WO2007023830A1 - Process for production of cytidine-diphosphate choline - Google Patents

Abstract

Disclosed is a process for production of CDP-choline comprising the steps of contacting a biocatalyst having an activity of producing CDP-choline from orotic acid or uracil and choline or phosphorylcholine with orotic acid or uracil and choline or phosphorylcholine in a medium to produce and accumulate CDP-choline in the medium, and collecting the CDP-choline from the medium. In the process, orotic acid or uracil is added to the medium in the form of an alkaline solution containing orotic acid or uracil.

Description

 Specification

 Method for producing cytidine diphosphate choline

 Technical field

 The present invention relates to a method for producing cytidine diphosphate choline useful as a pharmaceutical product.

 Background art

 [0002] Cytidine diphosphate choline (hereinafter abbreviated as CDP-choline) includes chemical synthesis, cytidine-5'-triphosphate (hereinafter abbreviated as CTP), cytidine- Known methods include 5′-diphosphate (hereinafter abbreviated as CDP) or cytidine-5 ′-phosphate (hereinafter abbreviated as CMP) (Patent Document 1, Patent Document 2, Patent Document 3), etc. However, there are problems such as low yield and expensive raw materials.

 [0003] In the production method using microorganisms, uridine-5 '-phosphate (hereinafter abbreviated as UMP) and choline or phosphorylcholine are used by using genetically modified microorganisms to improve productivity. (Patent Document 4), a process from CMP and choline or phosphorylcholine (Patent Document 5), a process from orotic acid and choline or phosphorylcholine (Patent Document 6), uracil and choline or phosphorylcholine (Patent Document 7) has been reported. However, in the method of production from orotate uracil, since orotate uracil has a low solubility in water, it is necessary to add these raw materials as powders when they are added to a medium for generating and accumulating CDP-choline. There is a lot of work. It is known that oloturaic acid uracil has high solubility in alkaline solution. However, it is known that CDP-choline can be efficiently produced by adding an alkaline solution containing olotic acid or uracil to the medium. ,,,.

 Patent Document 1: Japanese Patent Publication No. 48-2358

 Patent Document 2: Japanese Patent Publication No. 48-40758

 Patent Document 3: Japanese Patent Publication No. 48-2359

 Patent Document 4: Pamphlet of International Publication No.99 / 49073

 Patent Document 5: JP-A-2001-103973

Patent Document 6: Patent No. 3369236 Patent Document 7: International Publication No. 03/95660 Pamphlet

 Disclosure of the invention

 Problems to be solved by the invention

[0004] An object of the present invention is to provide an efficient process for producing CDP-choline.

 Means for solving the problem

[0005] The present invention relates to the following (1) to (5).

 (1) Contact orotate or uracil and choline or phosphorylcholine with a biocatalyst having the activity of producing CDP-choline in contact with orotic acid or uracil and choline or phosphorylcholine in the medium. In the method for producing CDP-choline, in which CDP-choline is collected from the medium, orotic acid or uracil is added to the medium as an alkaline solution containing orotic acid or uracil. And CDP-choline production method.

 (2) An alkaline solution containing orotic acid or uracil is added so as to maintain the pH of the medium at a pH suitable for the production of CDP-choline. Manufacturing method.

 (3) The method for producing CDP-choline according to (1) or (2) above, wherein the biocatalyst is a biocatalyst containing a microorganism culture or a treated product of the culture.

 (4) The microorganisms are Escherichia, Corvnebacterium, Bacillus, Streptococcus, Sinorhiz obium, Haemophilus and Saccharomyces (Saccharomyces). The method for producing CDP-choline as described in (3) above, which is a microorganism selected from the group consisting of the microorganisms belonging to the genus

(5) The method for producing CDP-choline according to the above (3), wherein the microorganism is selected from microorganisms belonging to the genus Corvnebacterium and the genus Escherichia.

(6) The method for producing CDP-choline according to the above (3), wherein the microorganism is a microorganism belonging to Corynebacterium ammoniagenes and Escherichia coli.

(7) The microorganism is Corynebatarum 'Ammonia Genes ATCC21170 and Escherichia' The method for producing CDP-choline of (3) above, which is a strain of E. coli MM294 / pCKG55 (FERM BP-3717). The invention's effect

 [0006] According to the present invention, a method for industrially advantageously producing CDP-choline is provided.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0007] The alkaline solution containing orotic acid or uracil used in the present invention includes a sodium hydroxide solution, a potassium hydroxide solution, a lithium hydroxide solution, a calcium hydroxide solution, a barium hydroxide solution or ammonia. Can give water. The normality of the alkaline solution is preferably 0.01-15, more preferably 0.1-12. The concentration of orotic acid or uracil in the alkaline solution is preferably 1 mmol / L to 2 mol / L, more preferably 10 mmol / L to 1.6 mol / L force S.

 [0008] An alkaline solution containing orotic acid or uracil is mixed with a biocatalyst having an activity to produce CDP-choline (hereinafter abbreviated as CDP-choline-generating activity) and choline or uracil and choline or phosphorylcholine. CDP-choline is produced and accumulated in the medium by adding to the medium containing phosphorylcholine and contacting the biocatalyst, orotic acid or uracil, and choline or phosphorylcholine in the medium. CDP-choline can be produced by collecting.

 [0009] The medium containing the biocatalyst and choline or phosphorylcholine may contain orotic acid or uracil or other components as necessary. When an alkaline solution containing orotic acid or uracil is added to the medium, it is preferable to add the medium so that the pH of the medium is maintained at a pH suitable for production of CDP-choline. The pH can be 5 to 10, preferably 6 to 8. The alkaline solution can be added to the medium continuously or intermittently.

 [0010] As the biocatalyst used in the present invention, any biocatalyst having CDP-choline producing activity can be used.

 Examples of such a biocatalyst include a culture of a microorganism having CDP-choline production activity, a treated product of the culture, and the like.

Microorganisms originally have a CDP-choline producing activity, and enzymes that have no CDP-choline producing activity but have a reaction to produce CDP-choline (CDP-choline biosynthesis). Examples include microorganisms transformed with DNA encoding abbreviated synthesizing enzymes, or microorganisms obtained by fusing cells of other microorganisms having the activity.

[0011] The microorganisms include the genus Escherichia, the genus Serratia, the genus Bacillus, the genus Brevibacterium, the genus Corvnebacterium, and the microbacterium ), Pseudomonas, Streptococcus, Sinorhizobium, Haemophilus, Arthrobacter, Aureobacte rium , Cellulomonas, Clavibacter, Curtobacterium, Pimerobacter, Sac charomvces J¾, Schizosaccharomes vc , Genus TrichosDoron, genus Schwaninioms, pich (Pichia) sp., Or it can be mentioned belong microorganisms to the genus Candida.

Cor vnebacterium acetoacidophilum ATCC 13870等の Corvnebacterium acetoacidophilum 等をあげることができる。ミクロパクテリゥム (Microbacterium)属に属する微生物として 【ま、 Microbactenum ammoniaphilum ATCし 15354等の iicrobactenum ammoniaphilu Microbacterium lactiumおよび Microbacterium lmperiale等をあ ること力 ²¹ eさる。 シユードモナス (Pseudomonas)属に属する微生物としては、 Pseudomonas putida等を あげることができる。ストレプトコッカス (Streptococcus)属に属する微生物としては、 eptococcus pneumoniae^ ¾r¾けること力 C、さる。ンノリゾビゥム (amorhizobium;属に 属する微生物としては、 Sinorhizobium mdi 等をあげることができる。へモフィラス（ Haemophilus)属に属する微生物としては、 Haemophilus Μ ^等をあげることが できる。アースロバクタ一 (Arthrobacter)属に属する微牛.物 しては、 Arthrobacter ci treusおよび Arthrobacter globiformis等をあげるこ ができる。オーレォバクテリゥム ( ureobacterium) に腐する fi¾牛.物とし飞は、 Aureobacterium flavescens. Aureobacter mm saperdaeおよひ AureoDactenum testaceum めげ こと力で^る セノレ口モナス (し ellulomonas) 〖こ す 1激牛.物としては、し ellulomonas flavigenaおよびし ellulomon as mla等をあげることができる。クラビバクタ一 (Clavibacter)属に属する微牛.物 し ては、 clavibacter michiganensisおよびし lavibacter rathavi等をあ hること力 ²¹ eさる。ク ノレトバクテリゥム（Curtobacterium)属に属する微牛.物 しては、 Curtobacterium albidu 2i、し urtobacterium citreumおよびし urtoDacterium lui§ l等をめげ Oこと力できる。ヒ メロパクター (Pimerobacter)属に属する微牛.物 しては、 Pimerobacter sinmlex等をあ げることができる。 [0012] A fine cattle belonging to the genus Escherichia, which includes Escherichia coli MM294, Escherichia coli XL1-Blue, Eschencnia coli XL2-Blue, Eschencnia coli DH 丄, Escherich ia coli MC1000, Escherichia coli KY3276, Examples include Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichiacoli W3110, Escherichinia coli NY49, Escherichia coli GI698 and Escherichia coli TBI. Examples of microorganisms belonging to the genus Serratia (ggimtk) include Serratia ficaria, Serratia fonticola, aerratia iiauefaciens, and aerratia marcescens. Examples of microorganisms belonging to the genus Bacillus (MU) include Bacillus subtilis and Bacillus a myloliauefaciens, etc. Ability to be ²¹ e. The microorganisms belonging to the genus Corynebacterium include Corvnebacterium glutamicum A D. Gi »¾032: ^ orvnepactenum glutamicum ATCC13869 and other Corvnebacterium glutamicum, Corynebacterium ammoniagenes ATCC6872 and Cory nebacterium ammoniagenes.

Cor Examples thereof include Corvnebacterium acetoacidophilum such as vnebacterium acetoacidophilum ATCC 13870. Micro Park Teri © beam (Microbacterium) [or as a microorganism belonging to the genus, Microbactenum ammoniaphilum ATC to iicrobactenum ammoniaphilu Microbacterium lactium and Microbacterium lmperiale etc. Oh Rukoto force ²¹ e monkey such as 15354. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida. Microorganisms belonging to the genus Streptococcus include C. eptococcus pneumoniae ^ ¾r¾. Examples of microorganisms belonging to the genus amorhizobium include Sinorhizobium mdi, etc. Examples of microorganisms belonging to the genus Haemophilus include Haemophilus Μ ^, which belong to the genus Arthrobacter. Artificial cattle, such as Arthrobacter ci treus and Arthrobacter globiformis, etc. Fi¾ cattle that rot to ureobacterium, and Aureobacterium flavescens. Aureobacter mm saperdae and AureoDactenum testaceum genulo genus (sell ellulomonas) 1 gigantic cattle.For example, ellulomonas flavigena and ellulomon as mla can be raised.Fine cattle belonging to the genus Clavibacter For example, clavibacter michiganensis and lavibacter rathavi, etc., have the power of ²¹ e. The cattle belonging to the genus (ium) can be used to cultivate Curtobacterium albidu 2i, urtobacterium citreum, urtoDacterium lui§l, etc. The cattle belonging to the genus Pimerobacter are Pimerobacter sinmlex etc. can be raised.

Examples of microorganisms belonging to the genus Saccharomvces include Saccharomvces cere visiae. Examples of microorganisms belonging to the genus Schizosaccharomvces include Schizosaccharomvces E2mk. Examples of microorganisms belonging to the genus Kluweromvces include Kluweromvces lactis. Examples of microorganisms belonging to the genus TrichosDoron include Trichospor on pullulans. Examples of microorganisms belonging to the genus Schwanniomvces include Schwanniomvces alluvius. Candida utilis etc. can be mention | raise | lifted as microorganisms which belong. [0014] Examples of microorganisms include the genus Escherichia, Corvnebacterium, Bacillus, Streptococcus, Sinorhizobium, which can include the above microorganisms, Microorganisms cultivated from the group of microorganisms belonging to the genus Haemophilus and Saccharomvces are preferably used, and the genus Escherichia, the genus Brevibac terium and the genus Cornepacterium (Corvnebacterium) More preferably, a microorganism selected from the group consisting of microorganisms belonging to the genus is used.

 [0015] Among the microorganisms described above, even if the microorganism originally has CDP-choline producing activity, if the CDP-choline producing activity is not sufficiently strong, DNA encoding CDP-choline producing enzyme is included in the microorganism. The ability to introduce a recombinant DNA having the above-mentioned activity may be prepared and used by fusing cells of other microorganisms having the activity.

 As the microorganism enhanced in activity and the microorganism imparted with the activity, a transformant obtained by introducing DNA encoding a CDP-collin producing enzyme into the microorganism by the method described below is preferably used.

[0016] Examples of DNA encoding CDP-choline synthase include, for example, orotate phosphoribosyltransferase having an activity to generate orotidine-5'-monophosphate (hereinafter abbreviated as OMP) from orotate [EC 2.4.2.10], orotidine-5, -monophosphate decarboxylase with activity to generate UMP from OMP [EC 4.1.1.23], uridine phosphorylase with activity to generate uridine [EC 2.4.2.3], Uridine kinase [EC 2.7.1.48] with the activity to generate uridine UMP, uridylate 'cytidylate kinase with the activity to generate uridine-5, -diphosphate (hereinafter abbreviated as UDP) from UMP [EC2.7.1.48], UDP force, etc. Nucleoside diphosphate kinase [EC 2.7.4.6], activity to generate uridine-5'-triphosphate (hereinafter abbreviated as UTP), activity to generate CTP from UTP With cytidine-5,- DNA encoding phosphate synthetase [EC 6.3.4.2] (hereinafter abbreviated as PyrG), DNA encoding choline kinase [EC 2.7.1.32] (hereinafter abbreviated as CKI) having the activity of producing phosphorylcholine from choline And choline phosphate cytidyltransferase [EC 2.7.7.15] (hereinafter abbreviated as CCT), which has the activity to produce CTP and phosphorylcholineca CDP-choline DNA can be raised.

[0017] As the DNA encoding CDP-choline synthase, DNA encoding the above-mentioned enzymes can be used, and DNA encoding PyrG, CK or CCT is preferably used.

 DNA encoding PyrG has been cloned from the Escherichia chromosome and its entire base sequence has been determined [J. Biol. Chem., 261, 5568 (1986)]. Recombinant DNA having DNA encoding PyrG includes Escherichia coli-derived PyrG at the Smal-Pstl site of the multicloning site of Escherichia coli vector pUC8 “Gene, 19, 25 (1982)”. Examples include pMW6 [Biosci. Biotechnol. Biochem., 61, 956 (1997)], which is a plasmid into which a 2426 base pair fragment (hereinafter abbreviated as bp) containing the DNA to be inserted is inserted.

[0018] DNA encoding CCT has its entire base sequence determined [Eur. J. Biochem., 169, 477 (1987)] ₀ For example, the plasmid PCC41 with a 1296 bp ml fragment containing DNA encoding CCT derived from yeast was inserted into the Smal site of the multicloning site of Escherichia ςθϋ vector pUC18 [Gene, 33, 103 (1985)]. Chemistry, £ Q, 701 (1988)].

[0019] The DNA encoding CKI is similarly cloned from the yeast chromosome and its entire base sequence has been determined [J. Biol. Chem., 264, 2053 (1989)] ₀ has DNA encoding CKI As recombinant DNA, a plasmid in which a 2692 bp I-Hindm fragment containing DNA encoding CKI derived from yeast was inserted into the yeast Escherichia ςοϋ shuttle vector YEpM4 [Mol. Cell. Biol, 7, 29 (1987)]. pCKlD [J. Biol. Chem., 264, 2053 (1989)].

 [0020] The plasmids described above can be isolated and purified according to a known method [Nu Acids Res., 7, 1513 (1979)] that carries these plasmids.

From the plasmid obtained as described above, for example, DNA encoding CDP-choline synthase is obtained according to Molecular Cloning, A Laboratory Manual, Third Edition, edited by Sambrook et al. And old Spring Harbor Laboratory (2001). Then, a biocatalyst having CDP-choline producing activity can be obtained by incorporating the DNA into an expression vector, preparing a recombinant DNA, and performing transformation using the microorganism as a host cell. [0021] For example, DNA encoding PyrG, CCT, or CKI is obtained from the plasmid pMW6, plasmid pCC41, or plasmid pCKID, and the polypeptide is encoded as necessary based on the obtained DNA. Prepare a DNA fragment of appropriate length containing the portion.

 If necessary, prepare a DNA in which the base sequence of the portion encoding CDP-choline synthase is replaced with a base so that it becomes the optimal codon for host cell expression. The DNA is useful for the efficient production of CDP-choline synthase.

 [0022] A recombinant vector is prepared by inserting the DNA fragment or full-length DNA downstream of the promoter of an appropriate expression vector. At this time, DNAs encoding CDP-choline synthase may be inserted into the expression vector separately, or a plurality of DNAs may be inserted into the same expression vector.

 The recombinant vector is introduced into a host cell compatible with the expression vector.

 [0023] Examples of host cells include the microorganisms described above.

 As an expression vector, one that can replicate autonomously in the host cell or can be integrated into a chromosome, and contains a promoter at a position where a DNA encoding an enzyme involved in CDP-choline production activity can be transcribed. Used.

 When prokaryotic organisms such as bacteria are used as host cells, the recombinant vector containing DNA encoding CDP-choline synthase is capable of autonomous replication in prokaryotes, and at the same time, is associated with a promoter and ribosome. It is preferable that the vector is composed of a sequence, the DNA, and a transcription termination sequence. Contains the genes that control the promoter!

[0024] Examples of expression vectors include pBTrp2, pBTacl, pBTac2 (all available from Boehringer Mannheim), PKK233-2 (manufactured by Pharmacia), pSE280 [manufactured by Invitrogen], pGEMEX- 1 [manufactured by Promega], pQE-8 [manufactured by QIAGEN], pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSAl [Agric. Biol. Chem., 53, 277 (1989)], pGELl [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescriptll SK (—) [Stratagene (Stratagene) ], P Trs30 (prepared from Escherichia coHJ 109 / pTrS30 (FERM BP-5407)), pTrs32 (prepared from Escherichia hiH coHJM109 / pTrS32 (FERM BP-5408)), pGHA2 (prepared from Escherichia coli IGHA2 (FERM BP-400) , JP 60-221091], pGKA2 [Escherichi coli IGKA2 ( FERM BP-6798), JP 60-221091), pTerm2 (US Patent 4686191, US Patent 4939094, US Patent 5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. BacterioL, 172, 2392 (1990)], pGEX (manufactured by Pharmacia), pET system revagen (Novagen)], and the like.

[0025] The promoter may be any as long as it functions in the host cell.

 For example, trp promoter (P)

 tip, lac promoter, P promoter, P promoter,

 L R

 Examples include promoters derived from Escherichia coli or phages such as T7 promoter. A promoter with two Ps connected in series (P X 2)

 trp trp, tac promoter, lac

Artificially designed and modified promoters such as T7 promoter and letl promoter can be used.

 [0026] It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (eg, 6 to 18 bases). In the recombinant vector of the present invention, a transcription termination sequence is not necessarily required for the expression of DNA encoding CDP-choline synthase, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.

 [0027] As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Laid-Open No. 63-248394), or the method described in Gene, H, 107 (1982), Molecular & General Genetics, Cliff, 111 (1979), etc. Can do.

 [0028] When yeast is used as a host cell, examples of expression vectors include YEP13 (ATC C37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like.

Any promoter can be used as long as it can be expressed in yeast strains. For example, promoters of glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP promoter, ADH promoter Gall promoter, gal 10 promoter, heat shock polypeptide promoter, MF al promoter, CUP 1 promoter and the like. [0029] As a method for introducing a recombinant vector, any method can be used as long as it introduces DNA into yeast. For example, the Elect Mouth Position Method [Methods EnzymoL, 194, 182 (1990)], Spheroplast (Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)), Lithium acetate method (J. Bacteriology, 153, 163 (1983)), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) ) The methods described can be mentioned.

 [0030] In the case where the microorganism has only a part of the CDP-choline producing activity, in order to obtain the CDP-choline producing activity, the microorganism has the CDP-choline producing activity by appropriately combining two or more kinds of microorganisms. It may be used as a biocatalyst. Even when the microorganism has CDP-choline production activity, two or more kinds of microorganisms can be combined.

 The combination of two or more kinds of microorganisms is selected from the above microorganisms! /, The combination of deviations is not limited to the genus Escherichia, the genus Serratia, the genus Bacillus, the genus Brevibacterium, Corvnebacterium spp., Microbacterium spp., Pseudomonas spp., Streptococcus spp., Sinorhizobium spp., HaemoDhilus spp. Arthrobacter, Aureobacterium, Cellulomonas, Clavibacter, Curtobacterium, Pimerobacter, Saccharomyces (Saccharomyces), Saccharomyces Schizosaccharomvces genus, Kluweromvces genus, Trichos polo Examples include microorganisms belonging to the same genus, or combinations of microorganisms belonging to different genera, selected from microorganisms belonging to the genus TrichosDoron, the genus Schwanniomvces, the genus Pichia and the genus Candida.

 [0031] For example, combinations of microorganisms belonging to the genus Corynepacteria and microorganisms belonging to the genus Escherichia can be mentioned. Specifically, Corvnebacterium ammoniagenes ATCC 21170 and Escherichia coli MM294 / pCKG55 strain (FERM BP- 3717) (Japanese Patent No. 3369236, US Pat. No. 6,387,667) and the like.

Examples of the culture of microorganisms having CDP-choline producing activity, which is one of the biocatalysts having CDP-choline producing activity, include those obtained by culturing the above microorganisms according to a conventional method. . [0032] When the microorganism is a prokaryotic organism such as a bacterium or a eukaryotic organism such as yeast, the culture medium for cultivating the microorganism contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the microorganism. As long as it is a medium that can efficiently culture the microorganism, either a natural medium or a synthetic medium may be used.

 As the carbon source, glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolyzate, organic acids such as acetic acid and propionic acid, ethanol, etc. are acceptable as long as the microorganism can assimilate. Alcohols such as propanol can be used.

 [0033] Examples of nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphates such as ammonium salts, and other acid salts. Nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.

 [0034] As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride salt, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. may be used. it can.

 The culture is performed under aerobic conditions such as shaking culture or aeration and agitation culture. The culture time is 15 to 50 ° C, and the culture time is usually 16 hours to 7 days. The pH during the culture is preferably maintained at 3-9. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

 [0035] When the microorganism is a transformant and the recombinant DNA used to transform the microorganism possesses an antibiotic resistance gene, a medium for culturing the microorganism In addition, an antibiotic corresponding to the antibiotic resistance gene possessed by the recombinant DNA may be added.

 When using a culture of two or more microorganisms or a treated product of the culture as a biocatalyst, a culture obtained by culturing each microorganism separately or in the same medium according to the above method may be used. it can.

[0036] When two or more types of microorganisms are cultured in the same medium, these microorganisms are cultured at the same time. However, the remaining microorganisms should be cultured in the medium during the cultivation of one microorganism or after the completion of the cultivation.

 Examples of the treated product of the microorganism culture include a treated product obtained by treating the cultured microorganism obtained by the above-mentioned method with a cell lytic enzyme such as a surfactant, an organic solvent or lysozyme. A surfactant, an organic solvent, or a cytolytic enzyme may be used alone to treat the culture, or a combination thereof may be treated. In addition, a concentrated or dried product of the culture obtained by concentrating or drying the microorganism culture obtained by the above-described method using a concentrator or a dryer, and the culture is solidified by filtration or centrifugation. Cells obtained by liquid separation, dried products of the cells obtained by drying the cells with a dryer, etc., cell lytic enzymes such as surfactants, organic solvents, or lysozyme, or combinations thereof The treated product of the cells obtained by treatment in this manner can also be mentioned as a treated product of the microorganism culture.

 [0037] After disrupting the cells with a homogenizer or the like, the cells are further subjected to usual enzyme purification means such as salting out, isoelectric precipitation, organic solvent precipitation, dialysis, and various chromatographic treatments. The obtained crude enzyme or purified enzyme can also be mentioned as a treated product of the culture.

 Alternatively, the treated product of the microorganism culture may be fixed to a water-insoluble simple substance or gel and used as a biocatalyst.

[0038] When a treated product of a culture of two or more microorganisms is used, the treated product may be used as a biocatalyst having CDP-choline producing activity separately. It can be used as a biocatalyst having

 The amount of biocatalyst used varies depending on the specific activity of the biocatalyst and the like. For example, even when a culture of microorganisms or a treated product of the culture is used as an enzyme source, the culture is centrifuged. It is preferable to use 3 to 300 mg, preferably 5 to 200 mg, as the wet cells obtained by isolation, per lmg of choline choline!

[0039] Choline or phosphorylcholine may be added to the medium as a salt thereof. Examples of the salt of choline or phosphorylcholine include choline halides such as choline choline, choline bromide, choline iodide, choline bicarbonate, methylcholine sulfate, choline dihydrogen citrate, heavy liquor. Examples thereof include phosphorylcholine halides such as choline tartrate and phosphorylcholine chloride. Choline or phosphorylcholine halides are preferably used, and saltycholine and saltyphosphorylcholine are more preferably used.

 [0040] Choline or phosphorylcholine or a salt thereof, orotic acid or uracil may be obtained by chemical synthesis, or may be obtained by biological methods by fermentation. Moreover, it does not necessarily have to be purely purified. All substrates are commercially available and can be easily obtained.

 The concentration of choline or phosphorylcholine is preferably 1 mmol / L to lmol / L, more preferably 10 to 100 mmol / L.

 [0041] Other components added to the medium as necessary include energy donors, phosphate ions, magnesium ions, ammonium ions, surfactants and organic solvents necessary for the production of CDP-choline. can give. These components do not need to be added if the required amount is brought from a biocatalyst or the like.

 As energy donors, sugars such as glucose, fructose and sucrose, molasses, starch hydrolysates, and amino acids such as glycine and alanine are used. These are preferably used at a concentration of 0.02 to 2.0 mol / L! /.

 [0042] Examples of phosphate ions include orthophosphoric acid, pyrophosphoric acid, tripolyphosphoric acid, tetrapolyphosphoric acid, etc., polyphosphoric acid, polymetaphosphoric acid, monopotassium phosphate, dipotassium phosphate, monosodium phosphate, disodium phosphate, etc. Inorganic phosphates can be used. These phosphate ions are preferably used at a concentration of 10 to 500 mmol / L! / ,.

 As the magnesium ions, inorganic magnesium salts such as magnesium sulfate, magnesium nitrate, and magnesium chloride, and organic magnesium salts such as magnesium citrate can be used. Magnesium ions are preferably used at a concentration of 5 to 200 mmol / L.

[0043] As the ammonia ion, aqueous ammonia, ammonia gas, various inorganic or organic ammonia salts, yeast extract, corn steep liquor and the like can be used. Organic nutrients such as glutamine, glutamine-containing peptides, and casamino acids can also be used instead of ammonia ions. The concentration of these ammonium ions is lOmmol / Preferably used at a concentration of L to 2 mol / L! /.

 [0044] Examples of surfactants include anionic surfactants such as sodium dioctylsulfosuccinate (for example, Rabizol B-80, manufactured by NOF Corporation), lauroyl sarcosinate, polyoxyethylene cetyl ether (for example, non-one). Non-ionic surfactants such as P-208 (manufactured by NOF Corporation) and tertiary amines such as alkyldimethylamine (for example, tertiary amine FB, NOF Corporation) promote CDP-choline production. If it is a thing, it can be used even with a gap. These are usually used in the range of 0.1 to 100 g / L, preferably 1 to 50 g / L.

 [0045] Examples of the organic solvent include xylene, toluene, aliphatic alcohols (such as methyl alcohol, ethyl alcohol, and butyl alcohol), acetone, ethyl acetate, and dimethyl sulfoxide. These are usually used at a concentration of 0.1 to 100 mL / L, preferably 1 to 50 mL / L.

 As a medium for contacting a biocatalyst, choline or phosphorylcholine or a salt thereof, orotic acid or uracil, a medium for culturing a microorganism to be used as a biocatalyst, a culture of the microorganism, a culture supernatant, and the like are used. Alternatively, an aqueous medium may be used.

 [0046] Examples of the aqueous medium include buffers such as water, phosphate buffer, HEPES (N-2-hydroxyethylpiperazine-N-ethanesulfonic acid) buffer, and tris [tris (hydroxymethyl) aminomethane] hydrochloric acid buffer. You can give liquid.

 If the reaction is not inhibited, an organic solvent may be added to the medium. Examples of the organic solvent include acetone, ethyl acetate, dimethyl sulfoxide, xylene, methyl alcohol, ethyl alcohol, butanol and the like.

[0047] When a biocatalyst, orotic acid or uracil, and choline or phosphocholine are contacted in a medium to produce and accumulate CDP-choline in the medium, the pH in the medium is usually 5 to 10, preferably 6 to Maintain at 8 and react at 20-50 ° C for 2-48 hours.

 By the method described above, CDP-choline can be produced and accumulated in the medium. Formation The accumulated CDP-choline can be isolated and purified by ordinary isolation and purification means such as ion exchange chromatography, adsorption chromatography, and salting out.

[0048] As a method for producing CDP-choline as described above, for example, Corvnebacterium ammoniagenes AT In the method of producing CDP-choline using CC21170 and Escherichia coli MM294 / pCKG55 strain (FERM BP-3717) as a biocatalyst (Japanese Patent No. 3369236, US Pat. No. 6387667), olotic acid or uracil is used. It can be added as an alkaline solution to produce CDP-choline.

[0049] Further, as another method, a biocatalyst having an activity of producing orotic acid or uracilca UMP can be obtained by the method for producing CDP-choline from UMP and choline described in WO99 / 49073. In addition, a method for producing CDP-collin by adding orotic acid or uracil as an alkaline solution can be mentioned.

 The force which shows the Example of this invention below This invention is not limited by this. Example 1

 [0050] Production of CDP-choline by adding orotate or uracil as alkaline solution

 Escherichia coli MM294 strain Escherichia coli MM2 94 / pCKG55 strain (Japanese Patent No. 3369236, FERM BP-3717) introduced with plasmid DCKG55 incorporating the CVR and CKI gene derived from Escherichia coli and the PvrG gene derived from Escherichia coli L medium containing ampicillin 50mg / L [Pactotryptone (Difco) 10g / L, yeast extract (Difco) 5g / L, NaCl 5g / L, pH adjusted to 7.2] 200mL Inoculated into a triangular flask with 2 L baffle and cultured at 25 ° C for 24 hours at 220 rpm with rotary shaking. 20 mL of this culture broth, 5 g / L of glucose (sterilized separately), 5 g / L of peptone (manufactured by Kyokuto Pharmaceutical), Na PO 6 g / L, KHP

 2 4 2

O 3g /, NH CI lg /, MgSO-7H O 250mg / L (disinfection) and vitamin Bl 4mg / L (

4 4 4 2

 Inoculate a 5L culture tank containing 2.5L of liquid medium (pH unadjusted), which is composed of a separate sterilizer), culture temperature 28 ° C, stirring 600rpm, aeration rate 2.5L / min, Culturing was performed using 14% ammonia water while adjusting the pH to 7.0.

[0051] When glucose in the supernatant of the seed culture solution is consumed, 250 mL of the culture is aseptically collected, and glucose 5 g / L (separately sterilized), peptone (manufactured by Kyokuto Pharmaceutical Co., Ltd.) 5 g / L, Na PO

 2

6 g / KH PO 3 g / NH CI lg / MgSO-7H O 250 mg / L (separately sterilized) and vitamins

4 2 4 4 4 2

Inoculate a 5L culture tank containing 2.5L liquid medium (pH unadjusted) consisting of Bl 4mg / L (separately sterilized), culture temperature 28 ° C, stirring 600rpm, aeration rate 2.5L / min Culturing was performed while adjusting the pH to 7.0 using 14% ammonia water. [0052] During the culture, a feed solution having a composition of glucose 167 g / L and peptone 167 g / L was added with a peristaltic pump at a rate of 30 mL / hour from the 11th to the 24th culture. On the other hand, orvneDacterium ammoniagenes ATCC21170 strain, Gunoleco ~~ su 50g / L, Horipepeptone (Daigo Nutrition Chemical) 10g / L, East Extract (Daigo Nutrition Chemical) 10g / L, Urine 5g / L , (NH) SO 5g / L, KH PO lg / L, K HPO 3g / L, MgSO-7H O lg / L ゝ CaC

 4 2 4 2 4 2 4 4 2

 1 -2H O 0.1g / L, FeSO-7H O 10mg / L, ZnSO-7H O 10mg / L, MnSO 4-6Η O 2

2 2 4 2 4 2 4 2

0 mg / L, L-cystine 20 mg / L, D-calcium pantothenate 10 mg / L, vitamin Bl 5 mg / L, nicotinic acid 5 mg / L, and piotin 30 g / L (using 10 N sodium hydroxide solution) Inoculated into a 2-L baffled triangular flask containing 200 mL of liquid medium with a composition of pH 7.2) and cultured at 28 ° C for 24 hours at 220 rpm. 20 mL of this culture was added to 100 g / L of dalcose, 10 g / L of polypeptone, KH PO lg / L, K HPO lg / L, MgSO-7H O lg

 2 4 2 4 4 2

CaCl -2H O 0.lg / L ゝ FeSO-7H O 20mg / L, ZnSO-7H O lOmg / L ゝ MnSO 4 ~

 2 2 4 2 4 2 4

6H O 20mg / L, β-alanine 15mg / L, L-cysteine 20mg / L, biotin 0.1mg / L, urine

2

 5L containing 2.5L liquid medium consisting of 2g / L of elemental (separately sterilized) and 5mg / L of vitamin Bl (separately sterilized) (adjusted to PH7.2 using 10N sodium hydroxide solution) After inoculating the culture tank, seed culture was performed while adjusting the pH to 6.8 with concentrated aqueous ammonia under the culture conditions of 32 ° C, stirring 600 rpm, and aeration rate 2.5 L / min.

[0053] When glucose in the supernatant of the seed culture is consumed, 350 mL of the culture solution is aseptically collected, and glucose 180 g / L, KH PO 10 g / L, K HPO 10 g / L, MgSO − 7H O lOg

 2 4 2 4 4 2

CaCl -2H O 0.lg / L ゝ FeSO-7H O 20mg / L, ZnSO-7H O lOmg / L ゝ MnSO 4 ~

 2 2 4 2 4 2 4

6H O 20mg / L, β-alanine 15mg / L, sodium glutamate lg / L, L-cysteine 20

2

 The compositional power of mg / L, piotin 0.1mg / L, urea 2g / L (separately sterilized), and vitamin Bl 5mg / L (separately sterilized) (adjusted to pH 7.2 using 10N sodium hydroxide solution) Inoculate a 5 L culture tank containing 2.5 L of liquid medium, and perform main culture while adjusting the pH to 6.8 with concentrated ammonia water under the culture conditions of 32 ° C, stirring 600 rpm, aeration rate 2.5 L / min. It was. The culture was terminated when the Darcos in the culture supernatant was consumed.

[0054] 500 mL of the culture solution of Escherichia coli MM294 / pCKG55 strain obtained in this way and 185 mL of the culture solution of £ ^ 11 nebacterium ammonia¾enes ATCC21170 strain each in 3 L Dispense into culture tanks and add 60% (w / v) glucose solution to each 80mL, 25% (w / v) MgSO

 4 2

25mL of soot, 160mL of 25% (w / v) KH PO, and 8.4g / L each of choline chloride and xylene (6

 2 4

 0 mM) and added to 20 ml / L. One bottle (pot 1) was supplemented with 35 mmol / L of powdered orotic acid. The mixture was stirred at 32 ° C with stirring at 800 rpm and aeration rate of 0.2 L / min, and the reaction was carried out while maintaining the pH at 7.2 with 10 N sodium hydroxide solution. Another one (pot 2) was maintained at pH 7.2 with 6 normal potassium hydroxide solution containing 800 mmol / L orotate and the same amount of orotate (35 mmol / L) as in pot 1 in the culture tank. From the time when was added, the pH adjuster was switched to a 10N sodium hydroxide solution, the pH was maintained at 7.2, and the reaction was carried out at 32 ° C with stirring at 800 rpm and aeration rate of 0.2 L / min. The remaining one (pot 3) was maintained at pH 7.2 with 6N potassium hydroxide solution containing 800mmol / L uracil, and the same amount of uracil (35mmol / When (L) is added, switch the pH adjuster to 10N sodium hydroxide solution, keep the pH at 7.2, stir at 32 ° C, reaction at 800 rpm, aeration rate 0.2 L / min Went. These mixed solutions were reacted at 32 ° C with stirring at 800 rpm and aeration rate of 0.2 L / min for 22 hours. After completion of the reaction, the reaction solution is centrifuged, the supernatant is diluted 100-fold with water, and the amount of CDP-choline produced is quantified by measuring the absorbance at 256 ° with a UV detector in high performance liquid chromatography analysis. did. The results are shown in Table 1.

[table 1]

 As described above, in the method for producing CDP-choline, by adding orotic acid or uracil as an alkaline solution containing orotic acid or uracil, the amount of CDP-choline produced increases, and CDP-choline is efficiently produced. did it.

Industrial applicability [0057] The present invention provides a method for producing CDP-choline in an industrially advantageous manner.

Claims

The scope of the claims  [1] chotidine diphosphate choline from orotic acid or uracil and choline or phosphorylcholine  (Hereinafter abbreviated as CDP-choline) is contacted with orotic acid or uracil, and choline or phosphorylcholine in a medium to form and accumulate CDP-collin in the medium, A method for producing CDP-choline, which comprises collecting CDP-choline, wherein olotic acid or uracil is added to the medium as an alkaline solution containing orotic acid or uracil.  [2] The method for producing CDP-choline according to claim 1, wherein an alkaline solution containing orotic acid or uracil is added so as to maintain the pH of the medium at a pH suitable for the production of CDP-choline. .  3. The method for producing CDP-choline according to claim 1 or 2, wherein the biocatalyst is a biocatalyst containing a culture of microorganisms or a processed product of the culture.  [4] Microorganisms are genus Escherichia, Corvnebacterium, Bacillus, Streptococcus, Sinorhizobi urn, Haemophilus and Saccharomyces 4. The method for producing CDP-choline according to claim 3, wherein the microorganism is selected from the group consisting of microbial power belonging to the genus.  [5] The process for producing CDP-choline according to claim 3, wherein the microorganism is a microorganism having a microbial force belonging to the genus Corvnebacterium and Escherichia. [6] The method for producing CDP-choline according to claim 3, wherein the microorganism is a microorganism selected from microbial power belonging to Corynebacterium ammoniagenes and Escherichia coH. [7] The microorganism is Corynebataterium 'ammoniagenes ATCC21170 and Escherichia' 4. The method for producing CDP-choline according to claim 3, which is MM294 / pCKG55 (FERM BP-3717) strain.