthosine to generate 5′-xanthylic acid, optionally allowing secretion of 5′-xanthylic acid into the culture medium by the bacterium, and collecting 5′-xanthylic acid. Furthermore, the method of the present invention includes a method for producing 5′-guanylic acid including the steps of cultivating the bacterium of the present invention in a culture medium, phosphorylating guanosine to generate 5′-guanylic acid, optionally allowing secretion of 5′-guanylic acid into the culture medium by the bacterium, and collecting 5′-guanylic acid. And the method of the present invention includes a method for producing 5′-guanylic acid including the steps of cultivating the bacterium of the present invention in a culture medium, phosphorylating xanthosine to generate 5′-xanthylic acid, optionally allowing secretion of 5′-xanthylic acid into the culture medium by the bacterium, aminating 5′-xanthylic acid to generate 5′-guanylic acid, and collecting 5′-guanylic acid.
In the present invention, the cultivation, collection, and purification of inosine from the medium and the like may be performed in a manner similar to conventional fermentation methods wherein inosine is produced using a microorganism. Furthermore, in the present invention, the steps of phosphorylating inosine, optionally allowing secretion into the culture medium by the bacterium, and collecting 5′-inosinic acid may be performed in a manner similar to conventional fermentation methods wherein a purine nucleotide such as 5′-inosinic acid is produced from a purine nucleoside such as inosine.
The phosphorylation of the purine nucleoside could be performed enzymatically using different phosphatases, nucleoside kinases, or nucleoside phosphotransferases, or chemically using phosphorylating agents such as POCl3 or the like. A phosphatase which is able to catalyze the C-5′-position selective transfer of a 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 utilizing the poly-phosphoric acid (salts), phenylphosphoric acid (salts) or carbamylphosphoric acid (salts) as a phosphoric acid donor (WO9637603A1) or the like may be used. Also, as an example of phosphatase, the phosphatase which is able to catalyze the transfer of a phosphoryl group to the C-2′, 3′, or 5′-position of nucleosides utilizing the p-nitrophenyl phosphate (Mitsugi, K., et al, Agric. Biol. Chem., 28, 586-600 (1964)), inorganic phosphate (Japanese Patent Application Nos or Japanese Patent Application Laid-Open No. JP42-1186) or acetyl phosphate (Japanese Patent Application Nos or Japanese Patent Application Laid-Open No. JP61-41555) as a substrate, or the like may be used. As an example of nucleoside kinase, guanosine/inosine kinase from E. coli (Mori, H. et. al. Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product. J. Bacteriol. 177:4921-4926 (1995); WO9108286) or the like may be used. As an example of nucleoside phosphotransferase, the nucleoside phosphotransferases 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, New York) or the like may be used. The chemical phosphorylation of nucleosides may be performed using a phosphorylation agent such as POCl3 (Yoshikawa, K. et. al. Studies of phosphorylation. III. Selective phosphorylation of unprotected nucleosides. Bull. Chem. Soc. Jpn. 42:3505-3508 (1969)) or the like.
The amination of the 5′-xanthylic acid can be performed enzymatically using, for example, GMP synthetase from E. coli (Fujio et. al. High level of expression of XMP aminase in Escherichia coli and its application for the industrial production of 5′-guanylic acid. Biosci. Biotech. Biochem. 1997, 61:840-845; EP0251489B1).
EXAMPLES
The present invention will be more concretely explained below with reference to the following non-limiting examples.
Example 1
Cloning of the ydhL Gene of B. Subtilis 
The entire nucleotide sequence of B. subtilis subsp. subtilis strain 168 has been determined (Kunst et al., Nature 390, 249-256 (1997)). Based on the reported nucleotide sequence, primers ydhL_N (SEQ ID No. 5) and ydhL_C (SEQ ID NO: 6) were synthesized and used to amplify the ydhL gene from B. subtilis strain 168 via PCR. The primer ydhL_N is identical to the sequence from 489 to 467 bp, which is upstream of the start codon of the ydhL gene, and having the restriction enzyme SalI recognition site introduced at the 5′-end thereof. The primer ydhL_C is complementary to a sequence from 58 to 80 bp, which is downstream of the termination codon of the ydhL gene, and having the restriction enzyme BglII recognition site introduced at the 5′-end thereof.
The chromosomal DNA of B. subtilis strain 168 was prepared by ordinary methods. PCR was carried out on a “Perkin Elmer GeneAmp PCR System 2400” under the following conditions: 30 sec. at 94° C., 45 sec. at 61° C., 3 min at 72° C., 25 cycles by means of PfuI polymerase (Fermentas). The resulting PCR fragment containing the ydhL gene with its own promoter was treated with BglI and SalI and inserted into the low copy-number vector pMW118, which had been previously treated with the BamHI and SalI enzymes. Thus, the plasmid pYDHL1 was obtained.
The pYDHL1 plasmid and the vector were used to transform the E. coli strain TG1 (Amersham Pharmacia Biotech) via standard techniques. Transformants were selected on L-broth agar plates with 100 μg/ml ampicillin. Thus, the E. coli strains TG1 (pYDHL1) and TG1 (pMW118) were obtained. Then the ability of these strains to grow in the presence of purine base analogues was determined on M9 glucose minimal agar plates containing graded concentrations of an inhibitor. The plates were spotted with 105 to 106 cells from an overnight culture grown in a minimal medium (supplemented with 100 μg/ml of ampicillin). The growth was estimated after 44 h incubation at 37° C.
The results are presented in Table 1.









TABLE 1









Growth on the minimal medium containing

















DAP, 400




DAP,
MP,
AzaA,
μg/ml +




400
400
300
MP,


Strain
-
μg/ml
μg/ml
μg/ml
400 μg/ml





TG1 (pMW118)
+
−
−
−
−


TG1 (pYDHL1)
+
+
+
+
+





Note.


DAP: 2,6-diaminopurine; MP: 6-mercaptopurine; AzaA: 8-azaadenine; +: good growth; −: no growth. Good growth means that the size of colonies which grew on the medium with purine analogues did not differ significantly from the size of colonies of the control strain which grew on the medium without purine analogues.






It can be seen from Table 1 that the ydhL gene amplification increased the resistance of the bacteria to 2,6-diaminopurine, 6-mercaptopurine, and 8-azaadenine.
The pYDHL1 plasmid and the pMW118 vector were also transformed into the E. coli strain TG1 deoD gsk3. The E. coli strain TG1 deoD gsk3 was an especially constructed parental strain which is used for checking the resistance to inosine and guanosine. This strain was obtained by sequential introduction of a deoD mutation, as described in PCT application WO9903988 and gsk3 mutation described by Petersen C. (J. Biol. Chem., 274, 5348-5356, 1999) using phage P1-mediated transduction into the known strain TG1 (VKPM B-5837), which is resistant to inosine and guanosine due to its ability to degrade purine. The strain TG1 deoD gsk3 cannot degrade guanosine and inosine due to a deoD mutation (WO9903988), and it is sensitive to nucleosides due to a gsk3 mutation (Petersen C. J. Biol. Chem., 274, 5348-5356, 1999). Thus, the strains TG1 deoD gsk3 (pYDHL1) and TG1 deoD gsk3 (pMW118) were obtained. Then the ability of these strains to grow in the presence of inosine and guanosine was determined as above.
The results are presented in Table 2.









TABLE 2









Growth on the minimal medium containing










Inosine
Guanosine











Strain
300 mg/l
1000 mg/l
10 mg/l
15 mg/l





E. coli deoD gsk3
−
−
−
−


(pMW118)


E. coli deoD gsk3
+
+
+
−


(pYDHLl)





Note.


+: good growth; −: no growth.






It follows from the Table 2 that ydhL gene overexpression conferred a high-level resistance to inosine and a low-level resistance to guanosine. Therefore, it can be predicted that the ydhL gene is involved in the secretion of purine nucleosides, and more specifically, in the secretion of inosine.
Example 2
Cloning of the ydhL Gene of Bacillus amyloliquefaciens 
The data presented in Example 1 show that overexpression of the ydhL gene of B. subtilis (hereafter: ydhLBs) conferred upon cells resistance to 2,6-diaminopurine and 6-mercaptopurine when introduced simultaneously into M9 glucose minimal agar (Table 1). On the basis of this fact, the cloning of an ortholog of the ydhLBs gene from B. amyloliquefaciens (hereafter: ydhLBa) was performed. The chromosomal DNA of B. amyloliquefaciens strain BA1(IAM1523) was prepared by ordinary methods. The chromosomal DNA was cut with EcoRI and ligated with the pMW118 vector which had been previously treated with the same enzyme. The resulting DNA was used to transform E. coli strain TG1. Transformants were selected on minimal agar containing 400 μg/ml 2,6-diaminopurine, 400 μg/ml 6-mercaptopurine, and 100 μg/ml ampicillin. Plasmid DNA was isolated from the transformants and analyzed. From these, the hybrid plasmid pYDHL2 containing a minimal inserted DNA fragment of about 1.6 kb was found. In contrast to the ydhLBs gene it contained no EcoRI site. The inserted fragment was sequenced from the universal primers, and was found to have high homology (more then 90%) with the ydhLBs gene (FIG. 1). The new gene from B. amyloliquefaciens was named ydhL gene (hereinafter, also labelled as ydhLBa gene). When introduced into the TG1 and TG1 deoD gsk3 strains, the pYDHL2 plasmid conferred upon cells resistance to purine base analogues and nucleosides, respectively, in the same way as the pYDHL1 plasmid did. It was concluded that the pYDHL2 plasmid indeed contains the ydhLBa gene.
Example 3
Effect of Amplification of the ydhLBs and ydhLBa Genes on Inosine Production by the E. coli Inosine-Producing Strain
The inosine-producing E. coli strain FADRaddedd (pMWKQ) was transformed with each of the pMW18 vector, the YDHL1 plasmid, and the pYDHL2 plasmid. The transformants were selected on L-agar containing 100 mg/l ampicillin and 75 mg/l kanamycin. Thus, the strain FADRaddedd (pMWKQ, pMW18), FADRaddedd (pMWKQ, pYDHL1), and FADRaddedd (pMWKQ, pYDHL2) were obtained. These strains were each cultivated at 37° C. for 18 hours in L-broth with 100 mg/l ampicillin and 75 mg/l kanamycin, and 0.3 ml of the obtained culture was inoculated into 3 ml of a fermentation medium containing 100 mg/l ampicillin and 75 mg/l kanamycin, in a 20×200 mm test tube, and cultivated at 37° C. for 72 hours with a rotary shaker.
The composition of the fermentation medium (g/l):
















Glucose
40.0



(NH4)2SO4
16.0



K2HPO4
0.1



MgSO4•7H2O
1.0



FeSO4•7H2O
0.01



MnSO4•5H2O
0.01



Yeast extract
8.0



CaCO3
30.0










Glucose and magnesium sulphate are sterilized separately. CaCO3 is dry-heat sterilized at 180° C. for 2 hours. pH is adjusted to 7.0. Antibiotic is introduced into the medium after sterilization.
After the cultivation, the amount of inosine which accumulated in the medium was determined by HPLC. A sample of the culture medium (500 μl) is centrifuged at 15,000 rpm for 5 minutes, and the supernatant is diluted 4 times with H2O and analyzed by HPLC.
Conditions for HPLC Analysis:
Column: Luna C18(2) 250×3 mm, 5u (Phenomenex, USA). Buffer: 2% v/v C2H5OH; 0.8% v/v thriethylamine; 0.5% v/v acetic acid (glacial); pH 4.5. Temperature: 30° C. Flow rate: 0.3 ml/min. Injection volume: 5 μl. Detection: UV 250 nm.
Retention time (min):
















Xanthosine
13.7



Inosine
9.6



Hypoxanthine
5.2



Guanosine
11.4



Adenosine
28.2










The results are presented in Table 3.









TABLE 3





Strain
OD540
Inosine, g/l







E. coli FADRaddedd (pMWKQ, pMW118)
9.0
1.5 ± 0.2


E. coli FADRaddedd (pMWKQ, pYDHL1)
8.6
2.3 ± 0.2


E. coli FADRaddedd (pMWKQ, pYDHL2)
8.4
2.2 ± 0.2









As it is seen from the Table 3, the overexpression of ydhLBs and ydhLBa genes improved inosine productivity of the FADRaddedd (pMWKQAp) strain.
Example 4
Cloning the ydhLBs gene into a low-copy number shuttle vector pMWMX1
The PCR fragment containing the ydhLBs gene under the control of its own promoter (obtained in Example 1) was treated with BglI and SalI and inserted into low-copy number shuttle vector pMWMX1, which had been previously treated with the BamHI and SalI enzymes. Thus, the plasmid pYDHL3 was obtained. The pMWMX1 vector (FIG. 2) is a derivative of two low-copy-number plasmids: pMW118 and pMX30. The pMX30 plasmid (Rabinovich et al., Mol. Biol. (Moscow), 18, 189-196, 1984) in turn, is a deletion derivative of pSM19035, a broad-host-range plasmid of the inc18 family (Brantl et al., Nucleic Acids Res., 18, 4783-4790). The pMW118 and pMX30 plasmids were each cut with EcoRI and ligated. Thus the vector pMWMX1 was obtained.
Example 5
Construction of the Low-Copy Number Shuttle Plasmid pYDHL3 Containing the ydhLBa Gene
The pYDHL2 plasmid was partially cut with EcoRI to obtain a single fragment, and ligated with pMX30 which had been cut with the same enzyme. Thus, a shuttle plasmid pYDHL4 containing the ydhLBa gene was obtained. Therefore, the pYDHL4 plasmid is equivalent to pYDHL3 plasmid, except for the replacement of the pMWMX1 vector containing the ydhLBs gene with the ydhLBa gene.
Example 6
Effect of the Amplification of ydhLBs or ydhLBa Genes on Resistance of the B. subtilis Strain 168 to Purine Base Analogues
The pYDHL3, pYDHL4 plasmids and the pMWMX1 vector were used to transform wild-type B. subtilis strain 168 via standard techniques. Transformants were selected on L-broth agar plates with 10 μg/ml erythromycin. Thus, the strains B. subtilis 168(pMWMX1), B. subtilis 168(pYDHL3) and B. subtilis 168(pYDHL4) were obtained. Then, the ability of these strains to grow in the presence of purine base analogues was determined on M9 glucose minimal agar plates (minimal medium M9, glucose-2%, vitamin assay casamino acids (Difco)—0.1%, tryptophan—100 μg/ml) containing graded concentrations of a purine analogue. The plates were spotted with 105 to 106 cells from an overnight culture grown on the above agar plates without the analogues. The growth was estimated after 48 h incubation at 34° C.
The results are presented in Table 4.









TABLE 4









Growth on the minimal medium containing














AzaG,
AzaG,
MePur,
AzaA,


Strain
-
2 μg/ml
10 μg/ml
20 μg/ml
2 mg/ml





B. subtilis 168
+
−
−
−
−


B. subtilis 168(pYDHL3)
+
+
n.d.
+
n.d.


B. subtilis 168(pYDHL4)
+
+
+
+
+





Note:


AzaG—8-azaguanine; MePur—6-methylpurine; AzaA—8-azaadenine; n.d.—no data; +: good growth; −: no growth. Good growth means that size of colonies grown on the medium with purine analogues did not differ significantly from size of colonies of the control strain grown on the medium without purine analogues.






It can be seen from Table 4 that the amplification of the ydhLBs or ydhLBa genes increased resistance of the bacteria to 8-azaguanine, 6-methylpurine, and 8-azaadenine.
Example 7
Effect of Amplification of the ydhLBs or ydhLBa Genes on Inosine Production by the B. subtilis Inosine-Producing Strain.
The inosine-producing B. subtilis strain KMBS16-1 was transformed with the pMWMX1 vector, pYDHL3, or pYDHL4 plasmids. Thus, the strains B. subtilis KMBS16-1(pMWMX1), B. subtilis KMBS16-1(pYDHL3), and B. subtilis KMBS16-1(pYDHL4) were obtained. These strains were each cultivated at 37° C. for 18 hours in L-broth with 10 mg/l erythromycin. 0.3 ml of the resulting culture was inoculated into 3 ml of a Bacillus fermentation medium containing 10 mg/l erythromycin, in a 20×200 mm test tube, and cultivated at 37° C. for 72 hours with a rotary shaker.
The composition of the Bacillus fermentation medium: (g/l)
















Glucose
80.0



KH2PO4
1.0



MgSO4
0.4



FeSO4•7H2O
0.01



MnSO4•5H2O
0.01



Mameno
1.35 of TN (total nitrogen)



(soybean hydrolysate)



DL-Methionine
0.3



NH4Cl
32.0



Adenine
0.1



Tryptophan
0.02



CaCO3
50.0










After the cultivation, the amount of inosine which had accumulated in the medium was determined by HPLC as above. The results are presented in Table 5.











TABLE 5







Strain
OD540
Inosine, g/l









B. subtilis KMBS16-1 (pMWMX1)
8.4
1.4 ± 0.1



B. subtilis KMBS16-1 (pYDHL3)
8.2
 1.6 ± 0.2.



B. subtilis KMBS16-1 (pYDHL4)
8.0
1.7 ± 0.2










As it is seen from the Table 5, the amplification of either of the ydhLBs or ydhLBa genes improved inosine productivity of the B. subtilis strain KMBS16-1.
Example 8
Modification of the Regulatory Region of the B. amyloliquefaciens ydhL Gene
It is known that modification of the B. subtilis ydhL regulatory region, namely modification of the G-box of 5′-untranslated RNA region, may increase expression of the gene (Mandal, M. and Breaker, R. R., Nat. Struct. Mol. Biol., 11 (1), 29-35 (2004)). Therefore, to enhance the ydhL gene expression level the same mutation (substitution of T by C) was introduced into the regulatory region of the ydhL gene from B. amyloliquefaciens (position “−77” from start point of the gene) using QuickChangeTM site-directed mutagenesis (Stratagene). For that purpose, two primers 1 and 2 (SEQ ID NO: 7 and 8, respectively) were designed based on the DNA sequence of the regulatory region of the ydhL gene from B. amyloliquefaciens. The DNA plasmid pYDHL2 (pMW118-ydhLBA) was used as a template. The whole plasmid was amplified with the aforementioned primers and several plasmids containing the ydhL gene were obtained. These plasmids were sequenced using the primer 3 (SEQ ID NO: 9) and the presence of the desired T to C substitution was confirmed. The new pMW118-derivative plasmid containing the ydhL gene from B. amyloliquefaciens with the mutation in the regulatory region was marked as pYDHL5. The pYDHL5 plasmid linearized by cutting with EcoRI restrictase and ligated with pMX30 previously linearized with the same restrictase. Thus, a shuttle plasmid pYDHL6 containing the ydhLBa gene having the mutation enhancing its expression level was obtained.
Example 9
Effect of Enhanced Expression of ydhLBa Gene on Inosine Production by the B. amyloliquefaciens Inosine-Producing Strain.
The pYDHL6 plasmid and the pMWMX1 shuttle vector each were transformed into B. subtilis 168 strain by a well-known method (Spizizen, J. Proc. Natl. Acad. Sci. USA, 44, 1072-1078 (1958)). The resulting strains, B. subtilis (pYDHL6) and B. subtilis (pMWMX1), were used as donors in phage E40-mediated transduction to introduce pYDHL6 and pMWMX1 plasmids into the strain B. subtilis G1136A (AJ1991, VKPM B-8994, ATCC19222) producing inosine and guanosine (U.S. Pat. No. 3,575,809). The strain B. subtilis G1136A has been re-identified by All-Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 113545 Moscow, 1st Dorozhny proezd, 1) as Bacillus amyloliquefaciens and deposited under accession number (VKPM B-8994). Therefore, the strain thereafter will be designated as Bacillus amyloliquefaciens G1136A. Phage E40 is a PBS 1-like phage (Takanishi, R., J. Gen. Microbiol., 31, 211-217, (1963)) capable of propagating on B. subtilis and B. amyloliquefaciens cells. After the transduction the strains B. amyloliquefaciens G1136A (pMWMX1) and B. amyloliquefaciens G1136A (pYDHL6) were obtained.
These strains were each cultivated at 37° C. for 18 hours in L-broth with 10 mg/l erythromycin. Then 0.3 ml of the resulting culture was inoculated into 3 ml of a Bacillus fermentation medium containing 10 mg/l erythromycin, in a 20×200 mm test tube, and cultivated at 37° C. for 72 hours with a rotary shaker.
The composition of the fermentation medium: (g/l)
















Glucose
80.0



KH2PO4
1.0



MgSO4
0.4



FeSO4•7H2O
0.01



MnSO4•5H2O
0.01



NH4Cl
15.0



Adenine
0.3



Total nitrogen (in the form of Mameno)
0.8



CaCO3
25.0










After the cultivation, the amount of inosine and guanosine that accumulated in the medium was determined by HPLC as above. The results are presented in Table 6.









TABLE 6





Strain
OD540
Inosine, g/l







B. amyloliquefaciens G1136A (pMWMX1)
16.7
1.8 ± 0.1


B. amyloliquefaciens G1136A (pYDHL6)
16.1
3.2 ± 0.1









As it is seen from the Table 6, the enhanced expression of the ydhLBa gene due to the regulatory mutation improved inosine productivity of the B. amyloliquefaciens strain G1136A.
While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. Each of the aforementioned documents is incorporated by reference herein in its entirety.

## Claims

1. A method for producing a purine nucleotide comprising:
(a) cultivating a bacterium belonging to the genus Bacillus in a culture medium to produce purine nucleoside,
(b) phosphorylating the purine nucleoside to generate a purine nucleotide, and
(c) collecting said purine nucleotide, wherein said bacterium has a purine nucleoside-producing ability and has been modified to have enhanced activity of a YdhL protein by a method selected from the group consisting of:
(A) transforming the bacterium with a multi-copy vector containing a ydhL gene and/or introducing multiple copies of the ydhL gene into chromosome of the bacterium,
(B) placing the ydhL gene under the control of a promoter capable of enhancing the expression of the ydhL gene, and
(C) combinations thereof.

2. The method according to claim 1, wherein said purine nucleotide is 5′-inosinic acid.

3. The method according to claim 1, wherein said purine nucleotide is 5′-xanthylic acid.

4. The method according to claim 1, wherein said purine nucleotide is 5′-guanylic acid.

5. The method according to claim 1, wherein said bacterium has been further modified to have enhanced expression of a purine nucleoside biosynthesis gene by a method selected from the group consisting of:
(i) transforming of the bacterium with a multi-copy vector containing the purine nucleoside biosynthesis gene and/or introducing multiple copies of the gene into chromosome of the bacterium,
(ii) placing the purine nucleoside biosynthesis gene under the control of a promoter capable of enhancing the expression of the purine nucleoside biosynthesis gene, and
(iii) combinations thereof.

6. A method for producing 5′-guanylic acid comprising
(a) cultivating a bacterium belonging to the genus Bacillus in a culture medium to produce xanthosine,
(b) phosphorylating xanthosine to generate 5′-xanthylic acid,
(c) aminating 5′-xanthylic acid to generate 5′-guanylic acid, and
(d) collecting 5′-guanylic acid,

wherein said bacterium has a purine nucleoside-producing ability and has been modified to have enhanced activity of a YdhL protein by a method selected from the group consisting of:
(A) transforming the bacterium with a multi-copy vector containing a ydhL gene and/or introducing multiple copies of the ydhL gene into chromosome of the bacterium,
(B) placing the ydhL gene under the control of a promoter capable of enhancing the expression of the ydhL gene, and
(C) combinations thereof.