WO1999049073A1 - Process for producing cytidine 5'-diphosphate choline - Google Patents

Abstract

A process for efficiently producing cytidine 5'-diphosphate choline usable in ameliorating consciousness disorder in association with heat injury or brain operation, cerebral stroke, etc. which comprises adding choline-phosphate citidyltransferase, choline kinase (unnecessary when phosphorylcholine is used) and cytidine 5'-triphosphate synthetase to a reaction system containing yeast cells, uridine 5'-monophosphate and choline or phosphorylcholine. By regulating the reaction temperature to about 20 to 30 °C and adding an energy source in portions, in particular, the productivity of the cytidine 5'-diphosphate choline can be further elevated.

Description

Bright fine manual cytidine 5 ¹ - production method art diphosphate choline

The present invention, head trauma, consciousness disorders associated with brain surgery, cytidine 5 ¹ are found using the improved treatment such as stroke - Ru der method of manufacturing a diphosphate choline (CDP-choline). Background art

Methods for producing CDP-choline using microorganisms such as yeast have been well known since ancient times. However, the conventional methods are low or the productivity of the CDP- co phosphorus is the object product, expensive cytidine as a substrate 5 ¹ - monophosphate (CMP) Ya cytidine 5'-diphosphate (CDP) and used like However, it was not a practically satisfactory method.

Further, recently, it has been reported a method of producing the CDP- choline from Oro' preparative acid and choline (Japanese Unexamined Patent Publication No. 76974), the method Urijin 5 Oro' DOO acid, Ichito Yi Lin acid (5 ¹ - A step of culturing microorganisms for producing UTP) and a step of culturing recombinant Escherichia coli that produces three enzymes involved in the synthesis of CDP-choline from UTP. This method is not always satisfactory from the viewpoint of labor and culture equipment. Disclosure of the invention

 The present inventors have conducted extensive research to establish a method for producing CDP-choline with high yield by a simple operation method.

To have an activity for converting monophosphate to efficiently 5'Application Benefits phosphoric acid, - (1) yeast cells nucleoside 5 ¹ (2) Choline phosphatidyltransferase is added to this reaction system.

(CCT), choline kinase (CKI) and cytidine 5, Ichito only Urijin 5 ¹ coexist Li phosphate synthetase (PyrG) - monophosphate (5 ¹ - UMP) and choline

CDP—the ability to produce choline,

(3) In particular, by controlling the reaction conditions in the range of about 20 ~ 30 ° C, significantly increasing the synthesis yield of CDP- co phosphorus, whereas Urijin 5 ¹ - monophosphate one glucose

(UDP-Dalcos) that the production of unintended products can be kept low.

 (4) Productivity of CDP-choline is further improved by adding energy sources such as glucose separately.

And completed the present invention.

That is, the present invention provides yeast cells, 5 ¹ - a UMP and choline, CCT, Ru der relates CDP- choline manufacturing method characterized by reacting in the presence of CKI and PyrG.

Further, the present invention is a yeast cell, 5 ¹ - a UMP and phosphorylcholine, Ru der relates CDP- choline manufacturing method characterized by reacting in the presence of CCT and PyrG. BRIEF DESCRIPTION OF THE FIGURES

 Figure 1 shows the effect of reaction temperature on CDP-choline productivity and the composition of the reaction mixture. BEST MODE FOR CARRYING OUT THE INVENTION

The yeast cells can be used in the present invention, 5 ¹ - UMP 5 ¹ - is not particularly limited as long as yeast can be converted into UTP, for example, Saccharomyces (Saccharomyces) genus, Ji Gosakkaromisesu (Zygosaccharomyces) genus Candida (Candida Genus, genus Torulopsis, genus, genus Hansenula, debaryomyces (Debaiyomyces) and the like. Among them, it is convenient to use commercially available baker's yeast, and any form of dried yeast cells and live yeast cells can be used.

 The concentration of yeast cells used is preferably about 10% by weight or less, more preferably about 2 to 5% by weight, and particularly preferably about 2 to 3% by weight in terms of dry weight.

51 ¹ -UMP, choline and phosphorylcholine used as substrates are all commercially available, and these commercially available products can be used. The use concentration of each substrate is preferably in the range of about 1 to 200 mM, particularly about 10 to 100 mM.

 CCT, CKI and PyrG coexisting in the reaction system are not limited to those of a specific origin such as those derived from animals, plants, and microorganisms, and all origins can be used. However, it is preferable to use a microorganism-derived enzyme from the viewpoint of simplicity of enzyme preparation and reaction efficiency. In addition, in order to simplify the preparation of various enzymes and to increase the preparation efficiency, the enzyme using a so-called recombinant DNA method for cloning the enzyme gene, expressing it in a large amount in a microorganism, and preparing the enzyme in large quantities. Production is most preferred.

 For example, yeast-derived enzymes can be exemplified as microbial-derived CCTs and CKIs. The yeast CCT gene has already been cloned and its entire nucleotide sequence has been determined (Eur. J. Biochem., 169, 477-486, 1987). The CKI gene has also been cloned from the yeast chromosome and its entire nucleotide sequence has been determined (J. Biol. Chem., 264, 2053-2059, 1989). Examples of PyrG include enzymes derived from Escherichia coli and Bacillus subtilis. The E. coli PyrG gene has already been cloned and its entire nucleotide sequence has been determined (J. Biol. Chem., 261, 5568-5574, 1982). Therefore, any of the genes can be cloned by the PCR method.

Sambrook et al., Cold Spring Harbor Laboratory (1989) ".

Examples of the enzyme preparation to be added to the reaction solution include microbial cells, processed products of the cells, enzyme preparations obtained from the processed products, and the like. The cells of the microorganism can be prepared by using a medium in which the microorganism can grow, culturing by a conventional method, and collecting cells by centrifugation or the like. To be more specific, using E. coli as an example, the culture media include bouillon medium, LB medium (1% tryptone, 0.5% list extract, 1% salt), 2XYT medium (1.6% tryptone, 1% salt). Yeast extract, 0.5% salt), etc., and after inoculating the medium with the inoculum, culturing at about 30 to 50 ° C for about 10 to 50 hours with stirring if necessary. Microbial cells having enzymatic activity can be prepared by centrifuging the culture solution and collecting the microbial cells.

 Microbial cell processed products include mechanical destruction of the above microbial cells (using a Waring blender, French press, homogenizer, mortar, etc.), freeze-thaw, autolysis, drying (freeze-drying, air-drying, etc.), enzymes Destruction of bacterial cells or denaturation of cell walls or cell membranes obtained by treatment according to general treatment methods such as treatment (eg, with lysozyme), ultrasonic treatment, or chemical treatment (eg, with acid or alkali treatment). Things can be exemplified.

 As the enzyme preparation, a fraction having the enzyme activity from the treated cells is purified by a conventional enzyme purification method (salt-out treatment, isoelectric point precipitation, organic solvent precipitation, dialysis, various types of chromatography) And the like).

 The synthesis reaction of CDP-choline is performed, for example, by adding yeast cells, 5'-UMP, choline or phosphorylcholine in an aqueous solution, preferably in a buffer solution, and adding CCT, CKI (not necessary when phosphorylcholine is used) and PyrG. Each enzyme preparation is present in an amount of about 0.01 unit / ml or more, preferably about 0.04 to 1.0 unit / ml, at about 5 to 30 ° C, preferably about 20 to 30, and more preferably about 23 to 28 °. The reaction can be carried out for about 1 to 60 hours at C with stirring if necessary.

The enzyme reaction is preferably performed by adding an inorganic phosphoric acid, a nitrogen source, and an energy source to the reaction system in addition to the yeast cells, the substrate, and the enzyme preparation. As the inorganic phosphoric acid to be used, a phosphate such as potassium phosphate can be used as it is. However, it is more preferable to use it in the form of a phosphate buffer. The working concentration of the inorganic phosphoric acid is preferably in the range of about 10 to 500 mM, particularly about 20 to 300 mM, and the pH of the phosphate buffer is preferably in the range of about 6.0 to 8.0. In addition, sugars such as glucose and fructoses; organic acids such as acetic acid and citric acid can be used as energy sources; amino acids such as glutamine and ammonium sulfate can be used as nitrogen sources. Each is preferably used in a range of about 10 to 500 mM, particularly about 20 to 400 mM. In particular, it is effective in terms of the productivity of CDP-choline to divide the energy source into smaller than 500 mM and add it to the reaction solution little by little.

 The CDP-choline thus obtained can be isolated and purified by usual isolation and purification means (ion-exchange chromatography, adsorption chromatography, salting out, etc.). Example

 Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

 In the examples, DNA preparation, restriction enzyme digestion, DNA ligation using T4 DNA ligase, and E. coli transformation were all performed by “Molecular cloning”.

(Maniatis et al., Cold Spring Harbor Laooratory, Cold Spring Harbor, New York (1982)), and quantification of CDP-choline in the reaction solution was performed by an HPLC method. Restriction enzymes, AmpliTaq DNA polymerase and T4 DNA ligase were obtained from Takara Shuzo.

 (1) Cloning of CCT gene

The chromosomal DNA of the yeast Saccharomyces cerevisiae DB746 (ATCC 44773) was prepared according to a known method, "Metnods in Yeast Genetics LaDoratory Manual J [Cold spring Harbor Laboratoiy, Cold Spring Harbor, New York (1983)]. The two types of primer DNAs shown in Table 1 were synthesized according to a conventional method, and the yeast CCT gene was amplified by the PCR method. Primer (A): 5 '-TACCATGGCAAACCCAACAAGGGA-3'

 Primer (B): 5 '-TATCTAGAGGGGCTCAGTTCGCTGATT-3'

Amplification of CCT gene by PCR is as follows: Reaction solution 10 (^ 1 [50 mM chloride, 10 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.001% gelatin, 0.5 g of temperate DNA, primer DNA (A) and (B) in each 0.2 _lambda M, AmpliTaq DNA polymerase La one peptidase 2.5 Yuni' preparative], using a Perkin-Elmer Cetus Instrument Co., Ltd. DNA thermal Cycler, thermal denaturation (94 ° C, 1 min), annealing (55 ° C, 1.5 min) and Polymerization (72 ° C, 1.5 min) were repeated 25 times.After gene amplification, the reaction mixture was treated with a phenol / cloth form (1: 1) mixture. The DNA was precipitated by adding 2 volumes of ethanol to the water-soluble fraction, separating the recovered DNA by agarose gel electrophoresis according to the method described in the literature (Molecular cloning, supra), and separating a DNA fragment equivalent to 1.8 kb. The DNA was digested with restriction enzymes Ncol and Xbal , Plasmid pTrc99A, which also was digested with restriction enzymes Ncol and Xbal

(Obtained from Pharmacia Biotech.) And T4 DNA ligase. Escherichia coli JM109 was transformed using the ligation reaction solution, and plasmid pTrc-CCT was isolated from the resulting ampicillin-resistant transformant. pTrc-CCT is obtained by inserting an Ncol-Xbal DNA fragment containing the yeast CCT gene into an Ncol-Xbal cleavage site downstream of the trc promoter of pTrc99A.

 (2) Cloning of CKI gene

 Using the chromosomal DNA of the yeast Saccharomyces cerevisiae DB746 (ATCC 44773) as a template, the following two types of primer DNAs were synthesized according to a conventional method, and the yeast CCT gene was amplified by the PCR method.

 Primer (C): 5 '-ATTCTAGAGGAGCAAAAGATGGTACAAGAATCA-3'

 Primer (D): 5'— ATCTGCAGGMTTCGTATACGTA ACA-3 '

The amplification of the CCT gene by PCR was performed using the reaction mixture 100 1 [50 mM potassium chloride, 10 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.001% gelatin, 0.5 g of template DNA, primer DNA (C) and ( D) 0.2 M each, AmpliTaq DNA polymer Lase 2.5 units), heat denaturation (94 ° C, 1 minute), annealing (55 ° C, 1.5 minutes), polymerisation (72 ° C) using a Perkin-Elmer Cetus Instrument DNA Thermal Cycler. , 1.5 minutes) was repeated 25 times. After gene amplification, the reaction solution was treated with a mixture of phenol nocroform-form (1: 1), and a two-fold solution of ethanol was added to the water-soluble fraction to precipitate DNA. The DNA collected by precipitation was separated by agarose gel electrophoresis according to the method described in the literature (Molecular cloning, described above), and a DNA fragment corresponding to 2.0 kb was purified. The DNA was cut with restriction enzymes Xbal and Pstl, and plasmid pTrc99A was digested with restriction enzymes Xbal and Pstl.

 (Obtained from Pharmacia Biotech.) And T4 DNA ligase. Escherichia coli JM109 was transformed using the ligation reaction solution, and plasmid pTrc-CKI was isolated from the resulting ampicillin-resistant transformant. pTrc-CKI is obtained by inserting an Xbal-Pstl DNA fragment containing the yeast CKI gene into an Xbal-Pstl cleavage site downstream of the trc promoter of pTrc99A.

 (3) Preparation of CCT and CKI

 Plasmid pTrc-CCT was digested with restriction enzymes Ncol and O ^ Xbal, and the Ncol-Xbal fragment containing the CCT gene was separated and purified. The fragment was ligated with plasmid pTrc-CKI DNA cut with Ncol and al using T4 DNA ligase, and Escherichia coli JM109 was transformed using the ligation reaction solution. Plasmid pTrc-CCK was isolated from the obtained ampicillin-resistant transformant. pTrc-CCK is obtained by ligating and inserting yeast CCT and CKI genes downstream of the trc promoter of pTrc99A.

Escherichia coli JM109 harboring plasmid pTrc-CCK was inoculated into 300 ml of 2 XYT medium containing 100 g / ml ampicillin and cultured with shaking at 37 ° C. 4 When 10 × 10 ⁸ bacteria / ml are reached, add isopropyl-/?-D-thiogalactovyranoside (IPTG) to the culture solution to a final concentration of 0.5 mM, and continue shaking culture at 37 ° C for 5 hours. Continued. After completion of the culture, the cells were collected by centrifugation (9,000 × g, 10 minutes), and 30 ml of buffer

After suspending in 50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, sonication was performed to disrupt the cells, followed by centrifugation (20,000 xg, 10 minutes) to remove cell residues. Removal did. The supernatant fraction thus obtained was used as an enzyme solution. The CCT activity in the enzyme solution was 18 units / ml, and the CKT activity was 1.6 units / ml.

 The unit (unit) of each enzyme was measured and calculated by the following method.

 (CCT activity measurement and unit calculation method)

 The enzyme preparation is added to 200 mM Tris-hydrochloric acid buffer (pH 7.8) containing 10 mM CTP, 10 mM phosphocholine and 10 mM magnesium chloride, and reacted at 37 ° C. After the reaction, heat treatment at 100 ° C for 1 minute,

 The amount of CDP-choline in the reaction solution is analyzed by (HPLC). The activity to produce 1 mole of CDP-choline per minute at 37 ° C is defined as 1 unit (unit).

 (CKI activity measurement and unit calculation method)

 The enzyme preparation was added to 200 mM Tris-HCl buffer (pH 7.8) containing lOmM CTP, lOmM choline chloride, lOmM ATP, lOmM magnesium chloride and 1 unit / ml CCT, and the reaction was performed at 37 ° C. The reaction is stopped by heat treatment at 100 ° C for 1 minute. Determine the amount of CDP-choline in the reaction solution by HPLC. The activity to produce 1 mole of CDP-choline per minute at 37 ° C is defined as 1 unit (unit).

 (4) Cloning of PyrG gene of Escherichia coli

 Using the chromosomal DNA of Escherichia coli JM109 as a template, the following two types of primer DNAs were synthesized according to a conventional method, and the Escherichia coli PyrG gene was amplified by PCR.

 Primer (E): 5'-TCGTCTTTTCAACCTAACTTCTCA-3 '

 Primer (F): 5'-CCGATGATTTTTACGArnTGGAC-3 '

Amplification of the PyrG gene by PCR was performed using the reaction mixture 100 1 (50 mM potassium chloride, 10 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.001% gelatin, 0.5 _{g of} template DNA, primer DNA (E) and (F) 0.2 M each, 2.5 units of AmpUTaq DNA Polymerase], heat denaturation (94 ° C, 1 minute), annealing (37 ° C, 1 minute) using Perkin-Elmer Cetus Instrument DNA Thermal Cycler. 2 minutes) and polymerisation (72 ° C, 3 minutes) were repeated 25 times. After gene amplification, the reaction solution was treated with a mixture of phenol and black form (1: 1), and twice the volume of ethanol was added to the water-soluble fraction to precipitate DNA. The DNA collected by precipitation was separated by agarose gel electrophoresis according to the method described in the literature (Molecular cloning, described above), and a DNA fragment corresponding to 1.8 kb was purified. The DNA was ligated with plasmid pUC18 (obtained from Takara Shuzo) digested with the restriction enzyme Smal using T4 DNA ligase. Escherichia coli JM105 was transformed using the ligation reaction solution, and plasmid pUC-pyrG was isolated from the obtained ampicillin-resistant transformant. pUC-PyrG has the E. coli pyrG gene inserted into the Smal cleavage site downstream of the lac promoter of pUC18.

 (5) Preparation of PyrG

Escherichia coli JM105 harboring plasmid pUC-PyrG was inoculated into 300 ml of 100 μg / ml ampicillin-containing soil and cultured at 37 ° C. with shaking. When the culture reached 4 × 10 ⁸ bacteria / ml, IPTG was added to the culture to a final concentration of 0.5 mM, and shaking culture was continued at 37 ° C. for 20 hours. After completion of the culture, the cells were collected by centrifugation (9,000 × _g , 10 minutes), suspended in 30 ml of buffer [50 mM Tris-HCl (pH7.5), 0.5 mM EDTA], and then sonicated. The cells were disrupted, and centrifugation (20,000 xg, 10 minutes) was performed to remove cell residues. The supernatant fraction thus obtained was used as an enzyme solution. The PyrG activity in the enzyme solution was 4.0 units / ml.

 The measurement of PyrG activity and the calculation of units were performed by the following methods. That is, an enzyme preparation was added to a 200 mM Tris-HCl buffer (pH 7.8) containing 5 mM UTP, 5 mM phosphocholine, 5 mM ATP, 10 mM magnesium chloride, 50 mM ammonium sulfate, and 1 unit / ml CCT. Perform the reaction at 37 ° C, and stop the reaction by heat treatment at 100 ° C for 1 minute. The amount of CDP-choline in the reaction solution is determined by the HPLC method. The activity to produce 1 μmol of CDP-choline per minute at 37 ° C is defined as 1 unit (unit).

 (6) Synthesis of CDP-choline I

lOOmM potassium phosphate (pH8.0), 30mM UMP, 25mM magnesium chloride, 25mM phosphorylcholine, 25mM glutamine, 0.2M glucose, 2% (w / v) 5 ml of a synthetic reaction solution containing dried yeast (Oriental Yeast), 0.5 unit / ml CCT, and 0.15 unit / ml PyrG was aerated and stirred in a test tube, and incubated at 17 to 32 ° C for 20 hours. . When the composition of the reaction solution was examined by HPLC method 20 hours after the start of the reaction, as shown in Fig. 1, the synthesis yield of CDP-choline significantly increased at 23-28 ° C, and undesired products such as UDP-glucose. It has been confirmed that the production of can be kept low.

 (7) Synthesis of CDP-choline II

 200 mM potassium phosphate (pH 8.0), 40 mM UMP, 40 mM magnesium chloride, 40 mM choline chloride, 40 mM glutamine, 0.4 M glucose, 2% (w / v) dried yeast (Oriental Yeast), 0.04 unit / ml CKI 5 ml of a synthesis reaction solution containing 0.45 units / ml CCT and 0.04 units / ml PyrG was stirred under aeration in a test tube, and kept at 28 ° C for 40 hours. 16 hours after the start of the reaction, glucose was added to a final concentration of 0.2M. After 40 hours of reaction, 22 mM CDP-choline was synthesized.

 When glucose was added from the beginning and reacted without dividing glucose into two, CDP-choline of 20 mM or more could not be produced.

 (8) Synthesis of CDP-choline III

 lOOmM potassium phosphate (pH8.0), 35mM UMP, 25mM magnesium chloride, 25mM phosphorylcholine, 40mM glutamine, 0.2M glucose, 2% (w / v) dried yeast (Oriental Yeast), 0.6unit / ml CCT, Then, 5 ml of the synthesis reaction solution containing 0.16 units / ml PyrG was stirred under aeration in a test tube, and kept at 28 ° C for 24 hours. Six hours after the start of the reaction, glucose was added to a final concentration of 0.15M. 24 hours after the reaction, 23 mM CDP-choline was synthesized. Industrial applicability

The present invention provides a simple and efficient method for coexisting CCT, CKI (not required when phosphorylcholine is used) and PyrG in a reaction system of yeast cells, 5 "-UMP, and choline or phosphorylcholine. Can be manufactured, extremely practical It is a way. In particular, by controlling the reaction temperature within the range of about 20-30 ° C, the synthesis yield of CDP-choline can be significantly increased, while the production of unintended products such as UDP-glucose can be kept low. Furthermore, by dividing and adding the energy source, it is possible to further improve the productivity of CDP-choline.

Claims

. Determined range yeast cells, Urijin 5'-monophosphate (5 ¹ - UMP) and choline, Korinhosu Hue one cytidyl trans Blow Ichize (CCT), choline kinase (CKI) and cytidine 5 ¹ - triphosphate Shinteta A method for producing cytidine 5'-choline diphosphate (CDP-choline), characterized in that the reaction is carried out in the co-presence of an enzyme (PyrG). . Yeast cells, Urijin 5'monophosphate (5 ¹ - UMP) and phosphorylcholine to choline phosphate Toshichijirutora port Nsufera Ichize (CCT) and cytidine 5 ¹ - reaction in the presence of preparative Li Lin synthetase (PyrG) Shichiji down 5 ^1, characterized in that to - diphosphate choline (CDP-choline) in the production method. 3. The method according to claim 1, wherein the reaction is carried out at a temperature of about 20 to 30 ° C. 3. The method according to claim 1, wherein the reaction is carried out under a temperature condition of about 23 to 28 ° C. 3. The method according to claim 1, wherein the energy source is divided and added.