Method of Purifying Cytidine Diphosphate Choline Verfahern Zur Reinigung Von Cytidin-Diphosphat-Cholin Procédé De Purification De La Cytidine Diphosphate Choline
(19) T ZZ_ ¥ _Z _T
(11) EP 1 939 210 B1
(12) EUROPEAN PATENT SPECIFICATION
(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07H 19/10 (2006.01) C12P 19/32 (2006.01) 03.12.2014 Bulletin 2014/49 (86) International application number: (21) Application number: 06782606.5 PCT/JP2006/315802
(22) Date of filing: 10.08.2006 (87) International publication number: WO 2007/018259 (15.02.2007 Gazette 2007/07)
(54) METHOD OF PURIFYING CYTIDINE DIPHOSPHATE CHOLINE VERFAHERN ZUR REINIGUNG VON CYTIDIN-DIPHOSPHAT-CHOLIN PROCÉDÉ DE PURIFICATION DE LA CYTIDINE DIPHOSPHATE CHOLINE
(84) Designated Contracting States: (74) Representative: Duckworth, Timothy John et al AT BE BG CH CY CZ DE DK EE ES FI FR GB GR J A Kemp HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI 14 South Square SK TR Gray’s Inn London WC1R 5JJ (GB) (30) Priority: 10.08.2005 JP 2005231958 (56) References cited: (43) Date of publication of application: EP-A1- 1 502 956 GB-A- 1 305 539 02.07.2008 Bulletin 2008/27 JP-A- 62 016 497
(73) Proprietor: Kyowa Hakko Bio Co., Ltd. • FUJIO TATSURO ET AL: "Construction of a Chiyoda-ku plasmid carrying both CTP synthetase and a Tokyo 100-8185 (JP) fused gene formed from cholinephosphate cytidylyltransferase and choline kinase genes (72) Inventors: and its application to industrial CDP-choline • MURATA, Hideki production: Enzymatic production of CDP- 1-1, Kyowa-cho, Hofu-shi, Yamaguchi 747-8522 choline from orotic acid (Part II)", BIOSCIENCE (JP) BIOTECHNOLOGY AND BIOCHEMISTRY, vol. 61, • MOKUDAI, Tsuyoshi no. 6, 1997, pages 960-964, XP55009405, ISSN: 1-1, Kyowa-cho, Hofu-shi, Yamaguchi 747-8522 0916-8451 (JP) • SHIOMI, Michio Yamabu-gun, Chiba 289-1726 (JP)
Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 939 210 B1
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Description prises contacting a CDP-choline solution containing a nucleic acid analogue and having a pH of not less [0001] The present invention relates to a method of than 0.5 and not more than 5.0 with an H-type strong- purifying cytidine diphosphate choline useful as a starting ly acidic cation exchange resin, and eluting CDP- material of pharmaceutical products and a starting ma- 5 choline adsorbed onto the resin with water or an terial of nutritious foods. aqueous solution having an ion concentration of not more than 0.1 mol/L to separate and purify CDP- Background Art choline. (2) The method of the above-mentioned (1), wherein [0002] As the method of purifying cytidine diphosphate 10 the CDP-choline solution is prepared from a resulting choline (hereinafter to be abbreviated as CDP-choline), medium containing CDP-choline, which has been chemical synthetic methods (patent reference 1, patent produced and accumulated therein by placing a bio- reference 2) and methods using a culture of a microor- catalyst having an activity to produce CDP-choline ganism or an enzyme (patent reference 3, patent refer- from a precursor of UTP and choline or phosphoryl- ence 4) are known. As the method of purifying CDP-15 choline in an aqueous medium together with the pre- choline produced by a chemical synthetic method, a cursor of UTP and choline or phosphorylcholine or method using an anion exchange resin (patent reference a salt thereof. 1) and a method using both of a strongly acidic ion ex- (3) The method of the above-mentioned (2), wherein change resin and a weakly basic ion exchange resin (pat- the biocatalyst comprises a culture or a treated cul- ent reference 2) are known. In the latter method, two20 ture of a microorganism capable of producing UTP kinds of ion exchange resins are used. Moreover, while from a precursor of UTP, and a culture or a treated phosphorylcholine and cytidine-5’-monophosphate culture of a microorganism capable of producing (hereinafter to be abbreviated as CMP) contained in a CDP-choline from UTP and choline or phosphoryl- CDP-choline solution can be separated, uracil or uridine- choline. 5’-triphosphate (hereinafter to be abbreviated as UTP) 25 (4) The method of the above-mentioned (2), wherein cannot be separated efficiently by this method. the biocatalyst comprises an enzyme that catalyzes a reaction to produce CDP-choline from a precursor [patent reference 1] Japanese Published Examined of UTP and choline or phosphorylcholine. Patent Application No. 6558/1988 (5) The method of the above-mentioned (4), wherein [patent reference 2] Japanese Published Examined 30 the enzyme that catalyzes a reaction to produce Patent Application No.31306/1994 CDP-choline is an enzyme selected from the group [patent reference 3] Japanese Patent No.3369236 consisting of orotate phosphoribosyl transferase, [patent reference 4] WO99/49073 orotidine-5’-monophosphate decarboxylase, uridine phosphorylase, uracil phosphoribosyltransferase, EP 1 502 956 discloses a process for producing cytidine 35 uridine kinase, uridylate and cytidylate kinases, nu- 5’-diphosphate choline. GB1305539 disloses a process cleoside diphosphate kinase, cytidine-5’-triphos- for the preparation of nucleoside diphosphate esters, phate synthase, choline phosphate cytidyltrans- which include cytidine diphosphate choline. Fujio et al ferase and choline kinase. (Bioscience Biotechnology and Biochemistry, 61 (6) (6) The method of any of the above-mentioned (1) 960-964, 1997) discloses a method for enzymatic pro- 40 to (5), wherein the nucleic acid analogue is selected duction of cytidine diphosphate choline from orotic acid from uracil and UTP. and choline chloride. Effect of the Invention Disclosure of the Invention 45 [0005] The present invention provides CDP-choline Problems to be Solved by the Invention and a salt thereof at a low cost.
[0003] An object of the present invention is to provide Best Mode for Carrying Out the Invention a method of purifying CDP-choline, in which method CDP-choline can be separated from a nucleic acid ana- 50 [0006] The CDP-choline solution to be used in the logue conveniently. present invention may be prepared by any method as long as it contains a nucleic acid analogue and has a pH Means of Solving the Problems of not less than 0.5 and not more than 5.0. When the prepared CDP-choline solution has a pH of 0.5 - 5.0, it [0004] The present invention relates to the following 55 is directly used. When the pH is higher than 5.0, an acid (1) - (6). such as hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid is added, and when the pH is lower than (1) A method of purifying CDP-choline, which com- 0.5, an alkali such as sodium hydroxide or potassium
2 3 EP 1 939 210 B1 4 hydroxide is added to adjust to pH 0.5 - 5.0, preferably may be subjected to an activated carbon treatment or 1.0 - 3.5, before use. decolorization treatment using a nonpolar porous syn- [0007] The H-type strongly acidic cation exchange res- thetic adsorbent, for example, Diaion HP series (e.g., in may be of a gel type or a porous type, as long as it is HP20, HP21 etc.) manufactured by Mitsubishi Chemical, an H-type strongly acidic cation exchange resin. Specific 5 Diaion SP800 series (e.g., SP825, SP850 etc.) manu- examples thereof include the Dowex series (e.g., HCR- factured by Mitsubishi Chemical, Diaion SP200 series S, HCR-W2, Marathon C, Monosphere 650C, MSC-1, (e.g., SP207 etc.) manufactured by Mitsubishi Chemical, Monosphere 88, 50Wx2, 50W34, 50W38 etc.) manu- Amberlite XAD series (e.g., XAD4, XAD7HP, XAD16HP, factured by Dow Chemical Company, Diaion SK series XAD1180, XAD2000 etc.) manufactured by Rohm and (e.g., SK1B, SK104, SK110, SK112 etc.) manufactured 10 Haas. by Mitsubishi chemical, Diaion PK series (e.g., PK208, [0014] The above-mentioned CDP-choline-containing PK212, PK220, PK228 etc.) manufactured by Mitsubishi solution or decolorized solution is adjusted to pH 2.0 - Chemical, Amberlite series (e.g., IR120B, IR124 etc.) 4.0 with acid or alkali. After concentration is carried out manufactured by Rohm and Haas. as necessary, the concentration of CDP-choline is ad- [0008] Degree of crosslinking of the H-type strongly 15 justed to 50 - 800 g/L, preferably 100 - 700 g/L, and crys- acidic cation exchange resin is not particularly limited as tals of CDP-choline can be obtained using an organic long as CDP-choline and nucleic acid analogue can be solvent, preferably a hydrophilic organic solvent such as separated at the degree of crosslinking. It is preferably acetone, ethanol, methanol or propanol. 2 - 10%, more preferably 4 - 6%. [0015] In addition, the above-mentioned CDP-choline- [0009] In the present invention, the H-type strongly 20 containing solution or decolorized solution is adjusted to acidic cation exchange resin is preferably used in the pH 5.0 - 9.5 with sodium hydroxide. After concentration form of a packed column, and as the column to be used is carried out as necessary, the concentration of CDP- in the present invention, any column may be used. choline is adjusted to 50 - 800 g/L, preferably 100 - 700 [0010] The CDP-choline solution containing a nucleic g/L, and crystals of CDP-choline sodium salt can be ob- acid analogue and having a pH of not less than 0.5 and 25 tained using an organic solvent, preferably a hydrophilic not more than 5.0 is contacted with the H-type strongly organic solvent such as acetone, ethanol, methanol or acidic cation exchange resin by applying the solution to propanol. a column packed with the resin to allow adsorption of [0016] Examples of the method of obtaining CDP- CDP-choline onto the resin. For example, when the so- choline crystals using an organic solvent include a meth- lution is applied to a column packed with an H-type30 od comprising adding an organic solvent to a CDP- strongly acidic cation exchange resin having a degree of choline solution to allow precipitation of crystals, and a crosslinking of 2 - 10%, the flow rate (SV) is 0.1 - 5.0, method comprising adding dropwise a CDP-choline so- preferably 0.2 - 3.0. lution to a large amount of an organic solvent to allow [0011] By the above-mentioned adsorption treatment, precipitation of crystals. a nucleic acid analogue contained in the solution, partic- 35 [0017] In the present invention, the CDP-choline solu- ularly a pyrimidine type nucleic acid substance such as tion containing a nucleic acid analogue may be any as orotic acid, orotidine-5’-monophosphate (hereinafter to long as it is a solution containing CDP-choline and a nu- be abbreviated as OMP), uridine-5’-monophosphate cleic acid analogue. Examples thereof include a solution (hereinafter to be abbreviated as UMP), uridine-5’-di- produced by a chemical synthetic method, a method us- phosphate (hereinafter to be abbreviated as UDP), UTP, 40 ing a biocatalyst having an activity to produce CDP- CMP, cytidine-5’-diphosphate (hereinafter to be abbre- choline from a precursor of UTP and choline or phospho- viated as CDP) or cytidine-5’-triphosphate (hereinafter rylcholine (hereinafter to be abbreviated as CDP-choline- to be abbreviated as CTP) can be efficiently separated producing activity). or removed from CDP-choline. Out of the nucleic acid [0018] Examples of the biocatalyst include a culture of analogues, uracil and UTP are particularly preferably 45 a microorganism having a CDP-choline-producing activ- separated and removed, since they hardly adsorb to or ity, a treated culture thereof or an enzyme that catalyzes do not at all adsorb to the resin, when a CDP-choline a reaction to produce CDP-choline. solution containing uracil and UTP is brought into contact [0019] As the microorganism, any microorganism can with the resin at a pH of not less than 0.5 and not more be used as long as it has a CDP-choline-producing ac- than 5.0. 50 tivity. A microorganism inherently having a CDP-choline- [0012] CDP-choline adsorbed on the H-type strongly producing activity can be used as it is for the production acidic cation exchange resin is eluted by applying an of CDP-choline, and a microorganism naturally having aqueous solution having an ion concentration of not more no CDP-choline-producing activity can be used for the than 0.1 mol/L, preferably not more than 0.03 mol/L, more production of CDP-choline by introducing a DNA encod- preferably water, whereby CDP-choline is eluted from 55 ing an enzyme that catalyzes a reaction to produce CDP- the resin, for the separation and purification. choline from a precursor of UTP and choline or phospho- [0013] Where necessary, the CDP-choline-containing choline. Preferable examples of the microorganism in- solution collected by the above-mentioned elution step clude microorganisms belonging to the generaEs-
3 5 EP 1 939 210 B1 6 cherichia, Serratia, Bacillus, Brevibacterium, Coryne- ganism belonging to the genus Curtobacterium include bacterium, Microbacterium, Pseudomonas, Streptococ- Curtobacterium albidum, Curtobacterium citreum and cus, Sinorhizobium, Haemophilus, Arthrobacter, Aureo- Curtobacterium luteum. Examples of the microorganism bacterium, Cellulomonas, Clavibacter, Curtobacterium, belonging to the genus Pimerobacter include Pimero- Pimerobacter, Saccharomyces, Schizosaccharomyces, 5 bacter simplex. Kluyveromyces, Trichosporon , Schwanniomyces, Pichia [0021] Examples of the microorganism belonging to or Candida. the genus Saccharomyces include Saccharomyces cer- [0020] Examples of the microorganism belonging to evisiae. Examples of the microorganism belonging to the the genus Escherichia include microorganisms belong- genus Schizosaccharomyces include Schizosaccharo- ing to Escherichia coli such as Escherichia coli MM294, 10 myces pombe. Examples of the microorganism belong- Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, ing to the genus Kluyveromyces include Kluyveromyces Escherichia coli DH1, Escherichia coli MC1000, Es- lactis. Examples of the microorganism belonging to the cherichia coli KY3276, Escherichia coli W1485, Es- genus Trichosporon include Trichosporon pullulans. Ex- cherichia coli JM109, Escherichia coli HB101, Es- amples of the microorganism belonging to the genus cherichia coli No.49, Escherichia coli W3110, Es- 15 Schwanniomyces include Schwanniomyces alluvius. Ex- cherichia coli NY49, Escherichia coli GI698 and Es- amples of the microorganism belonging to the genus cherichia coli TB1. Examples of the microorganism be- Pichia include Pichia pastoris. Examples of the microor- longing to the genus Serratia include Serratiaficaria , Ser- ganism belonging to the genus Candida include Candida ratia fonticola, Serratia liquefaciens and Serratia marc- utilis. escens. Examples of the microorganism belonging to the 20 [0022] In addition, more preferable examples of the mi- genus Bacillus include Bacillus subtilis, Bacillus mega- croorganism include microorganisms belonging to the terium and Bacillus amyloliquefaciens. Examples of the genera Escherichia, Bacillus, Brevibacterium, Coryne- microorganism belonging to the genusBrevibacterium bacterium and Saccharomyces, further preferably, mi- include Brevibacterium immariophilum, Brevibacterium croorganisms belonging to the generaEscherichia , saccharolyticum, Brevibacterium flavum and Brevibac- 25 Brevibacterium, Corynebacterium. terium lactofermentum. Examples of the microorganism [0023] Of the above-mentioned microorganisms, belonging to the genus Corynebacterium include micro- when a microorganism naturally having a CDP-choline- organisms belonging to Corynebacterium glutamicum producing activity has only an insufficient CDP-choline- such as Corynebacterium glutamicum ATCC130332 or producing activity, a recombinant DNA having a DNA en- Corynebacterium glutamicum ATCC13869, microorgan- 30 coding an enzyme that catalyzes a reaction to produce isms belonging toCorynebacterium ammoniagenes CDP-choline from a precursor of UTP and choline or such as Corynebacterium ammoniagenes ATCC6872 or phosphorylcholine may be introduced into the microor- Corynebacterium ammoniagenes ATCC21170, and mi- ganism according to a conventional method, or a cell of croorganisms belonging to Corynebacterium acetoaci- different microorganism having the activity may be fused dophilum such as Corynebacterium acetoacidophilum 35 therewith to prepare a microorganism having an en- ATCC13870, etc. Examples of the microorganism be- hanced activity. longing to the genus Microbacterium include microorgan- [0024] As the microorganism having an enhanced ac- isms belonging to Microbacterium ammoniaphilum such tivity or a microorganism imparted with the activity, a as Microbacterium ammoniaphilum ATCC15354, Micro- transformant obtained by introducing a DNA encoding bacterium lactium and Microbacterium imperiale . Exam- 40 an enzyme that catalyzes the reaction into a microorgan- ples of the microorganism belonging to the genus Pseu- ism according to the following method can be preferably domonas include Pseudomonas putida. Examples of the used. microorganism belonging to the genus Streptococcus in- [0025] Examples of the DNA encoding an enzyme that clude Streptococcus pneumoniae. Examples of the mi- catalyzes a reaction to produce CDP-choline from a pre- croorganism belonging to the genus Sinorhizobium in- 45 cursor of UTP and choline or phosphorylcholine (herein- clude Sinorhizobium meliloti. Examples of the microor- after to be abbreviated as a CDP-choline-producing en- ganism belonging to the genusHaemophilus include zyme) include DNAs encoding orotate phosphoribosyl Haemophilus influenzae. Examples of the microorgan- transferase [EC 2.4.2.10] having an activity to produce ism belonging to the genus Arthrobacter include Arthro- OMP from orotic acid, orotidine-5’-monophosphate de- bacter citreus and Arthrobacter globiformis. Examples of 50 carboxylase [EC 4.1.1.23] having an activity to produce the microorganism belonging to the genus Aureobacte- UMP from OMP, uridine phosphorylase [EC 2.4.2.3] hav- rium include Aureobacterium flavescens, Aureobacteri- ing an activity to produce uridine from uracil, uracil phos- um saperdae and Aureobacterium testaceum. Examples phoribosyltransferase [EC 2.4.2.9] having an activity to of the microorganism belonging to the genusCellulo- produce UMP from uracil, uridine kinase [EC 2.7.1.48] monas include Cellulomonas flavigena and Cellulo-55 having an activity to produce UMP from uridine, uridylate monas carta. Examples of the microorganism belonging and cytidylate kinases [EC 2.7.1.48] having an activity to to the genus Clavibacter include Clavibacter michigan- produce UDP from UMP, nucleoside diphosphate kinase ensis and Clavibacter rathayi. Examples of the microor- [EC 2.7.4.6] having an activity to produce UTP from UDP,
4 7 EP 1 939 210 B1 8 cytidine-5’-triphosphate synthetase [EC 6.3.4.2] (herein- [0033] Where necessary, moreover, a DNA wherein after to be abbreviated as PyrG) having an activity to nucleotide of the nucleotide sequence of a partial DNA produce CTP from UTP, choline kinase [EC 2.7.1.32] fragment corresponding to a CDP-choline-producing en- (hereinafter to be abbreviated as CKI) having an activity zyme are substituted so as to provide a codon optimal to produce phosphorylcholine from choline and choline 5 for the expression of a host cell is prepared. The DNA is phosphate cytidyltransferase [EC 2.7.7.15] (hereinafter useful for an efficient production of a CDP-choline-pro- to be abbreviated as CCT) having an activity to produce ducing enzyme. CDP-choline from CTP and phosphorylcholine. [0034] A recombinant vector is produced by inserting [0026] Preferable examples of the DNA encoding a theDNA fragmentor full-length DNA into the downstream CDP-choline-producing enzyme include DNAs encoding 10 of a promoter of a suitable expression vector. In this case, PyrG, CKI and CCT. DNA encoding a CDP-choline-producing enzyme may [0027] A DNA encoding PyrG has been cloned from be independently inserted into an expression vector, or the chromosome of Escherichia coli, and its entire nucle- plural DNAs may be inserted into the same expression otide sequence has been determined [J. Biol. Chem., vector. 261, 5568(1986)]. Examples of a recombinant having a 15 [0035] The recombinant vector is introduced into a host DNA encoding PyrG include pMW6 [Biosci. Biotechnol. cell suitable for the expression vector. Biochem., 61, 956 (1997)], which is a plasmid having a [0036] As the host cell, the above-mentioned microor- 2426 bp NruI-PstI fragment containing a DNA encoding ganisms can be mentioned. PyrG derived from Escherichia coli inserted into an SmaI- [0037] As the expression vector, one capable of au- PstI site of a multicloning site of vector pUC8 Es- of 20 tonomous replication or one capable of being incorporat- cherichia coli [Gene, 19, 259 (1982)]. ed into a chromosome in the host cell, and having a pro- [0028] The entire nucleotide sequence of the DNA en- moter at a site permitting transcription of a DNA encoding coding CCT has been determined [Eur. J. Biochem., 169, a CDP-choline-producing enzyme can be used. 477(1987)]. Examples of a recombinant DNA having a [0038] When prokaryote such as bacterium is used as DNA encoding CCT include plasmid pCC41 [Biochemi- 25 a host cell, a recombinant vector having a DNA encoding cal, 60, 701(1988)] having a 1296 bp DraI fragment con- a CDP-choline-producing enzyme is preferably autono- taining a DNA encoding CCT derived from yeast inserted mously replicatablein a prokaryote, as well as constituted into a SmaI site of a multicloning site of vector pUC18 of with a promoter, a ribosome binding sequence, the DNA Escherichia coli [Gene, 33, 103(1985)]. and a transcription termination sequence. A gene regu- [0029] A DNA encoding CKI has also been cloned from 30 lating the promoter may also be contained. a chromosome of yeast in the same manner, and the [0039] Examples of the expression vector include entire nucleotide sequence thereof has been determined pBTrp2, pBTac1, pBTac2 (all commercially available [J. Biol. Chem., 264, 2053(1989)]. Examples of a recom- from Boehringer Mannheim), pKK233-2 (manufactured binant DNA having a DNA encoding CKI include plasmid by Pharmacia), pSE280 (manufactured by Invitrogen), pCK1D [J. Biol. Chem., 264, 2053(1989)] having a 2692 35 pGEMEX-1 (manufactured by Promega), pQE-8 (manu- bp PstI-HindIII fragment containing a DNA encoding CKI factured by QIAGEN), pKYP10 (Japanese Published Un- derived from yeast inserted into a shuttle vector YEpM4 examined Patent Application No. 110600/58), pKYP200 of yeast and Escherichia coli, [Mol. Cell. Biol., 7, 29 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. (1987)]. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. [0030] The above-mentioned plasmids can be isolated 40 USA, 82, 4306 (1985)], pBluescript II SK(-) (manufac- and purified from Escherichia coli having these plasmids, tured by Stratagene), pTrs30 [prepared from Escherichia according to a known method [Nuc. Acids Res., 7, coli JM109/pTrS30 (FERM BP-5407)], pTrs32 [prepared 1513(1979)]. from Escherichia coli JM109/pTrS32 (FERM BP-5408)], [0031] A biocatalyst having a CDP-choline-producing pGHA2 [prepared from Escherichia coli IGHA2 (FERM activity can be obtained by obtaining a DNA encoding a 45 BP-400), Japanese Published Unexamined Patent Ap- CDP-choline-producing enzyme from the plasmid ob- plication No. 221091/60], pGKA2 [prepared fromEs- tained as mentioned above according to, for example, cherichia coli IGKA2 (FERM BP-6798), Japanese Pub- Molecular Cloning, A Laboratory Manual, Third Edition, lished Unexamined Patent Application No. 221091/60], Sambrook et al. edit, Cold Spring Harbor Laboratory pTerm2 (US Patent 4686191, US Patent 4939094, US (2001), incorporating the DNA into an expression vector 50 Patent 5160735), pSupex, pUB110, pTP5, pC194, to prepare a recombinant DNA, and transforming the pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (manu- above-mentioned microorganism as a host cell. factured by Pharmacia), and pET system (manufactured [0032] First, a DNA encoding PyrG, CCT or CKI is ob- by Novagen). tained from the above-mentioned plasmid pMW6, plas- [0040] As the promoter, any promoter can be used as mid pCC41 or plasmid pCK1D and, based on the ob-55 long as it is functionable in the host cell. Examples of the tained DNA, a DNA fragment having a suitable length promoter include those derived from Escherichia coli or and containing a part encoding the polypeptide is pre- on phage, such as trp promoter (Ptrp), lac promoter, PL pared as necessary. promoter, PR promoter or T7 promoter. In addition, a pro-
5 9 EP 1 939 210 B1 10 moter having an artificially altered design such as a pro- Haemophilus, Arthrobacter, Aureobacterium, Cellulo- moter (Ptrp32) in which two P trp are sequently connected monas, Clavibacter, Curtobacterium, Pimerobacter, in tandem, tac promoter, lac T7 promoter and let I pro- Saccharomyces, Schizosaccharomyces, Kluyveromy- moter can also be used. ces, Trichosporon, Schwanniomyces, Pichia and Cand- [0041] It is preferable to use a plasmid in which the 5 ida. distance between a Shine-Dalgarno sequence (ribos- [0049] For example, a combination of the microorgan- ome binding sequence) and an initiation codon is adjust- ism belonging to the genus Corynebacterium and a mi- ed to a suitable distance (e.g., 6 to 18 bases). croorganism belonging to the genus Escherichia can be [0042] In the recombinant vector of the present inven- mentioned. Specific example is a combination of Coryne- tion, a transcription termination sequence is not always 10 bacterium ammoniagenes ATCC21170 and Escherichia necessarily for the expression of a DNA encoding a CDP- coli MM294/pCKG55 strain (FERM BP-3717) (Japanese choline-producing enzyme. However, it is preferable to Patent No. 3369236, US Patent No. 6387667). placea transcription terminationsequence directly down- [0050] The example of the culture of a microorganism stream the structure gene. having a CDP-choline-producing activity, which is one of [0043] As a method of introducing the recombinant 15 the biocatalysts having a CDP-choline-producing activi- vector, any method can be used as long as a DNA can ty, is a culture obtained by culturing, according to a con- be introduced into the above-mentioned host cell. For ventional method, the microorganism obtained by the example, a method using a calcium ion [Proc. Natl. Acad. above-mentioned method. Sci. USA, 69, 2110 (1972)], a protoplast method (Japa- [0051] When the microorganism is a prokaryote such nese Published Unexamined Patent Application No.20 as bacteria or a eukaryote such as yeast, any of a natural 248394/1988), the method described in Gene, 17, 107 medium and a synthetic medium can be used as the me- (1982) or Molecular & General Genetics, 168, 111 (1979) dium used for culturing the microorganism as long as it can be used. contains a carbon source, a nitrogen source and/or inor- [0044] When a yeast is used as the host cell, for ex- ganic salts to be assimilated by the microorganism, and ample, YEp13 (ATCC37115), YEp24 (ATCC37051),25 enables efficient culturing of the microorganism. YCp50 (ATCC37419), pHS19 or pHS15 can be used as [0052] Any carbon source may be used as long as the the expression vector. microorganism can assimilate it, and glucose, fructose, [0045] As the promoter, any promoter can be used as sucrose, molasses containing them, carbohydrates such long as it permits expression in a yeast strain. Examples as starch and starch hydrolysate, organic acid such as of the promoter include promoters of genes of the glyc- 30 acetic acid or propionic acid, or alcohols such as ethanol olytic system such as hexose kinase, PHO5 promoter, or propanol can be used. PGK promoter, GAP promoter, ADH promoter, gal 1 pro- [0053] As the nitrogen source, ammonium salts of in- moter, gal 10 promoter, heat shock polypeptide promot- organic acid or organic acid such as ammonia, ammoni- er, MFα1 promoter and CUP 1 promoter. um chloride, ammonium sulfate, ammonium acetate or [0046] As the method of introducing the recombinant 35 ammonium phosphate, other nitrogen-containing com- vector, any method can be used as long as it introduces pounds, peptone, meat extract, yeast extract, corn steep the DNA into a yeast. Examples thereof include an elec- liquor, casein hydrolysate, soybean cake and soybean troporation method [Methods Enzymol., 194, 182 cake hydrolysate, or various bacterial cells obtained by (1990)], a spheroplast method [Proc. Natl. Acad. Sci. fermentation and digests thereof, can be used. USA, 75, 1929 (1978)], a lithium acetate method [J. Bac- 40 [0054] As the inorganic salt, monopotassium phos- teriology, 153, 163 (1983)], and the method described in phate, dipotassium phosphate, magnesium phosphate, Proc. Natl. Acad. Sci. USA, 75, 1929 (1978). magnesium sulfate, sodium chloride, ferrous sulfate, [0047] When the microorganism has only a part of the manganese sulfate, copper sulfate or calcium carbonate CDP-choline-producing activity, two or more kinds of mi- can be used. croorganisms may be combined as appropriate to afford 45 [0055] Culturing is performed under aerobic conditions a CDP-choline-producing activity, and used as the bio- by, for example, shaking culturing or aerobic stirred cul- catalyst having a CDP-choline-producing activity. Even turing. The culturing temperature is preferably 15°C - when the microorganism has a CDP-choline-producing 40°C and the culturing time is generally 16 hours - 7 days. activity, two or more kinds of microorganisms can be The pH is preferably maintained 3 - 9 during culturing. combined. 50 The pH is adjusted with inorganic or organic acid, alkali [0048] Two or more kinds of microorganisms to be solution, urea, calcium carbonate or ammonia. combined may be selected from any of the above-men- [0056] When the microorganism is a transformant, and tioned microorganisms. Examples of the combination in- the recombinant DNA used for transforming the micro- clude a combination of microorganisms belonging to the organism has an antibiotic-resistance gene, an antibiotic same genus or different genera selected from the micro- 55 corresponding to the antibiotic-resistance gene pos- organisms belonging to the genera Escherichia, Serratia, sessed by the recombinant DNA may be added to the Bacillus, Brevibacterium, Corynebacterium, Microbacte- medium used for culturing the microorganism. rium, Pseudomonas, Streptococcus, Sinorhizobium, [0057] When a culture of two or more kinds of micro-
6 11 EP 1 939 210 B1 12 organisms or a treated culture is used as the biocatalyst, depending on the specific activity of the biocatalyst. For respective microorganisms are cultured separately or in example, when the culture of the microorganism or the the same medium according to the above-mentioned treated product of the culture is used as the biocatalyst, method and the resulting product can be used. it is preferably used in an amount of 5 - 500 mg, preferably [0058] When two or more kinds of microorganisms are 5 10 - 300 mg, relative to 1 mg of choline chloride, as wet cultured in the same medium, these microorganisms may cells obtained by centrifuging the culture or the treated be cultured simultaneously, or one microorganism may culture. be cultured and the rest of the microorganisms may be [0065] Examples of choline, phosphorylcholine and cultured in the same medium during the culture of the salts thereof include choline, choline halides such as first microorganism or after completion of the culture10 choline chloride, choline bromide, choline iodide, choline thereof. bicarbonate, choline methylsulfate, choline dihydrogen [0059] Examples of the treated culture of the microor- citrate, choline bitartrate, phosphorylcholine, phospho- ganism include a surfactant-treated culture, organic sol- rylcholine halides such as phosphorylcholine chloride, vent-treated culture or cytolytic enzyme-treated culture etc., preferably choline or phosphorylcholine halide, obtained by treating the culture of the microorganism ob- 15 more preferably choline chloride or phosphorylcholine tained by the above-mentioned method with a surfactant, chloride. an organic solvent or a cytolytic enzyme such as lys- [0066] The precursor of UTP, choline, phosphorylcho- ozyme. A surfactant, an organic solvent or a cytolytic line and salts thereof may be obtained by chemical syn- enzyme may be used singly to treat the culture of the thesis, or obtained from an organism by a fermentation microorganism, or they may be combined to treat the 20 method. In addition, it is not necessarily to purify them culture of the microorganism. In addition, a concentrate completely. Furthermore, all of these substrates are com- or dried product of the culture of the microorganism, mercially available and can be obtained easily. which is obtained by concentrating or drying, in a con- [0067] The concentration of the precursor of UTP, centrating machine or a desiccator, a culture of the mi- choline, phosphorylcholine and salts thereof is preferably croorganism obtained by the above-mentioned method, 25 1 mmol/L - 1 mol/L, more preferably 10 - 100 mmol/L. cells obtained by solid-liquid separation of the culture of [0068] Examples of other necessary components in- the microorganism by filtration or centrifugation, and the clude energy donor necessary for producing CDP- dried product of the cells obtained by drying the cells in choline, phosphate ion, magnesium ion, ammonium ion, a desiccator can be mentioned. Furthermore, the sur- surfactant or organic solvent, Addition of these compo- factant-treated product, the organic solvent-treated prod- 30 nents is not necessary when they are provided in neces- uct and the cytolytic enzyme-treated product of the cells sary quantities from a biocatalyst and the like. can be mentioned, which are obtained by treating the [0069] As the energy donor, sugars such as glucose, cells with a surfactant, an organic solvent or a cytolytic fructose or sucrose, molasses, starch hydrolysate, and enzyme such as lysozyme, or a combination of these amino acids such as glycine or alanine can be used. They treated products. 35 are preferably used at the concentration of 0.02 - 2.0 [0060] When two or more kinds of microorganisms are mol/L. used, two or more kinds of treated cultures may be indi- [0070] As the phosphate ion, inorganic phosphate vidually used as biocatalysts having the CDP-choline- such as orthophosphoric acid, pyrophosphoric acid, producing activity, or a mixture obtained by mixing these polyphosphoric acid such as tripolyphosphoric acid, treated products of the cultures may be used as the bio- 40 tetrapolyphosphoric acid, polymetaphosphoric acid, mo- catalyst having a CDP-choline-producing activity. nopotassium phosphate, dipotassium phosphate, mono- [0061] CDP-choline can be produced by bringing the sodium phosphate or disodium phosphate, etc. can be above-mentioned biocatalyst in contact with a precursor used. These phosphate ions are preferably used at a of UTP and choline or phosphorylcholine or a salt thereof concentration of 10 - 500 mmol/L. in a medium to allow production and accumulation of45 [0071] As the magnesium ion, inorganic magnesium CDP-choline in the medium and recovering CDP-choline salt such as magnesium sulfate, magnesium nitrate or from the medium. magnesium chloride, and organic magnesium salt such [0062] Examples of the precursor of UTP include orotic as magnesium citrate can be used. Magnesium ion is acid, OMP, uracil, uridine, UMP and UDP, with prefer- preferably used at a concentration of 5 - 200 mmol/L. ence given to orotic acid and uracil. 50 [0072] As the ammonium ion, aqueous ammonia, am- [0063] A specific method of producing and accumulat- monia gas, various inorganic or organic ammonia salts, ing CDP-choline comprises mixing the above-mentioned yeast extract or corn steep liquor can be used. In addition, biocatalyst and the precursor of UTP and choline or phos- an organic nutrient source such as glutamine, peptide phorylcholine or a salt thereof in a medium, adding other containing glutamine or casamino acid can also be used components as necessary to the obtained mixture and 55 in place of ammonium ion. The concentration of these maintaining the mixture at 20 - 50°C for 2 - 48 hours while ammonium ions is preferably used at a concentration of keeping pH 5 - 11, more preferably 6 - 10. 10 mmol/L - 2 mol/L. [0064] The amount of the biocatalyst to be used varies [0073] As the surfactant, an anionic surfactant such as
7 13 EP 1 939 210 B1 14 sodium dioctylsulfosuccinate (e.g., Rapisol B-80, manu- above-mentioned enzyme in contact with the precursor factured by NOF Corporation) or lauroyl sarcosinate, a of UTP and choline or phosphorylcholine or a salt thereof non-ionic surfactant such as polyoxyethylene cetyl ether in the medium to produce and accumulate CDP-choline, (e.g., Nonion P-208, manufactured by NOF Corporation), and recovering CDP-choline from the medium. or tertiary amines such as alkyldimethylamine (e.g., ter- 5 [0083] A specific method of producing and accumulat- tiary amine FB, manufactured by NOF Corporation), etc. ing CDP-choline comprises mixing the above-mentioned can also be used as long as production of CDP-choline enzyme and the precursor of UTP and choline or phos- is promoted. These are used in the range of generally phorylcholine or a salt thereof in a medium, adding other 0.1 - 100 g/L, preferably 1 - 50 g/L. components as necessary to the obtained mixture and [0074] Examples of the organic solvent include xylene, 10 maintaining the mixture at 20 - 50°C for 2 - 48 hours while toluene, aliphatic alcohol (methyl alcohol, ethyl alcohol, keeping pH 5 - 11, more preferably 6 - 10. butyl alcohol, etc.), acetone, ethyl acetate and dimethyl [0084] The amount of the CDP-producing enzyme to sulfoxide. These are used at a concentration of generally be used varies depending on the specific activity of the 0.1 - 100 mL/L, preferably 1 - 50 mL/L. enzyme. For example, when the crude enzyme is used [0075] As the medium in which the biocatalyst is con- 15 as the enzyme, it is preferably used in an amount of 1 tacted with the precursor of UTP and choline or phos- mg - 500 mg, preferably 10 mg - 300 mg, relative to 1 mg phorylcholine or a salt thereof, the medium for culturing of choline chloride. the microorganism to be used as a biocatalyst, the culture [0085] The precursor of UTP, choline, phosphorylcho- of the microorganism or the supernatant of the culture line, a salt thereof to be added and other components to can be used, and an aqueous medium can also be used. 20 be added as necessary when producing and accumulat- [0076] Examples of the aqueous medium include wa- ing CDP-choline using the CDP-choline-producing en- ter and buffers such as phosphate buffer, HEPES (N-2- zyme are similar to those used for producing and accu- hydroxyethylpiperazine-N-ethanesulfonic acid) buffers mulating CDP-choline using the culture of the microor- or trips [tris(hydroxymethyl)aminomethane] hydrochlo- ganism as mentioned above. Furthermore, adenosine- ride buffer. 25 5’-triphosphate may be added as necessary as an energy [0077] Any organic solvent may be added to the me- donor, and 5-phosphoribosyl diphosphate may be further dium as long as the reaction is not inhibited. Examples added. of the organic solvent include acetone, ethyl acetate, [0086] As the medium in which the CDP-choline-pro- dimethyl sulfoxide, xylene, methyl alcohol, ethyl alcohol ducing enzyme is brought into contact with the precursor and butanol. 30 of UTP and choline or phosphorylcholine or a salt thereof, [0078] Examples of the method of producing CDP- the medium used for culturing the microorganism to be choline includes a method (Japanese Patent No. used as the biocatalyst, the culture or the supernatant of 3369236, US Patent No. 6387667) including producing the culture of the microorganism may be used, or an CDP-choline using Corynebacterium ammoniagenes aqueous medium may also be used. ATCC21170and Escherichiacoli MM294/pCKG55 strain 35 [0087] Examples of the aqueous medium include wa- (FERM BP-3717) as the biocatalyst. ter, buffers such as phosphate buffer, HEPES (N-2-hy- [0079] Examples of the CDP-choline-producing en- droxyethylpiperazine-N-ethanesulfonic acid) buffer and zyme include one or more enzymes selected from the tris [tris(hydroxymethyl)aminomethane] hydrochloride. group consisting of orotate phosphoribosyl transferase, [0088] Forpreparing theCDP-choline solutionfrom the orotidine-5’-monophosphate decarboxylase, uridine40 medium in which CDP-choline has been produced and phosphorylase, uracil phosphoribosyltransferase, urid- accumulated as mentioned above, a solid may be sep- ine kinase, uridylate and cytidylate kinases, nucleoside arated and removed from the medium according to a diphosphate kinase, PyrG, CCT and CKI. method using membrane separation, filtration or centrif- [0080] A crude enzyme or a purified enzyme obtained ugation. by disrupting the microorganism having the above-men- 45 [0089] Examples of the nucleic acid analogue con- tioned enzyme activity with a homogenizer, and further tained in the CDP-choline solution prepared by the applying a general enzyme purification method such as above-mentioned method include uracil and UTP. salting out, isoelectric point precipitation, organic solvent [0090] CDP-choline and nucleic acid analogs can be precipitation, dialysis or various chromatography treat- analyzed by a conventional method using high perform- ments can be used as the CDP-choline-producing en- 50 ance liquid chromatography (UV detection). zyme. In addition, the disrupted product of the microor- [0091] The present invention is explained in more de- ganism may be used as it is as the above-mentioned tail in the following by referring to Examples, which are enzyme. not to be construed as limitative. [0081] In addition, the above-mentioned disrupted product of microorganism, crude enzyme or purified en- 55 zyme may be immobilized on a water-insoluble carrier or gel, and used as the above-mentioned enzyme. [0082] CDP-choline can be produced by bringing the
8 15 EP 1 939 210 B1 16
Example 1 [0094] On the other hand, Corynebacterium·ammoni- agenes ATCC21170 strain having an activity to produce Purification of CDP-choline using strongly acidic cation UTP from orotic acid was inoculated to a test tube con- exchange resin taining a liquid medium (10 mL) having a composition of 5 glucose (50 g/L), polypeptone (10 g/L; manufactured by [0092] CDP-choline (50 g; manufactured by Wako Daigo Eiyo Chemicals), yeast extract (10 g/L; manufac- Pure Chemical Industries, Ltd.), uracil (2 g; manufactured tured by Daigo Eiyo Chemicals), urea (5 g/L), (NH 4)2SO4 by Nacalai Tesque) and UTP (1 g; manufactured by Na- (5 g/L), KH2PO4 (1 g/L), K2HPO4 (3 g/L), MgSO4·7H2O calai Tesque) were dissolved in water to prepare 5 L of (1 g/L), CaCl2·2H2O (0.1 g/L), FeSO4·7H2O (10 mg/L), 10 CDP-choline solution. The solution was adjusted to pH ZnSO4·7H2O (10 mg/L), MnSO4·4 - 6H2O (20 mg/L), L- 3.0 with sulfuric acid and applied to a column packed with cysteine (20 mg/L), calcium D-pantothenate (10 mg/L), a strongly acidic cation exchange resin Diaion PK208 (H- vitamin B1 (5 mg/L), nicotinic acid (5 mg/L) and biotin (30 type) (10 L) with a crosslinking degree of 4%. Subse- mg/L; adjusted to pH 7.2 with sodium hydroxide), and quently, water was applied, and a fraction wherein each cultured with reciprocal shaking at 300 rpm at 28°C for concentration of uracil and UTP was less than 0.1% (w/w) 15 24 hours. The culture thus obtained (20 mL) was inocu- relative to CDP-choline was obtained as a result of anal- lated to a 2 L conical flask with baffle containing a liquid ysis by high performance liquid chromatography. The medium (230 mL) having the same composition as the CDP-choline fraction was concentrated to 100 mL, and above-mentioned, and cultured with rotary shaking at ethanol (350 mL) was gradually added. The precipitated 190 rpm at 28°C for 24 hours. The culture thus obtained crystals were collected by filtration, washed with 100% 20 (250 mL) was inoculated to a 5 L culture bath containing ethanol solution, and dried under reduced pressure at a liquid medium (2.5 L) having a composition of glucose 20°C for 3 days. As the result, 40 g of CDP-choline crys- (100 g/L), meat extract (10 g/L), polypeptone (10 g/L), tals, wherein each concentration of uracil and UTP was KH2PO4 (1 g/L), K2HPO4 (1 g/L), MgSO4.7H2O (1 g/L), less than 0.1% (w/w) relative to CDP-choline, were ob- CaCl2·2H2O (0.1 g/L), FeSO4·7H2O (20 mg/L), 25 tained. ZnSO4·7H2O (10 mg/L), MnSO9·4 - 6H2O (20 mg/L), β- alanine (15 mg/L), L-cysteine (20 mg/L), biotin (100 Example 2 mg/L), urea (2 g/L; separately sterilized) and vitamin B1 (5 mg/L; separately sterilized) (adjusted to pH 7.2 with Purification of CDP-choline from culture of microorgan- sodium hydroxide) and seed-cultured with shaking at 600 ism having an activity to produce CDP-choline 30 rpm at 32°C under culture condition of airflow 2.5 L/minute while adjusting the mixture to pH 6.8 with con- [0093] Escherichia coli MM294/pCKG55 strain (FERM centrated aqueous ammonia. At the time point when glu- BP-3717) having enzyme activities of PyrG, CCT and cose in the supernatant of the above-mentioned seed- CKI was inoculated to a test tube containing L medium culture medium was consumed, the culture (350 mL) was (10 mL) [liquid medium containing bactotripton (10 g/L; 35 aseptically collected, inoculated to a 5 L culture bath con- manufactured by Difco), yeast extract (5 g/L; manufac- taining a liquid medium (2.5 L) having a composition of tured by Difco) and NaCl (5 g/L) and adjusted to pH 7.2] glucose (180 g/L), KH2PO4 (10 g/L), K2HPO4 (10 g/L), supplemented with ampicillin (50 mg/mL), and cultured MgSO4·7H2O (10 g/L), 2 CaCl·2H2O (0.1 g/L), with shaking at 25°C, 300 rpm for 24 hours. The culture FeSO4·7H2O (20 mg/L), ZnSO4·7H2O (10 mg/L), 40 thus obtained (20 mL) was inoculated to 2 L of conical MnSO4·4 - 6H2O (20 mg/L; separately sterilized),β - flask with baffles containing L medium (400 mL) supple- alanine (15 mg/L), L-cysteine (20 mg/L), sodium gluta- mented with ampicillin (50 mg/mL) and cultured with ro- mate (1 g/L), biotin (100 mg/L), urea (2 g/L; separately tary shaking at 25°C, 190 rpm for 16 hours. The culture sterilized) and vitamin B1 (5 mg/L; separately sterilized) thus obtained (125 mL) was inoculated to a 5 L culture (adjusted to pH 7.2 with sodium hydroxide) and main- bath containing a liquid medium (2.5 L; nonadjusted pH) 45 cultured with shaking at 600 rpm at 32°C under culture with a composition of glucose (5 g/L; separately steri- condition of airflow 2.5 L/minute while adjusting the mix- lized), peptone (5 g/L; manufactured by Kyokuto Phar- ture to pH 6.8 with concentrated aqueous ammonia. The maceutical Industrial), Na 2HPO4 (6 g/L), KH 2PO4 (3 g/L), culturing was ceased at the time point when glucose in NaCl (5 g/L), NH4Cl (1 g/L), MgSO4·7H2O (250 mg/L; the culture supernatant was consumed. separately sterilized) and vitamin B1 (4 mg/L; separately 50 [0095] The culture (360 mL) of Escherichia coli sterilized), and cultured with shaking at 600 rpm under MM294/pCKG55 strain and the culture (360 mL) of Co- culture condition of airflow 2.5 L/minute, at 25°C for 11 rynebacterium·ammoniagenes ATCC21170 strain thus hours and then at 32°C for 13 hours, while adjusting the obtained were placed in a 2 L culture bath, glucose (100 mixture to pH 7.0 with 14% aqueous ammonia. During g/L), orotic acid (10 g/L), choline chloride (8.4 g/L), 55 culturing, a feed solution having a composition of glucose MgSO4·7H2O (5 g/L) and xylene (20 mL/L) were added (167 g/L), peptone (167 g/L) was fed with a peristaltic thereto, and distilled water was added to allow the total pump at a rate of 30 mL/ hours during the period of from amount to be 800 mL. The mixture was reacted with shak- 11 hours to 24 hours from the start of the culturing. ing at 800 rpm at 32°C under the condition of airflow 0.8
9 17 EP 1 939 210 B1 18
L/minute while adjusting the mixture to pH 7.2 with 10N that CDP-choline or a salt thereof free from impurity can sodium hydroxide. During the reaction, KH2PO4 was be obtained conveniently by treating a CDP-choline so- added as appropriate to keep the concentration of phos- lution containing a nucleic acid analogue only once with phoric acid in the supernatant of the reaction mixture at a strongly acidic cation exchange resin. 5 1 - 5 g/L as KH 2PO4. The reaction was carried out for 23 hours to obtain 11.0 g/L of CDP-choline. Industrial Applicability [0096] Four batches of the above-mentioned reaction mixture were adjusted to pH 1.0 with sulfuric acid, cells [0099] According to the present invention, CDP- were separated by centrifugation (7000 rpm, 10 min), choline and a salt thereof are provided at a low cost. and water was added to the obtained supernatant to10 make the total amount 6 L (CDP-choline 6.0 g/L, uracil 0.5 g/L, UTP 1.0 g/L). This cytidinediphosphoric choline Claims solution was applied to a column packed with a strongly acidic cation exchange resin Diaion SK104 (H-type, 10 1. A method of purifying cytidine diphosphate choline L) having a crosslinking degree of 4%. Subsequently, 15 (hereinafter abbreviated as CDP-choline), which water was applied, and a fraction, in which each concen- comprises contacting a CDP-choline solution con- tration of uracil, UTP was less than 0.1%(w/w) relative taining a nucleic acid analogue and having a pH of to CDP-choline (analyzed by high performance liquid not less than 0.5 and not more than 5.0 with an H- chromatography) was collected. The CDP-choline frac- type strongly acidic cation exchange resin, and elut- tion was decolorized using activated carbon, and then 20 ing CDP-choline adsorbed onto the resin with water concentrated to 100 mL. Ethanol (350 mL) was gradually or an aqueous solution having an ion concentration added to the concentrated solution, and the precipitated of not more than 0.1 mol/L to separate and purify crystals were collected by filtration. The obtained crystals CDP-choline. were washed with 100% ethanol solution, and then dried under reduced pressure at 20°C for 3 days. As a result, 25 2. The method of claim 1, wherein the CDP-choline so- CDP-choline crystals (18 g), in which each concentration lutionis prepared froma resulting mediumcontaining of uracil, UTP was less than 0.1%(w/w) relative to CDP- CDP-choline, which has been produced and accu- choline, were obtained. mulated therein by bringing a biocatalyst having an activity to produce CDP-choline from a precursor of Example 3 30 uridine-5’-triphosphate (hereinafter abbreviated as UTP) and choline or phosphorylcholine in an aque- Purification of CDP-choline sodium salt from a culture of ous medium into contact with the precursor of UTP microorganism having an ability to produce CDP-choline and choline or phosphorylcholine or a salt thereof.
[0097] Four batches of the reaction mixture obtained 35 3. The method of claim 2, wherein the biocatalyst com- in the same manner as in Example 2 were adjusted to prises a culture or a treated culture of a microorgan- pH 1.0 with sulfuric acid, cells were separated by centrif- ism capable of producing UTP from a precursor of ugation (7000 rpm, 10 min), and water was added to the UTP, and a culture or a treated culture of a microor- obtained supernatant to make the total amount 6 L (CDP- ganism capable of producing CDP-choline from UTP choline 6.0 g/L, uracil 0.5 g/L, UTP 1.0 g/L). This cytidine 40 and choline or phosphorylcholine. diphosphate choline solution was applied to a column packed with a strongly acidic cation exchange resin Di- 4. The method of claim 2, wherein the biocatalyst com- aion SK104 (H-type, 10 L) having a crosslinking degree prises an enzyme that catalyzes a reaction to pro- of 4%. Subsequently, water was applied, and a fraction, duce CDP-choline from a precursor of UTP and in which each concentration of uracil, UTP was less than 45 choline or phosphorylcholine. 0.1%(w/w)relative to CDP-choline(analyzed by high per- formance liquid chromatography) was collected. The 5. The method of claim 4, wherein the enzyme that cat- CDP-choline fraction was adjusted to pH 7.5 with sodium alyzes a reaction to produce CDP-choline is an en- hydroxide, decolorized with activated carbon, and then zyme selected from orotate phosphoribosyl trans- concentrated to 100 mL. Ethanol (400 mL) was gradually 50 ferase, orotidine-5’-monophosphate decarboxylase, added to the concentrated solution, and the precipitated uridine phosphorylase, uracil phosphoribosyltrans- crystals were collected by filtration. The obtained crystals ferase, uridine kinase, uridylate and cytidylate kinas- were washed with 100% ethanol solution, and then dried es, nucleoside diphosphate kinase, cytidine-5’-tri- under reduced pressure at 20°C for 3 days. As a result, phosphate synthase, choline phosphate cytidyl- CDP-choline sodium salt crystals (20 g), in which each 55 transferase and choline kinase. concentration of uracil, UTP was less than 0.1%(w/w) relative to CDP-choline, were obtained. 6. The method of any of claims 1 to 5, wherein the nu- [0098] From the foregoing results, it has been clarified cleic acid analogue is selected from uracil and UTP.
10 19 EP 1 939 210 B1 20
7. A process for producing CDP-choline, which com- CDP-Cholin abzutrennen und zu reinigen. prises contacting a CDP-choline solution containing a nucleic acid analogue and having a pH of not less 2. Verfahren gemäß Anspruch 1, wobei die CDP-Cho- than 0.5 and not more than 5.0 with an H-type strong- lin-Lösung aus einem resultierenden Medium her- ly acidic cation exchange resin, and eluting CDP- 5 gestellt wird, das CDP-Cholin enthält, welches da- choline adsorbed onto the resin with water or an durch darin erzeugt und angereichert wurde, dass aqueous solution having an ion concentration of not man einen Biokatalysator, der eine Aktivität zur Er- more than 0.1 mol/L to separate and purify CDP- zeugung von CDP-Cholin aus einem Vorläufer von choline. Uridin-5’-triphosphat (im Folgenden als "UTP" abge- 10 kürzt) und Cholin oder Phosphorylcholin aufweist, in 8. The process of claim 7, wherein the CDP-choline einem wässrigen Medium in Kontakt mit dem Vor- solution is prepared from a resulting medium con- läufer von UTP und Cholin oder Phosphorylcholin taining CDP-choline, which has been produced and oder einem Salz davon bringt. accumulated therein by bringing a biocatalyst having an activity to produce CDP-choline from a precursor 15 3. Verfahren gemäß Anspruch 2, wobei der Biokataly- of UTP and choline or phosphorylcholine in an aque- sator eine Kultur oder eine behandelte Kultur eines ous medium into contact with the precursor of UTP Mikroorganismus, der UTP aus einem Vorläufer von and choline or phosphorylcholine or a salt thereof. UTP erzeugen kann, und eine Kultur oder eine be- handelte Kultur eines Mikroorganismus, der CDP- 9. The process of claim 8, wherein the biocatalyst com- 20 Cholin aus UTP und Cholin oder Phosphorylcholin prises a culture or a treated culture of a microorgan- erzeugen kann, umfasst. ism capable of producing UTP from a precursor of UTP, and a culture or a treated culture of a microor- 4. Verfahren gemäß Anspruch 2, wobei der Biokataly- ganism capable of producing CDP-choline from UTP sator ein Enzym umfasst, das eine Reaktion zur Er- and choline or phosphorylcholine. 25 zeugung von CDP-Cholin aus einem Vorläufer von UTP sowie Cholin oder Phosphorylcholin katalysiert. 10. The process of claim 8, wherein the biocatalyst com- prises an enzyme that catalyzes a reaction to pro- 5. Verfahren gemäß Anspruch 4, wobei das Enzym, duce CDP-choline from a precursor of UTP and das eine Reaktion zur Erzeugung von CDP-Cholin choline or phosphorylcholine. 30 katalysiert, ein Enzym ist, das aus Orotat-Phospho- ribosyltransferase, Orotidin-5’-monophosphat-De- 11. The process of claim 10, wherein the enzyme that carboxylase, Uridin-Phosphorylase, Uracil-Phos- catalyzes a reaction to produce CDP-choline is an phoribosyltransferase, Uridin-Kinase, Uridylat- und enzyme selected from orotate phosphoribosyl trans- Cytidylat-Kinase, Nucleosiddiphosphat-Kinase, Cy- ferase, orotidine-5’-monophosphate decarboxylase, 35 tidin-5’-triphosphat-Synthase, Cholinphosphat-Cyti- uridine phosphorylase, uracil phosphoribosyltrans- dyltransferase und Cholin-Kinase ausgewählt ist. ferase, uridine kinase, uridylate and cytidylate kinas- es, nucleoside diphosphate kinase, cytidine-5’-tri- 6. Verfahren gemäß einem der Ansprüche 1 bis 5, wo- phosphate synthase, choline phosphate cytidyl- bei das Nucleinsäure-Analogon aus Uracil und UTP transferase and choline kinase. 40 ausgewählt ist.
12. The process of any of claims 7 to 11, wherein the 7. Verfahren zum Herstellen von CDP-Cholin, umfas- nucleic acid analogue is selected from uracil and send das In-Kontakt-Bringen einer CDP-Cholin-Lö- UTP. sung, die ein Nucleinsäure-Analogon enthält und ei- 45 nen pH-Wert von nicht weniger als 0,5 und nicht mehr als 5,0 aufweist, mit einem stark sauren Kati- Patentansprüche onenaustauscherharz des H-Typs und Eluieren des an das Harz adsorbierten CDP-Cholins mit Wasser 1. Verfahren zum Reinigen von Cytidindiphosphatcho- oder einer wässrigen Lösung, die eine Ionenkonzen- lin (im Folgenden als "CDP-Cholin" abgekürzt), um- 50 tration von nicht mehr als 0,1 mol/1 aufweist, um fassend das In-Kontakt-Bringen einer CDP-Cholin- CDP-Cholin abzutrennen und zu reinigen. Lösung, die ein Nucleinsäure-Analogon enthält und einen pH-Wert von nicht weniger als 0,5 und nicht 8. Verfahren gemäß Anspruch 7, wobei die CDP-Cho- mehr als 5,0 aufweist, mit einem stark sauren Kati- lin-Lösung aus einem resultierenden Medium her- onenaustauscherharz des H-Typs und Eluieren des 55 gestellt wird, das CDP-Cholin enthält, welches da- an das Harz adsorbierten CDP-Cholins mit Wasser durch darin erzeugt und angereichert wurde, dass oder einer wässrigen Lösung, die eine Ionenkonzen- man einen Biokatalysator, der eine Aktivität zur Er- tration von nicht mehr als 0,1 mol/l aufweist, um zeugung von CDP-Cholin aus einem Vorläufer von
11 21 EP 1 939 210 B1 22
UTP und Cholin oder Phosphorylcholin aufweist, in biocatalyseur comprend une culture ou une culture einem wässrigen Medium in Kontakt mit dem Vor- traitée d’un micro-organisme capable de produire de läufer von UTP und Cholin oder Phosphorylcholin l’UTP à partir d’un précurseur d’UTP, et une culture oder einem Salz davon bringt. ou une culture traitée d’un micro-organisme capable 5 de produire de la CDP-choline à partir d’UTP et de 9. Verfahren gemäß Anspruch 8, wobei der Biokataly- choline ou de phosphorylcholine. sator eine Kultur oder eine behandelte Kultur eines Mikroorganismus, der UTP aus einem Vorläufer von 4. Méthode selon la revendication 2, dans laquelle le UTP erzeugen kann, und eine Kultur oder eine be- biocatalyseur comprend une enzyme qui catalyse handelte Kultur eines Mikroorganismus, der CDP- 10 une réaction pour produire de la CDP-choline à partir Cholin aus UTP und Cholin oder Phosphorylcholin d’un précurseur d’UTP et de la choline ou de la phos- erzeugen kann, umfasst. phorylcholine.
10. Verfahren gemäß Anspruch 8, wobei der Biokataly- 5. Méthode selon la revendication 4, dans laquelle l’en- sator ein Enzym umfasst, das eine Reaktion zur Er- 15 zyme qui catalyse une réaction pour produire de la zeugung von CDP-Cholin aus einem Vorläufer von CDP-choline est une enzyme sélectionnée parmi UTP sowie Cholin oder Phosphorylcholin katalysiert. l’orotate phoshoribosyl transférase, l’orotidine-5’- monophosphate décarboxylase, l’uridine phospho- 11. Verfahren gemäß Anspruch 10, wobei das Enzym, rylase, l’uracil phosphoribosyltransférase, l’uridine das eine Reaktion zur Erzeugung von CDP-Cholin 20 kinase, l’uridylate et la citidylate kinase, la nucléosi- katalysiert, ein Enzym ist, das aus Orotat-Phospho- de disphophate kinase, la cytidine-5’-triphosphate ribosyltransferase, Orotidin-5’-monophosphat-De- synthase, la choline phosphate citidyltransférase et carboxylase, Uridin-Phosphorylase, Uracil-Phos- la choline kinase. phoribosyltransferase, Uridin-Kinase, Uridylat- und Cytidylat-Kinase, Nucleosiddiphosphat-Kinase, Cy- 25 6. Méthode selon l’une quelconque des revendications tidin-5’-triphosphat-Synthase, Cholinphosphat-Cyti- 1 à 5, dans laquelle l’analogue d’acide nucléique est dyltransferase und Cholin-Kinase ausgewählt ist. sélectionné parmi l’uracil et l’UTP.
12. Verfahren gemäß einem der Ansprüche 7 bis 11, 7. Procédé de production de CDP-choline, qui com- wobei das Nucleinsäure-Analogon aus Uracil und 30 prend la mise en contact d’une solution de CDP- UTP ausgewählt ist. choline contenant un analogue d’acide nucléique et ayant un pH d’au moins 0,5 et d’au plus 5,0 avec une résine échangeuse de cation fortement acide Revendications de type H, et l’élution de CDP-choline adsorbée sur 35 la résine avec de l’eau ou une solution aqueuse 1. Méthode de purification de la cytidine diphosphate ayant une concentration en ion d’au plus 0,1 mol/L choline (ci-après abrégée en CDP-choline) qui com- pour séparer et purifier la CDP-choline. prend la mise en contact d’une solution de CDP- choline contenant un analogue d’acide nucléique et 8. Procédé selon la revendication 7, dans lequel la so- ayant un PH d’au moins 0,5 et d’au plus 5,0 avec 40 lution de CDP-choline est préparée à partir d’un mi- une résine échangeuse de cation fortement acide lieu obtenu contenant de la CDP-choline, qui a été de type H, et l’élution de la CDP-choline adsorbée produite et accumulée à l’intérieur en mettant un bio- sur la résine avec de l’eau ou une solution aqueuse catalyseur, ayant une activité pour produire de la ayant une concentration en ion d’au plus 0,1 mol/L CDP-choline à partir d’un précurseur d’UTP et de pour séparer et purifier la CDP-choline. 45 choline ou de phosphorylcholine dans un milieu aqueux, en contact avec le précurseur d’UTP et la 2. Méthode selon la revendication 1, dans laquelle la choline ou phosphorylcholine ou un de leurs sels. solution de CDP-choline est préparée à partir d’un milieu obtenu contenant de la CDP-choline, qui a été 9. Procédé selon la revendication 8, dans lequel le bio- produite et accumulée à l’intérieur en mettant un bio- 50 catalyseur comprend une culture ou une culture trai- catalyseur ayant une activité pour produire de la tée d’un micro-organisme capable de produire de CDP-choline à partir d’un précurseur d’uridine-5’-tri- l’UTP à partir d’un précurseur d’UTP, et une culture phosphate (ci-après abrégé en UTP) et de la choline ou une culture traitée d’un micro-organisme capable ou de la phosphorylcholine dans un milieu aqueux de produire de la CDP-choline à partir d’UTP et de en contact avec le précurseur d’UTP et la choline ou 55 choline ou de phosphorylcholine. la phosphorylcholine ou un de leurs sels. 10. Procédé selon la revendication 8, dans lequel le bio- 3. Méthode selon la revendication 2, dans laquelle le catalyseur comprend une enzyme qui catalyse une
12 23 EP 1 939 210 B1 24
réaction pour produire de la CDP-choline à partir d’un précurseur d’UTP et de choline ou phosphoryl- choline.
11. Procédé selon la revendication 10, dans lequel l’en- 5 zyme qui catalyse une réaction pour produire de la CDP-choline est une enzyme sélectionnée parmi l’orotate phoshoribosyl transférase, l’orotidine-5’- monophosphate décarboxylase, l’uridine phospho- rylase, l’uracil phosphoribosyltransférase, l’uridine 10 kinase, l’uridylate et la citidylate kinase, la nucléosi- de disphophate kinase, la cytidine-5’-triphosphate synthase, la choline phosphate citidyltransférase et la choline kinase. 15 12. Procédé selon l’une quelconque des revendications 7 à 11, dans lequel l’analogue d’acide nucléique est sélectionné parmi l’uracil et l’UTP.
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REFERENCES CITED IN THE DESCRIPTION
This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.
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