JP2007075109A6 - Purine nucleoside producing bacteria and method for producing purine nucleoside - Google Patents

Abstract

A production method for improving the efficiency of fermentation production of purine-based substances such as purine nucleosides and purine nucleotides, and a microorganism used for the production.
A bacterium belonging to the genus Bacillus which is modified by a mutation treatment or the like so as to be resistant under high osmotic pressure and has purine nucleoside-producing ability. A method for producing a purine nucleoside comprising culturing the bacterium and recovering purine nucleosides such as inosine, xanthosine, and guanosine from the bacterium or medium.
[Selection figure] None

Description

  The present invention relates to a process for producing purine nucleosides such as inosine, xanthosine, and guanosine, which are important as raw materials for the synthesis of 5'-inosinic acid, 5'-xanthylic acid, 5'-guanylic acid, and the production thereof. It relates to the novel microorganism used.

    Purine nucleosides such as inosine, guanosine, and xanthosine have adenine auxotrophy or genus Bacillus with drug resistance to various drugs such as purine analogs, sulfa drugs, methionine analogs, antifolates, and polymyxins ( It has been industrially produced by fermentation methods using Patent Documents 1 to 11, Non-Patent Document 1), or Escherichia (Patent Document 12) and other microorganisms.

The fermentation medium for nucleoside production contains high concentrations of glucose, sodium phosphate, and MgSO ₄ , and has an extremely high osmotic pressure compared to the natural world. In addition, a significant amount of purine nucleoside is accumulated in the medium during fermentation production. Accumulated nucleosides further increase the osmotic strength (osmotic pressure) of the medium. High osmotic pressure can negatively affect overall cellular activity and inhibit nucleoside biosynthesis.

  In response to proliferation at high osmotic pressure, cells are known to accumulate osmoprotectants, ie, osmolytes that are highly compatible with cell physiology (Non-Patent Document 2) Patent Document 3). In conjunction with its contribution to osmotic pressure balance, osmotic protectants are thought to be useful as stabilizers for enzymes and cellular components against the harmful effects of high ionic strength. Among the osmoprotective agents, the cyclic amino acid L-proline is thought to play a very important role in the bacterial adaptive response to high osmotic pressure, and a large amount of L-proline responds to proliferation under high osmotic pressure. However, it has been reported to be accumulated by cells through de novo synthesis. (Non-patent documents 2 to 4). Furthermore, Bacillus species are thought to involve many enzymes in proline biosynthesis, and there are reports that some of the genes encoding these enzymes are induced by high concentrations of salts. (Non-patent document 5). Furthermore, the high osmotic pressure induces the uptake of L-proline and enables cell growth under osmotic conditions where proliferation is inhibited by intracellular proline accumulation (Non-Patent Documents 3 to 6).

However, there are currently no reports of purine nucleosides such as inosine, adenosine, xanthosine, and guanosine using bacteria belonging to the genus Bacillus that are resistant to hyperosmolarity.
Japanese Examined Patent Publication No. 38-23039 Japanese Examined Patent Publication No. 54-17033 Japanese Patent Publication No.55-2956 Japanese Patent Publication No. 55-45199 JP 56-162998 A Japanese Patent Publication No.57-14160 Japanese Patent Publication No.57-41915 JP 59-42895 JP 2003-259861 A Japanese Patent Publication No.51-5075 Japanese Patent Publication No.58-17592 International Publication No. 9903988 Pamphlet Agric. Biol. Chem. 1978, 42, p. 399 Csonka, Microbiol. Rev., 1989, Vol. 53, p. 121-147; Kempf & Bremer, Arch. Microbiol. 1998, 170, p. 319-30 By Galinski & Truper, FEMS Microbiol. Rev., 1994, Vol. 29, p. 95-108 Yancey, PH, “Cellular and Molecular Physiology of Cell Volume Regulation” Edited by Strange, K., CRC Press, Boca Raton, USA, 1994, p. 81-109,

  An object of the present invention is to create a suitable Bacillus bacterium for producing a purine nucleoside by a fermentation method, and to provide a method for producing a purine nucleoside and a purine nucleotide using the bacterium.

In order to solve the above problems, the present inventor has conducted earnest research. As a result, the present inventors have found that a microorganism belonging to the genus Bacillus and imparting resistance to high osmotic pressure produces and accumulates many purine nucleosides in the medium, and has completed the present invention.
That is, the present invention is as follows.
(1) A Bacillus bacterium having resistance under high osmotic pressure and having the ability to produce purine nucleosides.
(2) The bacterium according to (1), which is a mutant derived from a parent strain belonging to the genus Bacillus and has a better growth than the parent strain when cultured in a medium containing a substance that increases osmotic pressure.
(3) The bacterium according to (1) or (2), wherein the substance that increases the osmotic pressure is sodium chloride.
(4) The bacterium according to (1) to (3), wherein the medium contains 2.0 M sodium chloride.
(5) The bacterium according to (1) to (4), wherein the bacterium belonging to the genus Bacillus is a bacterium selected from Bacillus subtilis or Bacillus amyloliquefaciens.
(6) The bacterium according to (1) to (5), wherein the purine nucleoside is selected from the group consisting of inosine, xanthosine, adenosine, and guanosine.
(7) The Bacillus bacterium described in (1) to (6) is cultured in a medium, and purine nucleosides are accumulated in the bacteria or the medium, and the purine nucleosides are recovered from the cells or the medium. A method for producing purine nucleosides.
(8) The method according to (7), wherein the purine nucleoside is selected from the group consisting of inosine, xanthosine, adenosine, and guanosine.
(9) A purine nucleoside is produced by the method according to (7) or (8), and a phosphoric acid donor selected from the group consisting of polyphosphoric acid, phenylphosphoric acid, and carbamylphosphoric acid, and nucleoside-5 '-A method for producing a purine nucleotide, characterized in that a purine nucleotide is produced by the action of a microorganism having the ability to produce a phosphate ester or acid phosphatase to produce the purine nucleotide.
(10) The method according to (9), wherein the purine nucleotide is selected from the group consisting of 5'-inosinic acid, 5'-xanthylic acid, 5'-guanylic acid, 5'-adenylic acid.

By using the microorganism of the present invention, purine nucleosides and purine nucleotides can be efficiently produced by fermentation.

(1) Bacillus bacteria of the present invention
The bacterium of the present invention is a bacterium belonging to the genus Bacillus that has been modified to have resistance under high osmotic pressure and has the ability to produce purine nucleosides.
In the present invention, “purine nucleoside-producing ability” refers to the ability to produce, secrete, and accumulate intracellularly or in a medium to the extent that purine nucleoside can be recovered from the cell or medium when the Bacillus bacterium of the present invention is cultured in the medium Say. Usually means the ability to accumulate purine nucleosides in the medium that do not fall below 50 mg / l, preferably not below 0.5 g / l.

  In the present invention, “purine nucleoside” includes inosine, xanthosine, adenosine, and guanosine. In addition, the Bacillus bacterium of the present invention may have two or more kinds of production ability among the purine nucleosides.

  In the present invention, “bacteria belonging to the genus Bacillus” means that the bacterium is classified as a genus Bacillus according to the classification method. Representative examples of bacteria belonging to the genus Bacillus used in the present invention are, but not limited to, Bacillus subtilis and B. amyloliquefaciens.

  Specific examples of bacteria belonging to the genus Bacillus include Bacillus subtilis 168 Marburg strain (ATCC6051), Bacillus subtilis PY79 (Plasmid, 1984, Vol. 12, p. 1-9), and Bacillus amyloliquefacii. Examples of ence include, but are not limited to, Bacillus amyloliquefaciens T (ATCC 23842), Bacillus amyloliquefaciens N (ATCC 23845) and the like. These strains can be obtained from the American Type Culture Collection address P.O BOX1549 Manassas VA 20108 USA).

Bacteria belonging to the genus Bacillus having the ability to produce purine nucleosides can be obtained, for example, by imparting resistance to drugs such as purine nucleoside requirement or further purine analogues to the Bacillus bacterium as described above.
For example, as strains obtained by such a method, Bacillus subtilis strain AJ12707 (FERM P-12951) (Japanese Patent Laid-Open No. 61-13876), Bacillus subtilis strain AJ3772 (FERM-P 2555) (Japanese Patent Laid-Open No. 62-55). 014794), Bacillus pumilus strain NA-1102 (FERM BP-289), Bacillus subtilis NA-6011 (FERM BP-291), Bacillus amyloliquefaciens ("Bacillus subtilis") G1136A (ATCC No. 19222) (US Pat. No. 3,575,809), Bacillus subtilis NA-6012 (FERM BP-292) (US Pat. No. 4,701,413), Bacillus. B. pumilis Gottheil No. 3218 (ATCC No. 21005) (US Pat. No. 3,616,206) Bacillus. Amiloliquefaciens strain AS115-7 (VKPM B-6134) (Russian Patent No. 20030378) and the like.

   Purine nucleoside-producing ability can also be imparted by increasing the intracellular activity of an enzyme involved in purine nucleoside biosynthesis. (US Patent Application 2004-0166575). “Increasing intracellular activity” means increasing the activity to a higher level than an unmodified Bacillus bacterium, such as a wild-type Bacillus bacterium. Examples include, but are not limited to, increasing the number of enzyme molecules per cell, increasing specific activity per enzyme molecule, and the like.

  Examples of enzymes involved in purine nucleoside biosynthesis include phosphoribosyl pyrophosphate (PRPP) amide transferase, phosphoribosyl pyrophosphate (PRPP) synthetase, and adenosine deaminase.

  Moreover, the intracellular activity of the enzyme involved in purine nucleoside biosynthesis can also be increased by increasing the expression level of the purine operon. Specifically, the expression level of the purine operon is increased and the purine nucleoside-producing ability can be improved by a method of releasing the regulation of feedback inhibition of an enzyme involved in inosine biosynthesis. (WO99 / 03988). Examples of the deregulation of the enzyme involved in purine nucleoside biosynthesis include deletion of purine repressor. (US Pat. No. 6,284,495). An example of a method for deleting the purine repressor includes disruption of the gene encoding purine repressor (purR SEQ ID NO: 5, GenBank accession number Z99104). (US Pat. No. 6,284,495)

  Furthermore, purine nucleoside-producing ability can be imparted by blocking the biosynthetic system branched from inosine biosynthesis, thereby reducing the production of metabolites (by-products) other than inosine (WO99 / 03988). Examples of reactions that branch from purine nucleoside biosynthesis and result in another metabolite are, for example, succinyl adenosine monophosphate (AMP) synthase (purA SEQ ID NO: 5), inosine-guanosine kinase, 6-phosphogluconic acid Examples include dehydrogenase and phosphoglucoisomerase. Succinyl-adenosine monophosphate (AMP) synthase is encoded by purA (GenBank accession number Z99104 SEQ ID NO: 5).

  Furthermore, purine nucleoside producing ability can also be increased by reducing or eliminating purine nucleoside degradation activity (WO 99/03998). The method for reducing or eliminating the degradation activity of purine nucleoside includes a method for destroying a gene encoding purine nucleoside phosphorylase (deoD SEQ ID NO: 3). (JP 2004-242610)

Reduction of the target enzyme activity can be achieved by the following method.
In order to decrease the activity of the target enzyme in the cells of the genus Bacillus, for example, the bacterium belonging to the genus Bacillus is irradiated with ultraviolet light, N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or EMS (ethyl methanesulfonate). And the like, and a method of selecting a mutant strain having a decreased activity of the target enzyme. Moreover, in addition to mutation treatment, Bacillus bacteria whose target enzyme activity has decreased include, for example, homologous recombination using genetic recombination (Experiments in Molecular Genetics, Cold Spring Harbor Laboratory press (1972); Matsuyama, According to S. and Mizushima, S., J. Bacteriol., 162, 1196 (1985), a gene encoding a target enzyme on a chromosome may be referred to as a gene that does not function normally (hereinafter referred to as a “destructive gene”). ).

In order to replace a disrupted gene with a normal gene on a host chromosome, for example, the following may be performed. In the following example, the purR gene will be described as an example, but other genes such as purA or deoD can be similarly disrupted.
In homologous recombination, when a plasmid or the like having a sequence homologous to a sequence on a chromosome is introduced into a microbial cell, recombination occurs at a certain frequency at a sequence having homology, and the entire introduced plasmid is Integrated on the chromosome. After this, when recombination occurs at the position of the homologous sequence on the chromosome, the plasmid falls off from the chromosome again, but at this time the gene destroyed by the position where recombination occurs is fixed on the chromosome, The original normal gene may fall off the chromosome along with the plasmid. By selecting such a strain, a strain in which the disrupted purR gene is replaced with a normal purR gene on the chromosome can be obtained.

  Such a gene disruption technique by homologous recombination has already been established, and a method using linear DNA, a method using a temperature-sensitive plasmid, and the like can be used. The purR gene can also be disrupted by using a plasmid containing a purR gene into which a marker gene such as drug resistance is inserted and which cannot be replicated in the target microorganism cell. That is, a transformant that has been transformed with the plasmid and has acquired drug resistance has a marker gene incorporated in the chromosomal DNA. Since this marker gene is highly likely to be incorporated by homologous recombination between the purR gene sequence at both ends and the purR gene on the chromosome, a gene-disrupted strain can be efficiently selected.

Specifically, a disrupted purR gene used for gene disruption specifically includes deletion of a certain region of the purR gene by restriction enzyme digestion and recombination, insertion of another DNA fragment (such as a marker gene) into the purR gene, or site-specificity. Mutation method (Kramer, W. and Frits, HJ,
Methods in Enzymology, 154, 350 (1987)) and treatment with chemical agents such as sodium hyposulfite and hydroxylamine (Shortle, D. and Nathans, D., Proc. Natl. Acad. Sci. USA, 75, 270 (1978) )) To cause substitution, deletion, insertion, addition or inversion of one or more bases in the base sequence of the purR gene coding region or promoter region, etc. It can be obtained by reducing or eliminating the activity or reducing or eliminating the transcription of the purR gene. Among these embodiments, a method of deleting a certain region of the purR gene by restriction enzyme digestion and recombination, or a method of inserting another DNA fragment into the purR gene is preferable from the viewpoint of certainty and stability.

  The purR gene can be obtained from a chromosomal DNA of a microorganism having a purine operon by a PCR method using an oligonucleotide prepared based on a known base sequence of the purR gene as a primer. The purR gene can be obtained from a chromosomal DNA library of a microorganism having a purine operon by a hybridization method using an oligonucleotide prepared based on the known base sequence of the purR gene as a probe. In the Bacillus subtilis 168 Marburg strain, the base sequence of the purR gene has been reported (GenBank accession No. D26185 (coding region is base number 180441-118898), DDBJ Accession No. Z99104 (coding region is base number 54439-55296) ). The nucleotide sequence of the purR gene and the amino acid sequence encoded by the gene are shown in SEQ ID NOs: 1 and 2 in the sequence listing. Any primer can be used as long as it can amplify the purR gene.

  The purR used in the present invention is not necessarily required to include the entire length because it is used for the preparation of each disrupted gene, and may have a length necessary to cause gene disruption. In addition, the microorganism used for obtaining each gene is not particularly limited as long as the gene has homology that causes homologous recombination with the homologous gene of the microorganism used to create the gene-disrupted strain. However, it is usually preferable to use a gene derived from the same bacterium as the target Bacillus bacterium.

  As DNA capable of causing homologous recombination with the purR gene of the genus Bacillus, DNA encoding an amino acid sequence containing substitution, deletion, insertion, or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 Is mentioned. The “several” is, for example, 2 to 50, preferably 2 to 30, more preferably 2 to 10.

  Specifically, the DNA capable of causing homologous recombination with the purR gene of the genus Bacillus is, for example, 70% or more, preferably 80% or more, more preferably 90%, with DNA having the base sequence shown in SEQ ID NO: 1. Examples thereof include DNA having the above homology. More specifically, a DNA that hybridizes with a DNA having the base sequence shown in SEQ ID NO: 1 under stringent conditions can be mentioned. The stringent conditions include conditions in which washing is performed at a salt concentration corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS.

  Examples of the marker gene include drug resistance genes such as a spectinomycin resistance gene and a kanamycin resistance gene. The spectinomycin resistance gene derived from Enterococcus faecalis was prepared by preparing plasmid pDG1726 from Escherichia coli ECE101 strain commercially available from Bacillus Genetic Stock Center (BGSC) and removing it from the plasmid as a cassette. Can get. For the erythromycin resistance gene of Staphylococcus aureus, prepare plasmid pDG646 from Escherichia coli ECE91 marketed by Bacillus Genetic Stock Center (BGSC), and take it out as a cassette from the plasmid. Can be obtained. Furthermore, the kanamycin resistance gene is a pDG783 plasmid containing a kanamycin resistance gene derived from Streptococcus faecalis (can be prepared from the Escherichia coli ECE94 strain commercially available from Bacillus Genetic Stock Center), and PCR is performed. It can be obtained by doing.

  When using a drug resistance gene as a marker gene, insert the gene into an appropriate site of the purR gene in the plasmid, transform the microorganism with the resulting plasmid, and select a transformant that has become drug resistant. A purR gene-disrupted strain is obtained. The disruption of the purR gene on the chromosome can be confirmed by analyzing the purR gene or marker gene on the chromosome by Southern blotting or PCR. Confirmation that the spectinomycin resistance gene, erythromycin resistance gene or kanamycin resistance gene has been incorporated into the chromosomal DNA can be carried out by PCR using primers capable of amplifying these genes.

In such a way, as a strain with increased purine nucleoside biosynthesis ability,
Bacillus subtilis KMBS16 (Japanese Patent Laid-Open No. 2004-242610)
Bacillus subtilis SB112K (Japanese Patent Laid-Open No. 11-346778)
Etc.

(2) Method for imparting resistance under high osmotic pressure in bacteria belonging to the genus Bacillus

In the present invention, “modified to have resistance under high osmotic pressure” is a mutant strain belonging to the genus Bacillus described in (1) and derived from a parent strain having purine nucleoside-producing ability, Means a bacterium that has been modified to grow better than the parent strain when cultured in a medium containing a substance that increases The parent strain may be a wild strain or a strain to which purine nucleoside-producing ability is imparted by imparting resistance under high osmotic pressure.
In the present invention, the “substance that increases the osmotic pressure” means a substance that increases the osmotic pressure of the medium (high osmotic pressure) by adding it to the medium. Sodium chloride, mannitol, sorbitol, etc. Can be mentioned.
In the present invention, under high osmotic pressure means a condition where the osmotic pressure is 2 osm / L or more, preferably 3 osm / L or more, more preferably 4 osm / L or more.

Here, the medium used for imparting resistance under high osmotic pressure is preferably a solid medium. That is, a parent strain that does not have resistance under high osmotic pressure does not grow or grows slower than when cultured in a medium that does not contain a substance that increases osmotic pressure. Specifically, for example, when cultured in a solid medium for a certain period of time, a colony diameter is smaller in a medium containing a substance that increases the osmotic pressure than in a medium not including a substance that increases the osmotic pressure. In contrast, the Bacillus bacterium of the present invention has been modified to reduce growth inhibition under high osmotic pressure, and is cultured in a medium containing an osmotically active substance such as NaCl, mannitol or sorbitol. It exhibits better growth than the parent strain, can grow on a medium under high osmotic pressure where an unmodified strain cannot grow, or the colony diameter increases.
For example, a bacterium that can form colonies in culture within 40 hours at 34 ° C. on M9 minimal medium containing 2.0 M NaCl or M9 minimal medium containing 2 M sorbitol has a high osmotic pressure. It can be said that it is resistant to the growth inhibition caused by.

  The selective medium is preferably a minimal medium, for example, a medium having the following composition. (Glucose 20 g / L, ammonium chloride 5 g / L, potassium dihydrogen phosphate 4 g / L, iron sulfate 0.01 g / L, manganese sulfate 0.01 g / L, sodium citrate 0.5 g / L (Sambrook, J. ), Fritsch, EF, and Maniatis T. "Molecular clonig: a laboratory manual", 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring, NY (See Harbor, 1989)

  In the present specification, the minimum medium may contain nutrients essential for growth as necessary. For example, most inosine-producing bacteria are adenine-requiring strains, and the medium contains a degree of adenine necessary for growth. However, if the amount of adenine is too large, the growth inhibitory effect of certain purine analogs decreases, so it is preferable to limit the amount of adenine. Specifically, a concentration of about 0.1 g / L is preferable.

  The bacterium of the present invention is obtained by mutation using normal mutagenesis, such as treatment with ultraviolet radiation, X-ray radiation, radiation, and chemical mutagens followed by selection by replica method. Is possible. A preferred mutagen is N-nitro-N'-methyl-N-nitrosoguanidine (hereinafter referred to as NTG). It can also be obtained by spontaneous mutation.

Furthermore, it can be confirmed that the liquid medium is modified to have resistance under high osmotic pressure. The cell suspensions of the modified and parent strains are each inoculated into a liquid medium containing a substance that increases osmotic pressure, and the cells are in the vicinity of the optimum growth temperature, for example, at a temperature of 34 ° C. for several hours to 1 day, preferably about Incubate for 18 hours. If the modified strain exhibits a higher optical density (OD) or turbidity of the medium compared to the parent strain, at least either in logarithmic growth phase or at stationary phase, it is assumed that growth has improved. . In particular, if the modified strain reaches the logarithmic growth phase compared to the unmodified strain or the parent strain, or if the maximum value of OD is higher, it is assumed that the growth is improved. The aforementioned logarithmic growth phase refers to a period in which the number of cells increases logarithmically on the growth curve. The stationary phase refers to a period in which the logarithmic growth phase ends, cell division and proliferation stop, and the increase in the number of cells can no longer be seen (Biochemical Encyclopedia, 3rd edition, Tokyo Kagaku Dojin).
When a liquid medium is used, it may be more difficult to detect a difference in growth inhibition due to high osmotic pressure than when a solid medium is used. In the present invention, even when a difference in growth inhibition due to high osmotic pressure is not detected by using a liquid medium, as long as it is evaluated that it shows more suitable growth compared to the parent strain on a solid medium, Included in the microorganism of the present invention.

  Furthermore, the degree of growth inhibition by hyperosmotic pressure is also applied to the suspension of the mutant strain on a solid medium containing the osmotically active substance and on a solid medium not containing the osmotically active substance, and It is also possible to assess the size of colonies appearing after culturing under the same conditions by comparing as described above. For example, when the triple-deficient strain KMBS16 in purR, purA, and deoD, derived from the Bacillus subtilis 168 Marburg strain, was cultured at high osmotic pressure in the medium, the relative growth rate was 5 for a culture time of about 18 hours. In contrast, the mutant strain obtained in the Examples section showed a relative growth rate of no less than 30% with an incubation time of about 18 hours and the same content of osmotically active substance. Therefore, by using the relative degree of growth as an index, growth inhibition due to high osmotic pressure can be evaluated without comparison with the parent strain. However, growth inhibition by hyperosmolarity can also be assessed by comparing the relative degree of growth between the parent strain and the mutant strain.

Typical examples obtained by the above method are Bacillus subtilis 51-53H (VKPM B-8997) and Bacillus amyloliquefaciens 23-68H (VKPM B-8996). The bacteria used in the present invention have the same bacteriological properties as the parental strain, except for resistance to hyperosmolarity and the ability to produce higher yields of purine nucleosides. Bacillus subtilis 51-53H and Bacillus amyloliquefaciens 23-68H, on March 10, 2005, Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhny proezd, 1), with accession numbers VKPM B-8997 and VKPM B-8996, respectively.
(3) Process for producing purine nucleosides and purine nucleotides

  The Bacillus bacterium of the present invention efficiently produces purine nucleosides. Therefore, by culturing the Bacillus bacterium of the present invention in a suitable medium, it is possible to produce and accumulate purine nucleosides in the bacterial cells or in the medium.

  The medium to be used for purine nucleoside production may be a normal medium containing a carbon source, a nitrogen source, inorganic ions, and other necessary organic components. Carbon sources include glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, and carbohydrates such as starch hydrolysates; glycerol, mannitol, and alcohols such as sorbitol; gluconic acid, Organic acids such as fumaric acid, citric acid, and succinic acid, and the like can be used. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen such as soybean hydrolysate; ammonia gas; aqueous ammonia and the like can be used. It is desirable that vitamins such as vitamin B1, necessary substances, for example, nucleic acids such as adenine and RNA, yeast extract and the like are included in appropriate amounts as trace amounts of organic nutrients. In addition to these, small amounts of calcium phosphate, magnesium sulfate, iron ions, manganese ions, and the like may be added if necessary.

  The culture is preferably performed under aerobic conditions for 16 to 72 hours, and the culture temperature during the culture is adjusted within 30 to 45 ° C. and the pH within 5 to 8. The pH can be adjusted by the use of inorganic or organic, acidic or alkaline materials, and ammonia gas.

Purine nucleosides can be recovered from the fermentation broth by any conventional method, such as techniques that utilize ion exchange resins and precipitation, or any combination.
It is also possible to produce a purine nucleotide (nucleoside-5′-phosphate ester) by phosphorylating the purine nucleoside produced using the microorganism of the present invention by allowing phosphotransferase to act. (Japanese Patent Laid-Open No. 2000-295996) For example, a method for producing a purine nucleotide using an Escherichia bacterium into which a gene encoding Escherichia coli innosing anosine kinase has been introduced (WO91 / 08286 pamphlet); A method for producing purine nucleotides using Corynebacterium ammoniagenes into which a gene encoding kinase is introduced (WO96 / 30501 pamphlet) can be employed.

  Moreover, the microorganism which has the capability to produce | generate the phosphoric acid donor selected from the group which consists of polyphosphoric acid, phenylphosphoric acid, carbamyl phosphoric acid, and nucleoside-5'-phosphate ester in the purine nucleoside produced using the microorganisms of this invention Alternatively, purine nucleotides (nucleoside-5′-phosphate esters) can be produced by the action of acid phosphatase (EC 3.1.3.2). The microorganism having the ability to produce a nucleoside-5′-phosphate ester is not particularly limited as long as it has the ability to convert purine nucleosides into purine nucleotides. For example, the microorganism as described in International Publication Pamphlet WO9637603 Is mentioned.

  In addition, Escherichia blattae JCM 1650, Serratia ficaria ATCC 33105, Klebsiella planticola IFO 14939 (ATCC 33531), as disclosed in JP-A-07-231793, Klebsiella pneumoniae IFO 3318 (ATCC 8724), Klebsiella terrigena IFO 14941 (ATCC 33257), Morganella morganii IFO 3168, Enterobacter aerogenes 2010 Bacter Aerogenes IFO 13534 (ATCC 13048), Chromobacterium fluviatile IAM 13652, Chromobacterium violaceum IFO 12614, Cedecea lapagei JCM 1684, Cedecea edea ec davi siae) JCM 1685, Cedecea neteri JCM 5909, and the like can also be used.

  As the acid phosphatase, for example, those disclosed in JP-A-2002-000289 can be used, and more preferably, acid phosphatase (see JP-A-10-201481) having increased affinity for nucleoside and nucleotidase activity. Mutant acid phosphatase (see WO9637603) having a reduced pH, mutant acid phosphatase (JP 2001-245676) having a reduced phosphate ester hydrolysis activity, and the like can be used.

Purine nucleotides can also be obtained by chemically phosphorylating purine nucleosides produced using the microorganism of the present invention. (Bulletin of the Chemical Society of Japan 42,3505) In addition, a method for obtaining GMP by coupling an XMP aminase activity with a microorganism having XMP production ability of the present invention using an ATP regeneration system possessed by the microorganism. In addition, a method of obtaining IMP by conjugating inosine kinase can also be adopted (Biosci. Biotech. Biochem., 51,840 (1997), JP-A-63-230094).
[Example]
Hereinafter, the present invention will be described more specifically with reference to examples.

Obtaining Bacillus bacteria resistant to high osmotic pressure Cells of Bacillus subtilis KMBS16 (Japanese Patent Laid-Open No. 2004-242610) were treated with 200 μg / ml N-methyl-N′-nitro-N-nitrosoguanidine (NTG). Treated for minutes to induce mutations. The treated cells were then treated with L-broth (Sambrook, J., Fritsch, EF), and Maniatis T containing 1.8 M, 2.2 M, or 2.4 M NaCl. .) "Molecular cloning: a laboratory manual" 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989) Agar plates (including agar 20 g / l) Plated on top. Inoculated plates were incubated at 34 ° C. for 5 days. Bacillus subtilis strain 51-53H (VKPM B-8997) was selected from the mutant colonies that appeared.
The resistance to sodium chloride of the Bacillus subtilis 51-53H strain and the parent strain KMBS16 strain was evaluated by the following method. Microbes were inoculated into tubes containing 3 ml of M9 glucose minimal medium containing 50 mg / l tryptophan, prepared by adding different sodium chloride concentrations, and grown in L-broth by shaking for 18 hours. About 10 ⁷ cells / ml of the test strain was inoculated, and shaking culture was performed at 37 ° C. for 18 hours. The resulting broth was diluted appropriately with water and the absorbance of the dilution at 540 nm was measured (SCOD ₅₄₀ ). The relative growth on NaCl-containing medium was indicated by (SCOD ₅₄₀ ) / (D ₅₄₀ ) × 100, where the degree of growth (D ₅₄₀ ) of the same strain on M9 medium without NaCl was 100. (Table 1)

Table 1 shows that Bacillus subtilis 51-53H is more resistant to high concentrations of sodium chloride: it can grow compared to the parent strain even at high concentrations of NaCl.

Confirmation of inosin production of hyperosmotic strains The Bacillus subtilis 51-53H strain and the parent strain Bacillus subtilis KMBS16 were each cultured in L-broth using 18 hours aeration for 20 hours at 34 ° C. 0.3 ml of the resulting culture was then inoculated into 3 ml of the fermentation medium of Example 2 in a 20 × 200 mm test tube and cultured on a rotary shaker at 34 ° C. for 72 hours.

After incubation, the amount of inosine accumulated in the medium was measured by HPLC. A sample of medium (500 μl) was centrifuged at 15,000 rpm for 5 minutes and the supernatant was diluted 100-fold with H ₂ O and analyzed by HPLC.
Analytical condition column: Luna C18 (2) 250 ^* 3 mm, 5u (Phenomenex, USA)
Buffer: 2% C ₂ H ₅ OH, 0.8% (v / v) triethylamine, 0.55 (v / v) acetic acid (ice), pH 4.5
Temperature: 30 ° C
Flow rate: 0.3 ml / min Detection: UV 250 nm
Retention time (minutes)
Xanthosine 13.7
Inosine 9.6
Hypoxanthine 5.2
Guanosine 11.4
Adenosine 28.2
The results are shown in Table 2.

As shown in Table 2, Bacillus subtilis strain 51-53H accumulated a larger amount of inosine than the parent strain.

Acquisition and evaluation of NaCl-resistant Bacillus amyloliquefaciens strains Bacillus amyloliquefaciens AJ1991 (ATCC19222) cells were treated with 200 μg / ml NTG and treated with 2M, 2.2M or 2.4M NaCl. On LB agar plates containing The inoculated plate was incubated at 34 ° C. for 5 days. Bacillus amyloliquefaciens strain 23-68H (VKPM B-8996) was selected from the mutant colonies that appeared.

Bacillus amyloliquefaciens strain 23-68H and the parent strain, Bacillus amyloliquefaciens AJ1991, were each cultured in L-broth for 18 hours at 34 ° C. Then 0.3 ml of the resulting culture was inoculated into 3 ml of fermentation medium in a 20 × 200 mm test tube and cultured on a rotary shaker at 34 ° C. for 72 hours.
Composition of fermentation medium: (g / l)
glucose 80.0
KH ₂ PO ₄ 1.0
MgSO ₄ 0.4
FeSO ₄ ^* 7H ₂ O 0.01
MnSO ₄ ^* 5H ₂ O 0.01
NH ₄ Cl 15.0
Adenine 0.3
Soy hydrolyzate (nitrogen content) 0.8
CaCO ₃ 25.0
After incubation, the amount of inosine and guanosine accumulated in the medium was measured by HPLC as described above. The results are shown in Table 3.

As shown in the table, the Bacillus amyloliquefaciens 23-68H strain resistant to high osmotic pressure accumulated more inosine and guanosine than the parent strain.

Claims (10)

A Bacillus bacterium modified to have resistance under high osmotic pressure and having the ability to produce purine nucleosides. A modified strain derived from a parent strain belonging to the genus Bacillus, characterized in that it is modified to grow better than the parent strain when cultured in a medium containing a substance that increases osmotic pressure. The bacterium of claim 1. The bacterium according to claim 1 or 2, wherein the substance that increases the osmotic pressure is sodium chloride. The bacterium according to any one of claims 1 to 3, wherein the medium contains 2.0 M sodium chloride. The bacterium according to any one of claims 1 to 4, wherein the Bacillus bacterium is a bacterium selected from Bacillus subtilis or Bacillus amyloliquefaciens. The bacterium according to any one of claims 1 to 5, wherein the purine nucleoside is selected from the group consisting of inosine, xanthosine, adenosine, and guanosine. The Bacillus bacterium according to any one of claims 1 to 6 is cultured in a culture medium, purine nucleosides are accumulated in the bacteria or the culture medium, and the purine nucleosides are recovered from the cells or the culture medium. A method for producing purine nucleosides. 8. The method of claim 7, wherein the purine nucleoside is selected from the group consisting of inosine, xanthosine, adenosine, and guanosine. A purine nucleoside is produced by the method according to claim 7 or 8, and a phosphoric acid donor selected from the group consisting of polyphosphoric acid, phenylphosphoric acid, and carbamylphosphoric acid is added to the purine nucleoside, and a nucleoside-5'-phosphate ester. A method for producing a purine nucleotide, characterized in that a purine nucleotide is produced by the action of a microorganism or acid phosphatase having the ability to produce the purine nucleotide, and the purine nucleotide is collected. 10. The method of claim 9, wherein the purine nucleotide is selected from the group consisting of 5'-inosinic acid, 5'-xanthylic acid, 5'-guanylic acid, 5'-adenylic acid.