Phosphorylcholine cytidyltransferase and application thereof in preparation of citicoline

Abstract

The invention belongs to the field of bioengineering, and particularly relates to phosphorylcholine cytidyltransferase and application thereof in preparation of citicoline, and the specific application is that the phosphorylcholine cytidyltransferase can efficiently catalyze phosphorylcholine and CTP to react to generate citicoline. The phosphorylcholine cytidyltransferase is prepared by the following steps: synthesizing a coding gene of the phosphorylcholine cytidyltransferase, selecting pHT01 as an expression vector, co-expressing with choline kinase, transforming into host bacteria, and inducing protein expression. According to the choline phosphate cytidyltransferase disclosed by the invention, under the same reaction condition and within the same reaction time, the yield of a citicoline product obtained by conversion is higher than that of choline phosphate cytidyltransferase (ScCCT) which is most commonly used in the market at present and is derived from saccharomyces cerevisiae S288c. Specifically, the yield of the MsCCT001 is 10 times higher than that of the ScCCT, the yield of the MbCCT006 is 6 times higher than that of the ScCCT, and the yield of the MeCCT008 is 8 times higher than that of the ScCCT.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and particularly relates to three phosphocholine cytidylyltransferases and applications thereof in preparing citicoline. Background Art

[0002] Citicoline was first discovered in the 1950s and successfully synthesized by chemical methods. Chemical synthesis is currently the main method for preparing Citicoline, which mainly uses cytosine nucleoside phosphate morpholine and phosphorylcholine hydrochloride to condense in acetonitrile or pyridine to form Citicoline. However, the raw materials and condensing agents used in this method are expensive, and the reaction needs to be completed in toxic solvents, resulting in low product yields and high production costs. Chemical synthesis methods are often criticized for their potential negative impact on the environment. In order to promote sustainable development, we are constantly seeking more environmentally friendly alternative technologies to reduce the pollution and resource waste that may be generated during chemical synthesis.

[0003] At present, the synthesis pathway of Citicoline has been confirmed to exist in many microorganisms, especially in Saccharomyces cerevisiae, where there is a complete synthesis mechanism. Researchers have successfully cloned the CCT gene from S.cerevisiae into a specific vector and transformed it into S.cerevisiae to construct the corresponding genetically engineered bacteria. Using this engineered bacteria, with choline phosphate and CMP as raw materials, after a certain period of reaction, the molar conversion rate of Citicoline is high. In addition, the expression of related genes from S.cerevisiae has also been achieved in Pichia pastoris. After the recombinant bacteria are cultured to a specific stage, they are inoculated into a culture medium containing choline chloride or choline phosphate and CMP for fermentation, and a certain amount of Citicoline can be obtained. However, these yeast-based fermentation schemes generally face the problems of low fermentation density and low production efficiency.

[0004] In recent years, the biotransformation method has dominated the reported methods for synthesizing citicoline. This method uses choline phosphate cytidylyltransferase (CCT) in microorganisms, with choline chloride, cytidine triphosphate (CTP) and adenosine triphosphate (ATP) as substrates for biosynthesis, and has many advantages such as mild reaction conditions, low cost and simple process. Given that Escherichia coli originally lacks a complete citicoline synthesis pathway, researchers successfully catalyzed the synthesis of citicoline in Escherichia coli by introducing and expressing the codon-optimized Saccharomyces cerevisiae CCT gene, opening up new strategies and pathways for the efficient synthesis of citicoline. However, the existing biotransformation method also faces problems such as low conversion rate, low product purity and limited production scale.

[0005] Therefore, if choline cytidylyltransferase (CCT) with stronger catalytic ability can be further developed, the industrial preparation efficiency of Citicoline will be greatly improved, which is of great significance to promoting the development of the Citicoline market. Summary of the invention

[0006] In order to solve the above technical problems, the present invention discloses an enzyme preparation capable of efficiently catalyzing a reaction to prepare cytidine diphosphate choline, wherein the active ingredient of the enzyme preparation is phosphocholine cytidylyltransferase, and also provides the nucleotide sequence, amino acid sequence, recombinant expression plasmid, genetic engineering strain and preparation method of the phosphocholine cytidylyltransferase.

[0007] The technical solution in the present invention is: The enzyme preparation for preparing citicoline is prepared by the following steps: Construction of engineered bacteria Bacillus subtilis 168 MsCCT001-CKI, expressed from yeast Maudiozymasaugeensis Phosphocholine cytidylyltransferase MsCCT001 and Saccharomyces cerevisiae Saccharomyces cerevisiae S288c Choline kinase from Saccharomyces cerevisiae Maudiozymasaugeensis The gene of phosphocholine cytidylyltransferase was used as the initial gene, and the gene of phosphocholine cytidylyltransferase MsCCT001 was obtained through gene comparison on the NCBI website; Alternatively, constructing engineered bacteria Bacillus subtilis 168 MbCCT006-CKI, expressed in yeast Maudiozymabarnettii Phosphocholine cytidylyltransferase MbCCT006 and Saccharomyces cerevisiae Saccharomyces cerevisiae S288c Choline kinase from Saccharomyces cerevisiae MaudiozymabarnettiiThe gene of phosphocholine cytidylyltransferase was used as the initial gene, and the gene of phosphocholine cytidylyltransferase MsCCT006 was obtained through gene comparison on the NCBI website; Alternatively, constructing engineered bacteria Bacillus subtilis 168 MeCCT008-CKI, expressed from yeast Maudiozymaexigua Phosphocholine cytidylyltransferase MeCCT008 and Saccharomyces cerevisiae Saccharomyces cerevisiae S288c Choline kinase from Saccharomyces cerevisiae Maudiozymaexigua The gene of phosphocholine cytidylyltransferase was used as the initial gene, and the gene of phosphocholine cytidylyltransferase MsCCT008 was obtained through gene comparison on the NCBI website; The three engineered bacteria constructed above are expanded and cultured, and after the bacteria are collected by centrifugation, the bacteria are broken to obtain a crude enzyme solution, i.e., an enzyme preparation.

[0008] Preferably, phosphocholine cytidylyltransferases MsCCT001, MbCCT006 and MeCCT008 are prepared by the following steps: (1) Construction of expression vectors: The genes of phosphocholine cytidylyltransferase ScCCT on the existing plasmid pHT01-ScCCT-CKI (co-expressing choline kinase and phosphocholine cytidylyltransferase from Saccharomyces cerevisiae) were replaced with MsCCT001, MbCCT006, and MeCCT008 respectively; The above genes were synthesized by a whole gene synthesis method, and ScCCT, a phosphocholine cytidylyltransferase in pHT01-ScCCT-CKI was replaced to obtain vectors pHT01-MsCCT001-CKI, pHT01-MbCCT006-CKI, and pHT01-MeCCT008-CKI; (2) Transformation into protein expression host bacteria: Transform the constructed vector into Bacillus subtilis competent cells. The specific steps are as follows: Take the BS168 strain and inoculate it on the culture medium and culture it overnight; pick a single colony and inoculate it into a test tube containing 5 mL of GMⅠ liquid culture medium, and culture it in a shaking incubator at 37 °C and 200 r / min for 14-16 h; Take 1 mL of GMⅠ cultured bacterial liquid, place it in a 1.5 mL eppendorf tube, centrifuge at 12000 r / min for 1 min, discard 500 µL of supernatant, keep 500 µL to resuspend the remaining bacteria, inoculate it in a test tube containing 4.5 mL GMⅠ liquid culture medium, and culture for 4.5 h; Take the cultured bacterial solution, centrifuge it, resuspend the remaining bacteria, inoculate it into the culture solution, and culture it to obtain Bacillus subtilis competent cells. Dispense them into ep tubes, and transfer 1µL of pHT01-MsCCT001-CKI, pHT01-MbCCT006-CKI, and pHT01-MeCCT008-CKI plasmids into 1 mL of competent cells, respectively. Incubate, centrifuge after completion, resuspend the bacteria, and culture overnight to complete the transformation; (3) Inducing protein expression: Take a single bacterial plaque, inoculate it into the culture medium, add chloramphenicol to make the final concentration of chloramphenicol 5 µg / mL, and culture overnight to obtain the seed solution; The seed solution was inoculated into the culture medium, and chloramphenicol was added to make the final concentration of chloramphenicol 5 µg / mL. The culture was cultured until the OD600 value of the bacterial solution reached between 0.6 and 0.8. Then, isopropylthiogalactoside (IPTG) was added at a final concentration of 1 mM to induce the expression of the target protein.

[0009] A gene encoding any one of phosphocholine cytidylyltransferase MsCCT001, phosphocholine cytidylyltransferase MbCCT006, and phosphocholine cytidylyltransferase MeCCT008.

[0010] Preferably, the amino acid sequence of the gene encoding choline cytidylyltransferase MsCCT001 is shown as SEQ ID NO: 1, and the nucleotide sequence is shown as SEQ ID NO: 2; the amino acid sequence of the gene encoding choline cytidylyltransferase MbCCT006 is shown as SEQ ID NO: 3, and the nucleotide sequence is shown as SEQ ID NO: 4; the amino acid sequence of the gene encoding choline cytidylyltransferase MeCCT008 is shown as SEQ ID NO: 5, and the nucleotide sequence is shown as SEQ ID NO: 6.

[0011] The expression cassette, vector or recombinant bacteria containing any of the above genes is also one of the key contents protected by the present invention.

[0012] The application of choline phosphate cytidyl transferase in the preparation of cytidylcholine mainly comprises taking choline chloride, cytidine triphosphate and adenosine triphosphate as substrates, carrying out a catalytic reaction under the action of choline phosphate cytidyl transferase, and synthesizing cytidylcholine; Preferably, the above-mentioned choline phosphate cytidylyltransferase is selected from any one of choline phosphate cytidylyltransferase MsCCT001, choline phosphate cytidylyltransferase MbCCT006, and choline phosphate cytidylyltransferase MeCCT008.

[0013] Preferably, the reaction conditions of phosphocholine cytidylyltransferase in preparing citicoline are: 30-37°C, pH=7.0-8.0, 0.5 mM choline chloride, 1 mM cytidine triphosphate, 1 mM adenosine triphosphate, 40 mM magnesium chloride, 50 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, and a reaction system volume of 10 mL.

[0014] A catalyst, the active ingredients of which include choline phosphate cytidylyltransferase MsCCT001, choline phosphate cytidylyltransferase MbCCT006, and choline phosphate cytidylyltransferase MeCCT008.

[0015] Preferably, the catalyst is used in the preparation of CDPcholine with choline chloride, cytidine triphosphate and adenosine triphosphate as substrates, and the catalytic reaction is carried out at 30-37°C, pH=7.0-8.0, 0.5 mM choline chloride, 1 mM cytidine triphosphate, 1 mM adenosine triphosphate, 40 mM magnesium chloride and 50 mM tris(hydroxymethyl)aminomethane hydrochloride buffer.

[0016] Compared with the prior art, the present invention has the following advantages and effects: (1) This invention reports for the first time that Maudiozymasaugeensis and yeast Maudiozymaexigua ,yeast Maudiozymabarnettii The phosphocholine cytidylyltransferase can be used for the synthesis of citicoline. Compared with the phosphocholine cytidylyltransferase from Saccharomyces cerevisiae commonly used in existing reports, the enzyme activities of the three enzymes reported in the present invention are higher; the yield of the transferase MsCCT001 obtained in the present invention is 10 times higher than that of ScCCT, the yield of MbCCT006 is 6 times higher than that of ScCCT, and the yield of MeCCT008 is 8 times higher than that of ScCCT; (2) The catalytic ability of phosphocholine cytidylyltransferase is stronger, which greatly improves the industrial preparation efficiency of citicoline. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The schematic diagram is a principle diagram of producing citicoline from choline chloride substrate by using choline kinase and choline phosphocytidyl transferase catalyzed reaction in the present invention; Figure 2 is a high performance liquid chromatogram of the citicoline standard product in the present invention; Figure 3 is a high performance liquid chromatogram of the reaction solution sample of the experimental group in the present invention; Figure 4The figure is a bar graph comparing the yields of citicoline produced by the three phosphocholine cytidylyltransferases of the present invention and the most widely used phosphocholine cytidylyltransferase ScCCT from Saccharomyces cerevisiae S288c under the same reaction conditions at the same time. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0019] 1. Main reagents and consumables: The pHT01 plasmid is a known Bacillus subtilis expression vector with a vector size of 7956 bp, a Pgrac promoter, and vector resistance to chloramphenicol, purchased from Wuhan Miaoling Biotechnology Co., Ltd.; BS168 Bacillus subtilis strain was purchased from Beijing Bio-Bowei Biotechnology Co., Ltd.; Agar powder (Cat. No. A8190) was purchased from Solarbio; TRYPTONE (Cat. No.: LP0042) and YEAST EXTRACT (Cat. No.: LP0021) were purchased from OXOID; Reagents such as NaCl (Cat. No.: 10019318), K2HPO4·3H2O (Cat. No.: 10017518), KH2PO4 (Cat. No.: 10017618) and propylene glycol (Cat. No.: 10010618) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0020] 2. Prepare LB medium: Each 100 mL of LB medium contains: 1 g tryptone, 0.5 g yeast extract and 1 g NaCl, pH 7.0-7.2.

[0021] Preparation method: Dissolve 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 1 L of distilled water, do not adjust the pH value, and sterilize at 121°C with high pressure steam for 20 min. If preparing solid culture medium, add 1 g of agar powder to 100 mL of culture medium.

[0022] 3. Prepare 2×SR medium: Each 100 mL of 2×SR medium contains: 5 g tryptone, 3 g yeast extract and 0.6 g potassium dihydrogen phosphate, pH = 7.0-7.5.

[0023] Preparation method: Dissolve 50 g of tryptone, 30 g of yeast extract, and 6 g of dipotassium phosphate in 1 L of distilled water, do not adjust the pH value, and sterilize with high-pressure steam at 121°C for 20 min.

[0024] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure grade. Unless otherwise specified, the methods used in the following examples are all conventional methods in the art, and the experimental conditions used are all conventional experimental conditions in the art, and reference can be made to the relevant experimental manuals or manufacturer's instructions.

[0025] Example 1 Preparation of enzyme preparation 1.1 Discovery of phosphocholine cytidylyltransferase Through gene mining, the phosphocholine cytidylyltransferase genes MsCCT001, MbCCT006, and MeCCT008 were screened and obtained from the NCBI database. The NCBI number of MsCCT001 is: SMN20780.1. The open reading frame of the gene is 1332 bp in length. The carbonyl reductase encoded by it consists of 444 amino acids. Its amino acid sequence (444 aa) is shown in SEQ ID NO:1, and its nucleotide sequence is shown in SEQ ID NO:2.

[0026] The NCBI number of MbCCT006 is: XP-041406747.1. The open reading frame of the gene is 1335 bp in length. The carbonyl reductase encoded by it consists of 334 amino acids. Its amino acid sequence (445aa) is shown in SEQ ID NO:3, and its nucleotide sequence is shown in SEQ ID NO:4.

[0027] The NCBI number of MeCCT008 is: KAG0656917.1. The open reading frame of the gene is 1329 bp in length. The carbonyl reductase encoded by the gene consists of 334 amino acids. The amino acid sequence (443aa) is shown in SEQ ID NO:5, and the nucleotide sequence is shown in SEQ ID NO:6.

[0028] The present invention obtained samples with higher enzyme activity through screening, namely, three samples of choline phosphotransferase provided by the present invention, which showed better catalytic performance and enzyme activity.

[0029] 1.2 Acquisition of phosphocholine cytidylyltransferase MsCCT001, MbCCT006, and MeCCT008 genes The genes of phosphocholine cytidylyltransferases with Genbank IDs SMN20780.1, XP-041406747.1, and KAG0656917.1 can be obtained by a total gene synthesis method or a molecular cloning method. In the present invention, Nanjing GenScript Biotech Co., Ltd. synthesizes the above genes by a total gene synthesis method, and the obtained genes are MsCCT001, MbCCT006, and MeCCT008, respectively, and replace the phosphocholine cytidylyltransferase ScCCT in pHT01-ScCCT-CKI (co-expressing phosphocholine cytidylyltransferase and choline kinase from Saccharomyces cerevisiae), to obtain pHT01-MsCCT001-CKI, pHT01-MbCCT006-CKI, and pHT01-MeCCT008-CKI.

[0030] 1.3 Heterologous expression of phosphocholine cytidylyltransferase Construction of engineered bacteria Bacillus subtilis 168 MsCCT001-CKI, expressed from yeast Maudiozymasaugeensis Phosphocholine cytidylyltransferase MsCCT001 and Saccharomyces cerevisiae Saccharomyces cerevisiae S288c of choline kinase; Alternatively, constructing engineered bacteria Bacillus subtilis 168 MbCCT006-CKI, expressed in yeast Maudiozymabarnettii Phosphocholine cytidylyltransferase MbCCT006 and Saccharomyces cerevisiae Saccharomyces cerevisiae S288c of choline kinase; Alternatively, constructing engineered bacteria Bacillus subtilis 168 MeCCT008-CKI, expressed in yeast Maudiozymaexigua Phosphocholine cytidylyltransferase MeCCT008 and Saccharomyces cerevisiae Saccharomyces cerevisiae S288c of choline kinase.

[0031] The specific experimental steps are as follows: Take out the laboratory stored in -80℃ freezer Bacillus subtilis 168 For the BS168 strain, use an inoculation loop to dip the bacterial solution from the glycerol tube, inoculate it on a non-resistant LB solid medium, and invert it in a 37°C incubator for overnight culture; Pick a single colony on the culture medium after overnight culture, inoculate it into a test tube containing 5mL GMⅠ liquid culture medium, and culture it in a shaker at 37℃ and 200 r / min for 14-16 h; take 1 mL of GMⅠ cultured bacterial solution, place it in a 1.5 mL ep tube, centrifuge it at 12000 r / min for 1 min, discard 500 µL of supernatant, keep 500 µL to resuspend the remaining bacteria, and inoculate it into a test tube containing 4.5 mL GMⅠ liquid culture medium, and culture it in a shaker at 37℃ and 200 r / min for 4.5 h; take 1.5mL of bacterial solution after 4.5 h of culture, centrifuge it at 12000 r / min for 1 min, discard 750 µL of supernatant, keep 750 µL to resuspend the remaining bacteria, and inoculate it into a test tube containing 4.25mL GMⅡ liquid culture medium, and culture it in a shaker at 37℃ and 240 r / min for 1.5 h. The competent cells of Bacillus subtilis were obtained. Each 1 mL of competent cells was dispensed into ep tubes. 1 μL of pHT01-MsCCT001-CKI, pHT01-MbCCT006-CKI, and pHT01-MeCCT008-CKI plasmids were transferred into 1 mL of competent cells, respectively, and incubated at 37 °C, 200 r / min in a shaker for 1 h. After the incubation was completed, centrifuged at 12000 r / min for 1 min, 900 μL of supernatant was discarded, and the remaining 100 μL of supernatant was used to resuspend the cells, which were all applied to LB solid culture medium, so that the final concentration of chloramphenicol was 5 μg / mL, and placed in a 37 °C incubator for overnight culture.

[0032] Take the plate cultured overnight, pick out a single plaque grown on it, inoculate it into a test tube containing 5 mL of LB liquid culture medium, add chloramphenicol to make the final concentration of chloramphenicol 5 µg / mL, and culture overnight in a shaking incubator at 200 r / min and 37 ℃ to obtain the seed solution.

[0033] 1.4 Protein expression Prepare 2×SR liquid culture medium, inoculate the seed liquid with 1% inoculation into a conical flask containing 50 mL 2×SR liquid culture medium, the final concentration of chloramphenicol is 5 µg / mL, and culture in a shaker at 37°C and 200r / min until the bacterial liquid OD 600 value reaches between 0.6-0.8, add isopropylthiogalactoside (IPTG) with a final concentration of 1 mM to induce the expression of the target protein, adjust the induction temperature to 28°C, and collect the cells after induction culture for 20 h. Collect the cells by centrifugation at 6000 r / min and 4°C for 15 min, then take Tris-HCl buffer and shake to resuspend and wash the cells twice, and finally centrifuge and store in a -20°C refrigerator for use.

[0034] Resuspend the cells with 50 mM Tris-HCl (pH=7.0-8.0), measure the OD600 of the cells after resuspending, and then break them to obtain a crude enzyme solution, i.e., an enzyme preparation. The conditions for high-pressure homogenization are: 4 ℃, 1000-1200 bar.

[0035] Example 2 Preparation of Citicoline (1) Synthetic citicoline is prepared by using phosphocholine cytidylyltransferase and choline kinase. The specific steps are as follows: The enzyme preparation obtained in Example 1 was used as a catalyst, and choline chloride, adenosine triphosphate (ATP), and cytidine triphosphate (CTP) were used as substrates. The enzyme preparation was mixed with choline chloride, ATP, CTP, magnesium chloride, and Tris-HCl buffer to carry out an enzyme-catalyzed reaction. The reaction was carried out using a metal bath, the temperature was controlled at 37° C., the rotation speed was controlled at 300 r / min, and the sample was taken for inactivation after the reaction for 15 min.

[0036] In the above reaction system, the concentration of choline chloride is 0.5 mM, the concentration of ATP is 1 mM, the concentration of CTP is 1 mM, the concentration of magnesium chloride is 40 mM, the concentration of Tris-HCl buffer is 50 mM, the pH is 7.0-8.0, the concentration of enzyme preparation is 10 g / L, and the volume of the reaction system is controlled at 10 mL.

[0037] (2) Detection of Citicoline The detection method of Citicoline in the reaction solution is as follows: the reaction solution after 15 minutes of reaction is inactivated in a boiling water bath, and centrifuged at 13000 r / min for 3 minutes. The supernatant is mixed with an equal volume of 10% formic acid water, and then centrifuged at 13000 r / min for 15 minutes. The supernatant is taken and filtered with a 0.22 µm sterile membrane into a liquid phase bottle for analysis; The content of Citicoline was determined by high performance liquid chromatography with an injection volume of 20 µL, a chromatographic column of PCHILIC 5µm 4.6×250 nm, a mobile phase of 0.05% formic acid in water, acetonitrile-methanol (8:2), a column temperature of 30 °C, a flow rate of 1 mL / min, a detection wavelength of 280 nm, and a retention time of Citicoline of 8.9 min.

[0038] The test shows that the three choline phosphate cytidylyltransferases discovered in the present invention have a higher yield of choline diphosphate products under the same reaction conditions and within a reaction time of 15 minutes than the choline phosphate cytidylyltransferase from Saccharomyces cerevisiae S288c, which is currently the most commonly used choline phosphate cytidylyltransferase on the market. Specifically: Table 1 Actual production of citicoline Sample Name Actual Yield ScCCT 0.02 ± 0.005 mg / mL MsCCT001 0.20 ± 0.005 mg / mL MbCCT006 0.12 ± 0.007 mg / mL MeCCT008 0.16 ± 0.004 mg / mL The yield of MsCCT001 is 10 times higher than that of ScCCT, the yield of MbCCT006 is 6 times higher than that of ScCCT, and the yield of MeCCT008 is 8 times higher than that of ScCCT.

[0039] In summary, the present invention provides an enzyme preparation capable of efficiently catalyzing a reaction to prepare citicoline, wherein the active ingredient choline phosphate cytidylyltransferase has high activity.

[0040] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. An enzyme preparation for preparing citicoline, characterized in that Prepared by the following steps: Construction of engineered bacteria Bacillus subtilis 168 MsCCT001-CKI, expressed from yeast Maudiozyma saugeensis Phosphocholine cytidylyltransferase MsCCT001 and Saccharomyces cerevisiae Saccharomyces cerevisiae S288c of choline kinase; Alternatively, constructing engineered bacteria Bacillus subtilis 168 MbCCT006-CKI, expressed in yeast Maudiozyma barnettii Phosphocholine cytidylyltransferase MbCCT006 and Saccharomyces cerevisiae Saccharomyces cerevisiae S288c of choline kinase; Alternatively, constructing engineered bacteria Bacillus subtilis 168 MeCCT008-CKI, expressed in yeast Maudiozyma exigua Phosphocholine cytidylyltransferase MeCCT008 and Saccharomyces cerevisiae Saccharomyces cerevisiae S288c of choline kinase; The three engineered bacteria constructed above are expanded and cultured separately, and after the bacteria are collected by centrifugation, the bacteria are broken to obtain their respective crude enzyme solutions, thereby obtaining enzyme preparations in the form of crude enzyme solutions.

2. The enzyme preparation for preparing citicoline according to claim 1, characterized in that Phosphocholine cytidylyltransferases MsCCT001, MbCCT006 and MeCCT008 are all prepared by the following steps: constructing an expression vector, transforming a protein expression host bacterium, and inducing protein expression.

3. A gene encoding any one of the choline phosphotransferase MsCCT001, choline phosphotransferase MbCCT006, and choline phosphotransferase MeCCT008 according to claim 1.

4. The gene according to claim 3, characterized in that The amino acid sequence of the gene encoding phosphocholine cytidylyltransferase MsCCT001 is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2; The amino acid sequence of the gene encoding phosphocholine cytidylyltransferase MbCCT006 is shown in SEQ ID NO:3, and the nucleotide sequence is shown in SEQ ID NO:4; The amino acid sequence of the gene encoding choline phosphocholine cytidylyltransferase MeCCT008 is shown in SEQ ID NO:5, and the nucleotide sequence is shown in SEQ ID NO:

6.

5. An expression cassette, vector or recombinant bacterium containing any one of the genes described in claim 3.

6. Use of phosphocholine cytidylyltransferase in the preparation of citicoline, characterized in that: Using choline chloride, cytidine triphosphate and adenosine triphosphate as substrates, a catalytic reaction is carried out under the action of phosphocholine cytidylyltransferase to synthesize citicoline; The choline phosphate cytidylyltransferase is selected from any one of the choline phosphate cytidylyltransferase MsCCT001, choline phosphate cytidylyltransferase MbCCT006 and choline phosphate cytidylyltransferase MeCCT008 as claimed in claim 1.

7. Use of phosphocholine cytidylyltransferase according to claim 6 in the preparation of citicoline, characterized in that: The catalytic reaction is carried out at 30-37° C., pH=7.0-8.0, 0.5 mM choline chloride, 1 mM cytidine triphosphate, 1 mM adenosine triphosphate, 40 mM magnesium chloride, 50 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, and the reaction system volume is 10 mL.

8. A catalyst, characterized in that The active ingredients thereof comprise the choline phosphate cytidylyltransferase MsCCT001, choline phosphate cytidylyltransferase MbCCT006 and choline phosphate cytidylyltransferase MeCCT008 as described in claim 1.

9. Use of the catalyst according to claim 8 in the preparation of citicoline, characterized in that: With choline chloride, cytidine triphosphate and adenosine triphosphate as substrates, the catalytic reaction is carried out at 30-37°C, pH=7.0-8.0, 0.5 mM choline chloride, 1 mM cytidine triphosphate, 1 mM adenosine triphosphate, 40 mM magnesium chloride and 50 mM tris(hydroxymethyl)aminomethane hydrochloride buffer.

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