A choline kinase mutant, polynucleotide, vector, genetically engineered bacteria and application of the mutant in cytidine diphosphate choline synthesis

Abstract

This invention discloses a choline kinase mutant, polynucleotide, vector, genetically engineered bacteria, and the application of the mutant in the synthesis of citicoline, belonging to the field of biomedical catalysis technology. In a reaction solution composed of cytidine, choline chloride, disodium ATP, magnesium sulfate heptahydrate, and acetyl phosphate, the addition of multiple enzyme solutions, including a choline kinase mutant, cytidine kinase, cytidine kinase, nucleoside diphosphate kinase, acetate kinase, and phosphocholine cytidine transferase, enables one-pot catalytic production of citicoline. This choline kinase mutant exhibits high enzyme activity and substrate tolerance. The optimal choline kinase mutant obtained by this invention has 30.16 times the enzyme activity of the original mutant, solving the problem of key enzyme limitation on the reaction, resulting in an approximately 59% increase in the final yield of citicoline, an approximately 35% increase in conversion rate, a significant improvement in production efficiency, and a significant reduction in production costs. The new choline kinase mutant is suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biomedical catalysis technology and relates to a choline kinase mutant, polynucleotide, vector, genetically engineered bacteria, and the application of the mutant in the synthesis of cytidine diphosphate choline. Background Technology

[0002] Citicoline (CDP-choline) is a nucleotide derivative composed of ribose, CTP, a phosphate group, and choline. It is readily soluble in water but insoluble in ethanol and acetone, with a relative molecular mass of 488.32. It is typically a white or off-white crystalline powder. Citicoline is an essential nutrient that enhances the excitability of the nervous system and plays an important role in the treatment of patients in rehabilitation. It can also synthesize acetylcholine in the body, improving a range of cognitive impairments such as memory loss and language decline. After ingestion, Citicoline promotes the synthesis of neurotransmitters such as dopamine and norepinephrine, thereby stabilizing the nervous system and promoting energy metabolism in the brain. Furthermore, Citicoline has been proven to be a highly safe drug. Its pharmacological activity and mechanism of action suggest that Citicoline may have guiding significance in the treatment of cerebrovascular diseases, such as depression, Parkinson's syndrome, and Alzheimer's disease.

[0003] The production of citicoline is mainly carried out through two methods: chemical synthesis and enzymatic synthesis. Chemical synthesis involves a chemical condensation reaction of the corresponding raw materials, along with protection and deprotection reactions of certain functional groups, ultimately yielding citicoline. The problems with this method include high raw material costs, complex reaction steps, difficulty in separation and purification, low purification yield, and significant environmental pollution. Enzymatic synthesis mainly includes microbial fermentation synthesis and in vitro enzymatic synthesis. Microbial fermentation synthesis, by selecting suitable microbial strains and utilizing inexpensive raw materials such as glucose and cellulose, allows microorganisms to synthesize citicoline under appropriate culture conditions. However, this method involves highly complex processes, including the selection of microbial strains, modification of key enzymes in the synthetic pathway, and optimization of culture conditions, making it time-consuming and labor-intensive. In vitro enzymatic synthesis, on the other hand, is relatively easier. It only requires the heterologous expression of several key enzymes in citicoline synthesis, such as choline kinase and phosphocholine cytidine transferase, using suitable host bacteria. These enzymes are then reacted with the substrates required for citicoline synthesis under specific conditions, achieving a one-pot synthesis with high production efficiency, controllable costs, and environmental friendliness. Patent application CN118667792A discloses a phosphocholine cytidine transferase and its applications. The relative enzyme activity of the phosphocholine cytidine transferase mutant is 3.53 times that of the wild type, and the concentration of cytidine diphosphate choline produced is 19.0 g / L with a conversion rate of 97.4%. Patent application CN121182776A discloses a phosphocholine cytidine transferase mutant and its applications. The obtained mutant exhibits a catalytic efficiency 1.90 to 2.41 times that of the initial phosphocholine cytidine transferase in the reaction of phosphocholine with cytidine triphosphate to produce cytidine diphosphate choline. Patent application CN118581179A discloses a one-pot method for the synthesis of cytidine sodium from cytidine using a multi-enzyme system. The one-pot method using a mixed enzyme system in an aqueous phase can catalyze the synthesis of cytidine sodium from cytidine. Patent application CN104774799A discloses a genetically engineered bacterium expressing choline kinase and phosphorylated choline cytidine transferase, its construction method, and its application. The *E. coli* strain in this invention can efficiently express choline kinase, with an induced enzyme activity of 1.94 U / mg and a cytidine choline content of 19 g / L in the catalytic system. Patent application CN116240193A discloses a choline kinase mutant and its application in cytidine choline production, using the mutant recombinant fusion protein inKN-CKI. KG79-80DE, S368D, K569E The highest yield of citicoline reached 130 mM after 6 hours of reaction, with a conversion rate of 92.8%. In summary, the yield levels of citicoline synthesized by in vitro enzymatic methods are generally low, with low activities of key enzymes such as choline kinase and phosphocholine cytidine transferase, resulting in high production costs. Moreover, most studies focus on mutations of phosphocholine cytidine transferase, while studies on mutations of choline kinase are relatively few.

[0004] To address the issues of low activity and high cost of choline kinase, a key enzyme in the industrial production of citicoline, this invention provides a choline kinase mutant. This mutant enzyme exhibits higher enzyme activity and substrate tolerance in the one-pot synthesis of citicoline, significantly improving conversion rate and yield, making it more suitable for industrial applications compared to the unmutated form. Summary of the Invention

[0005] The primary objective of this invention is to provide a choline kinase mutant, which is a mutation of the amino acid sequence shown in SEQ ID NO.1, wherein the mutation mode is any one of the following: V69T single mutation, S116R single mutation, E165A single mutation, R272L single mutation, L411G single mutation, V535I single mutation, S116R and E165A double mutation, L411G and V535I double mutation, S116R and R272L double mutation, S116R and R272L and L411G triple mutation, S116R and R272L and L411G quadruple mutation, S116R and R272L and L411G and E165A pentamutation, and S116R and R272L and L411G and E165A and V535I pentamutation, and S116R and R272L and L411G and E165A and V535I and V69T hexamutation. The choline kinase mutant provided by this invention exhibits higher enzyme activity and substrate tolerance in the synthesis of citicoline compared to the unmutated form, which can significantly improve conversion rate and yield, reduce production costs, and is suitable for industrial applications.

[0006] Furthermore, the mutation mode is any one of the following: S116R single mutation, S116R and E165A double mutation, S116R and R272L double mutation, S116R and R272L and L411G triple mutation, S116R and R272L and L411G and E165A quadruple mutation, S116R and R272L and L411G and E165A and V535I pentamutation, and S116R and R272L and L411G and E165A and V535I and V69T hexamutation.

[0007] Furthermore, the mutation mode is any of the following: double mutation of S116R and E165A, double mutation of S116R and R272L, triple mutation of S116R and R272L and L411G, quadruple mutation of S116R and R272L and L411G and E165A, pentamutation of S116R and R272L and L411G and E165A and V535I, and hexamutation of S116R and R272L and L411G and E165A and V535I and V69T.

[0008] The preferred mutation mode is any one of the following: three mutations of S116R, R272L, and L411G; four mutations of S116R, R272L, L411G, and E165A; and five mutations of S116R, R272L, L411G, E165A, and V535I.

[0009] A second objective of this invention is to provide a polynucleotide encoding the choline kinase mutant described above.

[0010] A third objective of this invention is to provide a carrier containing the said polynucleotide.

[0011] A fourth object of the present invention is to provide recombinant engineered bacteria containing the said vector, particularly Escherichia coli BL21(DE3).

[0012] A fifth objective of this invention is to provide the application of the choline kinase mutant in the synthesis of cytidine diphosphate choline.

[0013] Specifically, a reaction solution consisting of cytidine, choline chloride, disodium ATP, magnesium sulfate heptahydrate, and acetyl phosphate is added to produce cytidine phosphocholine by adding choline kinase mutant, cytidine kinase, cytidine kinase, nucleoside diphosphate kinase, acetate kinase, and phosphocholine cytidine transferase enzyme solution.

[0014] Further, the catalytic reaction conditions are as follows: cytidine, choline chloride, disodium ATP, magnesium sulfate heptahydrate, and acetyl phosphate are thoroughly dissolved in 500-600 mL of 100-120 mM pH 7.0 phosphate buffer to prepare a reaction solution with concentrations of 145-150 mM cytidine, 145-150 mM choline chloride, 5.0-5.2 mM disodium ATP, 0.6-1.0 mM magnesium sulfate heptahydrate, and 480-520 mM acetyl phosphate. Then, 1800-2100 U of choline kinase mutant, 1800-2000 U of cytidine kinase, 250-300 U of cytidine kinase, 130-180 U of nucleoside diphosphate kinase, 130-180 U of acetate kinase, and 80-120 U of phosphorylated cytidine transferase are added to the reaction solution. The reaction is carried out at 25°C and pH 7.0 for 7-10 hours, with a total reaction volume of 1000 mL.

[0015] The beneficial effects of this invention are that, in the one-pot catalytic production of citicoline using the choline kinase mutant of this invention, the highest yield of citicoline is 67.98 g / L, and the conversion rate is 98%. Compared with the original yield, the yield and conversion rate are increased by about 59% and 35% respectively, which greatly reduces the production cost. Moreover, the highly active choline kinase mutant can tolerate high concentrations of acetyl phosphate, which solves the rate-limiting problem of key enzymes in the reaction process and enables large-scale industrial production. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the synthesis route using the cytidine diphosphate choline enzyme method.

[0017] Figure 2 This is a diagram showing the construction of the pET30a-CKI expression vector in Example 1;

[0018] Figure 3 This is a flowchart illustrating the construction of the choline kinase CKI mutant in Example 3.

[0019] Figure 4 The image shows the detection results of the conversion of wild-type choline kinase CKI to produce citicoline in Example 6.

[0020] Figure 5 The graph shows the detection results of the conversion of the mutant choline kinase CKI-11 in Example 6 into citicoline.

[0021] Figure 6 The image shows the detection results of the mutant choline kinase CKI-12 from Example 6 being converted to produce citicoline. Detailed Implementation

[0022] To further illustrate the present invention, the technical solutions provided by the present invention are described in detail below with reference to embodiments. The described embodiments are only some embodiments of the present invention, and not all embodiments. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention and without creative effort are within the scope of protection of the present invention.

[0023] Example 1: Construction and fermentation of genetically engineered strains

[0024] The choline kinase CKI (amino acid sequence as shown in SEQ ID NO. 1, nucleotide sequence as shown in SEQ ID NO. 2, from *Saccharomyces cerevisiae*, NCBI Reference Sequence: NP_013234.1), cytidine kinase CDK (mouse source, GenBank: AAK14052.1), cytidine kinase CMPK (from *Dendrobium distichum*, NCBI Reference Sequence: XP_637196.1), nucleoside diphosphate kinase NDK (from *Saccharomyces cerevisiae*, UniProt: P36010.1), acetate kinase ACK (from *Escherichia coli*, GenBank: CAK1350630.1), and phosphorycholine cytidine transferase CCT (from *Saccharomyces cerevisiae*, UniProt: P36010.1) of this invention are used. P13259.2), respectively sent to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis and E. coli codon optimization, and cloned into the NdeI and XhoI restriction sites of the prokaryotic expression vector pET30a(+) to obtain the corresponding expression vector pET30a-CKI (e.g., P13259.2). Figure 2 The following gene-engineered expression strains (pET30a-CDK, pET30a-CMPK, pET30a-NDK, pET30a-ACK, and pET30a-CCT) were sequentially introduced into *E. coli* BL21(DE3) via electroporation to obtain the corresponding genetically engineered expression strains BL21(DE3) / pET30a-CKI, BL21(DE3) / pET30a-CDK, BL21(DE3) / pET30a-CMPK, BL21(DE3) / pET30a-NDK, BL21(DE3) / pET30a-ACK, and BL21(DE3) / pET30a-CCT. Fermentation of the genetically engineered expression strains was carried out in shake flasks using conventional TB medium, initially cultured at 37℃ and 200 r / min. 600 The concentration was increased to 0.6-0.8. Then, expression was induced for 10 h at 25 °C using 0.5 mM IPTG or 0.1 g / L lactose. The cells were collected by centrifugation at 10000 r / min for 5 min, the supernatant was removed, and an appropriate amount of 0.01 M pH 8.0 Tris-HCl buffer was added to prepare a cell suspension. After stirring and mixing, the cells were sonicated and centrifuged at 10000 r / min for 5 min. The precipitate was removed, and the supernatant was used for enzyme activity analysis or transformation verification, as in Example 6.

[0025] Example 2: Construction of a choline kinase mutant library

[0026] Using pET30a-CKI plasmid as a template, the CKI gene was amplified by error-prone PCR using universal T7F / R primers (primer sequences: T7F: 5'-TAATACGACTCACTATAGGG-3' and T7R: 5'-GCTAGTTATTGCTCAGCGG-3', as shown in SEQ ID NO. 3 and 4, respectively). The Mg content in the PCR amplification reaction system was adjusted. 2+ Mn 2+ The concentrations of dCTP and dTTP oligonucleotides ensure that the base mismatch rate of the mutant library is only two per thousand, which means that only one or two amino acids in a mutant are mutated.

[0027] Table 1: Error-prone PCR reaction systems

[0028] Error-prone PCR reaction conditions: First, pre-denature at 95℃ for 5 min; then denature at 94℃ for 30 s, anneal at 56℃ for 1 min, extend at 72℃ for 2 min, for a total of 30 cycles; finally, extend at 72℃ for 10 min.

[0029] Two μL of the error-prone PCR product was sampled and analyzed by agarose gel electrophoresis. After verification, the product was purified using a PCR product purification kit. At 37°C, the purified PCR product and the prokaryotic expression vector pET30a(+) were double-digested with NdeI and XhoI restriction endonucleases, respectively. The digested products were recovered from the gel (the recovered PCR product fragment was approximately 1800 bp, and the recovered pET30a(+) vector fragment was approximately 5400 bp). The mixture was then incubated at a 3:1 molar ratio of error-prone PCR product to pET30a(+) prokaryotic expression vector, and T4 DNA ligase was added. The mixture was then incubated overnight at 16°C. The next day, the ligation product was transformed into *E. coli* BL21(DE3) using electroporation to construct an engineered bacterium, resulting in a large-capacity random mutant library.

[0030] Example 3: Establishment of a method for measuring choline kinase activity

[0031] The method for determining the activity of choline kinase and its mutant enzymes is as follows: Weigh 0.70 g of choline chloride, 2.76 g of disodium ATP, and 1.02 g of magnesium chloride, add 80 mL of 0.1 mol / L pH 7.00 phosphate buffer, adjust the pH to 7.00 with sodium hydroxide solution, and bring the volume to 100 mL. Take 0.5 mL of enzyme solution (or diluted enzyme solution) into a 1.5 mL centrifuge tube (pre-fill the centrifuge tube with a certain amount of glass beads, preferably to the 1.0 mL mark). Shake on an MS3 shaker for 20 min (shake frequency set to 1500 r / min). Dilute the sample (with deionized water) to a suitable linear range for detection (1-11 enzyme units). Take an appropriate amount of diluted enzyme solution into a 50 mL test tube, add physiological saline to make up to 5 mL, add 10 mL of substrate solution preheated to 25 °C, stir and react in a 25 °C water bath for 10 min, then add 2 mL of 25% trichloroacetic acid solution to stop the reaction. After shaking and centrifuging, take 0.5 mL of the sample into a 50 mL volumetric flask, dilute to volume with water, and then perform liquid chromatography analysis.

[0032] Substrate blank: In a 50 mL test tube containing 5 mL of physiological saline, add 10 mL of substrate preheated to 25 °C, stir and react in a 25 °C water bath for 10 min, then add 2 mL of 25% trichloroacetic acid solution.

[0033] Enzyme activity unit: Under certain reaction conditions, one unit U is defined as the amount of enzyme that produces 1 μmol of ADP per minute.

[0034] HPLC analysis conditions: Column: Diamosil C18(2) 5μm 4.6×250mm. Mobile phase: Weigh 6.8g potassium dihydrogen phosphate, dissolve in 950mL ultrapure water, adjust pH to 6.2 with 1mol / L NaOH solution, filter through a 0.45μm aqueous microporous membrane, add 50mL chromatographic grade methanol, mix well, and degas for 20min. Flow rate: 1mL / min. Column temperature: 25℃. Detection: Wavelength 254nm. Injection volume: 20μL.

[0035] Reference solution: Accurately weigh 40 mg of ADP standard, dissolve it in the mobile phase and dilute to 200 mL, filter and take 20 μL sample for liquid phase analysis.

[0036] Enzyme activity calculation:

[0037] Liquid enzyme activity at 25℃ = (u / ml)

[0038] W 标 ADP reference standard weight, mg;

[0039] p 标 ADP reference standard content, %

[0040] A 样 Peak area of ​​ADP;

[0041] A 标 Peak area of ​​ADP reference standard;

[0042] M: ADP molecular weight; 427.2

[0043] T: Reaction time, in minutes;

[0044] V: Enzyme solution sample volume, mL.

[0045] Example 4: High-throughput screening and superimposed mutation construction of choline kinase mutant libraries

[0046] Using sterilized toothpicks, carefully pick single colonies from the mutant library (one colony per toothpick) and inoculate them into different wells of a 96-well cell culture plate (each well already containing LB liquid medium with 50 μg / mL kanamycin). Incubate the 96-well cell culture plate at 37°C and 700 rpm for 6 hours using a shaker. Then, using an 8-channel pipette, transfer 50 µL to a new 96-well plate as seed culture. Add lactose to each well to a final concentration of 1% (m / v) and incubate at 25°C and 250 rpm for 8 hours. After induction, freeze the 96-well cell culture plate at -86°C for 2 hours, then allow it to stand at room temperature for half an hour. Centrifuge at 4000 rpm and 4°C for 20 minutes, and transfer 50 μL of the supernatant from each well to another empty 96-well plate. Add 100 μL of reaction solution (concentration: 25 mM choline chloride, 25 mM disodium ATP, 25 mM magnesium chloride, 0.02% bromothymol blue-phenol red sodium indicator (m:m=1:3), 0.01 M pH 7.00 phosphate buffer) to 50 μL of supernatant in each well, and incubate on a horizontal shaker at 37°C for 30-120 min. Observe the color change. As the reaction proceeds, the pH of the solution gradually decreases, and the solution color gradually changes from dark red to yellow. The faster the color change of the reaction solution, the higher the activity of cytidine kinase.

[0047] Through initial and secondary screening of 60,000-80,000 clones, enzyme activity assays and sequencing analysis yielded four mutant strains with 2-10 times the activity of wild-type CKI, named CKI-1, CKI-2, CKI-3, and CKI-4. Sequencing revealed that each strain had one or two amino acid mutations on the original sequence, resulting in six new mutation sites (V69T, S116R, R272L, L411G, E165A, and V535I). Specifically, valine (Val) at position 69 was mutated to threonine (Thr), serine (Ser) at position 116 was mutated to arginine (Arg), arginine (Arg) at position 272 was mutated to leucine (Leu), leucine (Leu) at position 411 was mutated to glycine (Gly), glutamate (Glu) at position 165 was mutated to alanine (Ala), and valine (Val) at position 535 was mutated to isoleucine (Ile).

[0048] To obtain mutants with higher vigor, in addition to the two single mutants CKI-1 and CKI-3, single mutant strains with the other four mutation sites were constructed using PCR and verified by sequencing. Then, these six single mutant strains were subjected to shake-flask fermentation. The single mutant strain with the highest vigor was screened and then subjected to stacked mutations at the remaining five mutation sites one by one, completing the second round of double mutant construction. After verification by sequencing and shake-flask fermentation, the double mutant strain CKI-9 with the best vigor was selected. Similarly, based on CKI-9, the remaining four mutation sites were subjected to a third round of triple mutant stacked construction. After verification by sequencing and shake-flask fermentation, the triple mutant strain CKI-10 with the best vigor was selected. This process was repeated to successively construct and screen the four mutant strains CKI-11, five mutant strains CKI-12, and six mutant strains CKI-13 with the best vigor (mutant construction process as follows). Figure 3 As shown in Table 2). Comparative analysis of the viability of all mutant strains revealed that the five-mutant CKI-12 strain exhibited the highest shake-flask viability, which was 30.16 times that of the wild-type CKI (as shown in Table 2).

[0049] Table 2: Comparison of choline kinase and mutant strain activity during shake-flask fermentation

[0050] Example 5: Tolerance test of choline kinase and mutants to acetyl phosphate

[0051] After obtaining multiple mutants, from CKI-1 to CKI-13, this invention conducted tolerance experiments on high concentrations of acetyl phosphate to these mutants and wild-type enzymes. In the one-pot production of citicoline, acetyl phosphate is used as the energy substrate for the ATP cycle. A high concentration of 500 mM acetyl phosphate is required to generate sufficient ATP for the phosphorylation reaction of cytidine and choline. Insufficient ATP affects the reaction rate and conversion yield. Therefore, screening for choline kinases tolerant to high concentrations of acetyl phosphate is crucial for achieving high conversion rates of citicoline. In the acetyl phosphate tolerance experiment, equal volumes of mutant and wild-type enzymes were taken, and enzyme activity was measured at different acetyl phosphate concentrations. The smaller the decrease in enzyme activity, the better the enzyme's tolerance to that concentration of acetyl phosphate.

[0052] According to the method for determining choline kinase activity described in Example 3, acetyl phosphate activity was detected under conditions of 50 mM, 150 mM, and 500 mM, and the results are summarized in Table 3.

[0053] Table 3: Comparison of tolerance of choline kinase and mutants to acetyl phosphate

[0054] As shown in Table 3, when the concentration of acetyl phosphate was increased from 50 mM to 500 mM, the activity of wild-type CKI decreased to about 42% of its original value. Except for CKI-1, the residual activity of mutants CKI-2 to CKI-13 was 46-91% of the original value. Compared with the wild type, they all showed improved tolerance to high concentrations of acetyl phosphate. Among them, mutants CKI-11 and CKI-12 performed better, with relatively smaller decreases in activity, at 83% and 91% of the original value, respectively. They were able to tolerate a concentration of 500 mM acetyl phosphate, which can maximize the energy supply of the reaction system and is suitable for industrial production.

[0055] Example 6: Application of choline kinase mutant in one-pot production of citicoline

[0056] To realize the industrial application of choline kinase mutants in the synthesis of citicoline, mutants CKI-11 and CKI-12, which have better activity and tolerance to acetyl phosphate, were selected for a one-pot catalytic synthesis of citicoline. Prepare reaction solutions using 600 mL of 100 mM pH 7.0 phosphate buffer to achieve final concentrations of 148 mM cytidine, 148 mM choline chloride, 5.08 mM ATP disodium, 0.8 mM magnesium sulfate heptahydrate, and 500 mM acetyl phosphate. Then, add 2000 U of choline kinase CKI, mutant enzyme CKI-11, or mutant enzyme CKI-12 to each of the reaction solutions. Next, add 1900 U of cytidine kinase, 260 U of cytidine kinase, 150 U of nucleoside diphosphate kinase, 150 U of acetate kinase, and 100 U of phosphocholine cytidine transferase to each of the three systems (all enzyme solutions were obtained by lysing after shake-flask culture in Example 1). React at 25°C, pH 7.0, and 200 rpm for 7-10 hours with stirring. The total reaction volume is approximately 1000 mL. All enzyme activity units (U) refer to the production of 1 μmol of product per minute per unit enzyme amount under specific reaction conditions.

[0057] The entire reaction was monitored by HPLC. After the reaction was completed, the yield of cytidine in each reaction solution was analyzed, and the conversion rate was calculated (molar yield (%) = (moles of cytidine produced ÷ moles of cytidine initially added) × 100%). The final conversion results are shown in Table 4, and the HPLC detection results are as follows. Figure 4 , Figure 5 and Figure 6 As shown.

[0058] HPLC analysis conditions: Column: Agilent SB-C18 5μm 4.6×250mm. Mobile phase: Weigh 1.36g potassium dihydrogen phosphate and 0.475g tetrabutylammonium dihydrogen phosphate, dissolve in 950mL deionized water, adjust pH to 3.2 with phosphoric acid, filter through a 0.22μm aqueous filter membrane, add 50mL of chromatographic grade methanol, mix thoroughly, and degas for 20min. Flow rate: 1.0 mL / min. Column temperature: 25℃. Injection volume: 10uL. Detection wavelength: 276nm.

[0059] Peak times: cytidine 4.3 min, cytidine choline 6.4 min.

[0060] Table 4: Comparison of choline kinase conversion in the production of citicoline

[0061] As shown in Table 4 above, the mutant enzymes CKI-11 and CKI-12 of choline kinase significantly improved the yield and conversion rate of cytidine diphosphate choline (CDP-choline) under the same conditions compared with wild-type CKI. The yields were 63.82 g / L and 67.98 g / L, respectively, and the conversion rates were 92% and 98%, respectively. These are the highest levels of CDP-choline production using the in vitro one-pot enzyme method, indicating that the mutant choline kinases CKI-11 and CKI-12 have great industrial application value.

[0062] The amino acid sequence of choline kinase derived from Saccharomyces cerevisiae:

[0063] SEQ ID NO. 1 .

[0064] Nucleotide sequence of choline kinase derived from Saccharomyces cerevisiae:

[0065] SEQ ID NO. 2

Claims

1. A choline kinase mutant, characterized in that it is Mutations are made in the amino acid sequence shown in SEQ ID NO.1, and the mutation mode is any one of the following: single mutation of S116R, double mutation of S116R and E165A, double mutation of S116R and R272L, triple mutation of S116R and R272L and L411G, quadruple mutation of S116R and R272L and L411G and E165A, pentamutation of S116R and R272L and L411G and E165A and V535I, and hexamutation of S116R and R272L and L411G and E165A and V535I and V69T.

2. A polynucleotide encoding the choline kinase mutant of claim 1.

3. A vector containing the polynucleotide of claim 2.

4. Recombinant engineered bacteria containing the vector described in claim 3.

5. The application of the choline kinase mutant according to claim 1 in the synthesis of citicoline.

6. The application according to claim 5, characterized in that, In a reaction solution composed of cytidine, choline chloride, disodium ATP, magnesium sulfate heptahydrate, and acetyl phosphate, choline kinase mutant, cytidine kinase, cytidine kinase, nucleoside diphosphate kinase, acetate kinase, and phosphocholine cytidine transferase enzyme solution are added to catalyze the reaction to produce cytidine choline.

7. The application according to claim 6, characterized in that, The catalytic reaction conditions are as follows: Cytidine, choline chloride, disodium ATP, magnesium sulfate heptahydrate, and acetyl phosphate are thoroughly dissolved in 500-600 mL of 100-120 mM pH 7.0 phosphate buffer to prepare reaction solutions with concentrations of 145-150 mM cytidine, 145-150 mM choline chloride, 5.0-5.2 mM disodium ATP, 0.6-1.0 mM magnesium sulfate heptahydrate, and 480-520 mM acetyl phosphate. Then, 1800-2100 U of choline kinase mutant, 1800-2000 U of cytidine kinase, 250-300 U of cytidine kinase, 130-180 U of nucleoside diphosphate kinase, 130-180 U of acetate kinase, and 80-120 U of phosphorylated cytidine transferase are added to the reaction system. The reaction is carried out at 25°C and pH 7.0 for 7-10 hours, with a total reaction volume of 1000 mL.

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