Phosphocholine cytidylyltransferase mutants and uses thereof
By mutating the amino acid sequence of cytidine diphosphate choline (CDP-CCT001), a highly active CDP-C mutant was obtained, which solved the problems of environmental pollution from chemical synthesis and insufficient catalytic capacity from biotransformation, and realized the efficient and environmentally friendly preparation of CDP-C.
Patent Information
- Application Number
- CN202511734679.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-25
- Publication Date
- 2026-02-03
- Estimated Expiration
- 2045-11-25
AI Technical Summary
Existing chemical synthesis methods for preparing cytidine diphosphate choline suffer from problems such as expensive raw materials, harsh reaction conditions, low product yield, and environmental pollution. While biotransformation methods have advantages, their catalytic capacity is insufficient. Therefore, it is necessary to develop highly efficient bioenzymes for the preparation of cytidine diphosphate choline.
By mutating the amino acid sequence of the phosphocholine cytidine transferase MsCCT001 of the yeast Maμdiozyma saµgeensis, highly active phosphocholine cytidine transferase mutants, such as K91E, G98S, C156V, S148A, and N194D, were obtained. These mutants were used to catalyze the synthesis of cytidine diphosphate choline from phosphocholine and cytidine triphosphate.
It improves the catalytic efficiency of cytidine diphosphate choline, increasing the relative enzyme activity by 2.02 to 2.41 times, reducing production costs, and is environmentally friendly.
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Figure CN121182776B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to a phosphocholine cytidine transferase mutant and its applications. Background Technology
[0002] Citicoline, also known clinically as CDP-choline, is an endogenous compound with extremely high application value in the pharmaceutical field. Its core functions are primarily focused on the protection and repair of the nervous system. It not only promotes the repair of nerve cell membranes after brain injury but also increases the levels of key neurotransmitters in the brain, thus demonstrating significant efficacy in improving cognitive impairment after Alzheimer's disease and stroke. Recent research also indicates its positive potential in regulating mood disorders and treating optic neuropathy such as glaucoma. In recent years, the market demand for citicoline has been increasing.
[0003] Chemical synthesis is currently the main method for preparing cytidine diphosphate choline (CDP-choline), primarily involving the condensation of cytosine nucleoside phosphate morpholine and phosphate choline hydrochloride in acetonitrile or pyridine to form CDP-choline. However, this method uses expensive raw materials and condensing agents, requires the reaction to be carried out in toxic solvents, and suffers from low product yields and high production costs. Chemical synthesis is often criticized for its potential negative environmental impacts. To promote sustainable development, researchers are continuously seeking more environmentally friendly alternative technologies to reduce the pollution and resource waste that may occur during chemical synthesis.
[0004] In recent years, biotransformation has become the dominant method for synthesizing cytidine diphosphate choline (CDP-choline). This method utilizes cytidine diphosphate cytyltransferase (CCT) from microorganisms, using cytidine triphosphate and phosphate choline as substrates for biosynthesis. It offers numerous advantages, including mild reaction conditions, low cost, and simple process. Compared to chemical methods, the biosynthesis of CDP-choline is safer, more efficient, less expensive, and more environmentally friendly.
[0005] Therefore, if a more powerful bio-enzyme can be developed for the production of cytidine diphosphate choline, it will greatly improve the efficiency of industrial preparation of cytidine diphosphate choline and will be of great significance to the market development of cytidine diphosphate choline. Summary of the Invention
[0006] To address the aforementioned technical problems, this invention provides a phosphate choline cytidine transferase mutant capable of efficiently catalyzing the reaction of phosphate choline to prepare cytidine diphosphate choline.
[0007] The phosphocholine cytyltransferase mutant provided by this invention uses ScCCT from Saccharomyces cerevisiae s288C as a starting probe. BLAST analysis was performed using the NCBI database, and enzyme activity was detected to confirm its origin from the yeast.Maμ diozyma saµgeensis The amino acid sequence of the phosphorycholine cytidine transferase MsCCT001 is shown in SEQ ID NO:1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:2.
[0008] This invention involves mutating the phosphocholine cytyltransferase MsCCT001 and screening for its mutants to identify key sites affecting the initial enzymatic properties of phosphocholine cytyltransferase. Simultaneously, a highly active phosphocholine cytyltransferase mutant was obtained. The phosphocholine cytyltransferase mutant provided by this invention is obtained by mutating any one or more of the following mutations in phosphocholine cytyltransferase MsCCT001: K91E, G98S, C156V, S148A, N194D.
[0009] Preferably, the mutant is obtained by performing an N194D mutation on the amino acid sequence of phosphocholine cytyltransferase MsCCT001, followed by any combination of mutations shown below:
[0010] (1) K91E, G98S;
[0011] (2) K91E, S148A;
[0012] (3) G98S, C156V;
[0013] (4) G98S, S148A;
[0014] (5) C156V, S148A;
[0015] (6) S148A.
[0016] As a further preferred embodiment, the mutant is obtained by simultaneously mutating the amino acid sequence of phosphocholine cytyltransferase MsCCT001 by the following mutations: K91E, G98S, and N194D.
[0017] Furthermore, the gene encoding the above-mentioned phosphocholine cytidine transferase mutant, the recombinant expression vector containing the gene, and the recombinant engineered bacteria are also within the scope of protection of this invention.
[0018] The present invention provides a catalyst in which the active ingredient comprises the phosphate choline cytidine transferase mutant described above.
[0019] Furthermore, the application of the aforementioned mutants or catalysts in the preparation of cytidine diphosphate choline is also a key technical aspect protected by this invention.
[0020] Preferably, the application specifically involves: using phosphocholine and cytidine triphosphate as substrates, adding the mutant or the catalyst, and conducting a catalytic reaction under the action of magnesium chloride at 25-40°C and pH 5.0-8.0 to synthesize cytidine diphosphate choline; wherein the molar ratio of phosphocholine to cytidine triphosphate is 1:1-2, and the molar ratio of phosphocholine to magnesium chloride is 1:15-30.
[0021] The beneficial effects of this invention are as follows:
[0022] This invention mutates phosphocholine cytyltransferase MsCCT001 by N194D mutation, followed by multiple combinations of mutations such as K91E / G98S, C156V / S148A, or S148A, to obtain highly active phosphocholine cytyltransferase mutants. In the reaction catalyzing the preparation of cytidine diphosphate choline from phosphocholine and cytidine triphosphate, the relative enzyme activities of the obtained mutants are as high as 241.2%, 202.7%, and 216.2%, respectively, with catalytic efficiency 2.02 to 2.41 times that of the initial phosphocholine cytyltransferase. This invention provides a more potent catalytic enzyme preparation for the preparation of cytidine diphosphate choline through genetic engineering. Attached Figure Description
[0023] Figure 1 A schematic diagram illustrating the reaction mechanism of phosphoric acid cytidine transferase catalyzing the conversion of the substrate phosphoric acid choline and cytidine triphosphate into cytidine diphosphate choline.
[0024] Figure 2 This is a high-performance liquid chromatogram of the phosphoric acid choline cytidine transferase mutant MsCCT001-K91E / G98S / N194D catalyzing the production of cytidine diphosphate choline in Example 2 of the present invention.
[0025] Figure 3 This is a high-performance liquid chromatogram of cytidine diphosphate choline standard. Detailed Implementation
[0026] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.
[0027] The main reagents and consumables used in this invention are:
[0028] High-fidelity DNA polymerase (Phanta Max Sμper-Fidelity DNA polymerase P505) was purchased from Nanjing Novizan Biotechnology Co., Ltd.; FastDigest DpnI DNA endonuclease was purchased from Thermo Fisher Scientific; pET28(a+) plasmid was a known E. coli expression vector with a size of 5369 bp, T7 promoter, N-6×His tag, and Kanamycin (Kanamycin) antibiotic, purchased from Novogen; E. coli DH5α competent cells and BL21 (DE3) competent cells were purchased from Angyu Biotechnology Co., Ltd.; agar powder was purchased from Solarbio; tryptophan and yeast extract were purchased from OXOID.
[0029] LB medium
[0030] Each 100 mL LB medium contains: 1 g tryptone, 0.5 g yeast extract, 1 g NaCl, pH 7.0~7.2.
[0031] Preparation method: Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1 L of distilled water. Do not adjust the pH. Autoclave at 121°C for 20 min. If preparing a solid culture medium, add 1.8 g agar powder to 100 mL of the medium.
[0032] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be commercially available or prepared according to conventional methods in the art, and are of laboratory purity 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. Please refer to relevant experimental manuals or manufacturer's instructions.
[0033] Example 1: Preparation of phosphate choline cytidine transferase mutant
[0034] I. Design of Phosphocholine Cytidine Transferase Mutants
[0035] Through gene mining, the gene of phosphocholine cytidine transferase MsCCT001 was screened from the NCBI database. The open reading frame of this gene is 1332 bp in length, and the phosphocholine cytidine transferase it encodes consists of 444 amino acids. Its amino acid sequence (444 aa) is shown in SEQ ID NO:1.
[0036] The nucleotide sequence encoding the phosphocholine cytyltransferase MsCCT001 is shown in SEQ ID NO:2.
[0037] This embodiment employs a zero-shot prediction method using protein language models. Three protein language models (Saprot, proSST, and progen) with different advantages are used to perform zero-shot predictions on MsCCT001. Mutations that are mutually agreed upon by the three models are selected. These mutations include: K91E, S148A, C156V, G98S, N194D, G98A, I87E, A88E, R85D, L95K, V220A, A193E, L84E, G98E, T183S, M234A, D206Q, L86Q, P397K, V220S, K94E, L86R, G98K, L86E, A271K, Q90K, D206E, L84D, V308K, A80K, Q366E, T183R, R186K, and L8. 4R, Q366D, K367S, K367E, A193S, G98R, A376E, F89I, N92Q, L84N, N244E, D206K, C145A, Q404F, S113K, G98D, T183E, A376D, A193P, T183Q, R85A, K367D, Q110K, F89A, Q274K, L10 The mutants 0I, A88N, P159T, P159S, A80E, K102E, A88Q, and T183K were experimentally verified, and five mutants with high enzyme activity, K91E, S148A, C156V, G98S, and N194D, were obtained. Full combination iterations were performed on these mutants to obtain the mutants provided in this invention. Each mutant exhibits good catalytic performance and enzyme activity.
[0038] II. Obtaining the Phosphocholine Cytidine Transferase Mutant Gene
[0039] The genes of the phosphocholine cytidine transferase mutants obtained above can be obtained through whole-genome synthesis or molecular cloning.
[0040] After obtaining the phosphorylcholine cytidine transferase MsCCT001 gene through whole-gene synthesis in our laboratory, we further obtained the phosphorylcholine cytidine transferase mutant gene using PCR. The specific steps are as follows:
[0041] 1. Obtaining the initial complete gene of phosphocholine cytyltransferase
[0042] Our laboratory has synthesized phosphocholine cytyltransferase MsCCT001, which was synthesized by Nanjing Genscript Biotech Co., Ltd.
[0043] 2. Site-directed mutagenesis of phosphocholine cytidine transferase
[0044] (1) Design of site-directed mutagenesis primers
[0045] (2) Site-directed mutagenesis of phosphocholine cytidine transferase
[0046] Using the pET28a(+) plasmid containing the initial phosphocholine cytyltransferase gene as a template, the phosphocholine cytyltransferase mutant gene was amplified according to the following PCR system and procedure using upstream and downstream primers containing the mutation site designed based on the template.
[0047] PCR system: Phanta Max Sμper-Fidelity DNA polymerase P520 20 μL, template plasmid 1 μL, upstream primer-F (10 μM) 1 μL, downstream primer-R (10 μM) 1 μL, dd H2O 2 μL.
[0048] The PCR program was as follows: a. Pre-denaturation at 98℃ for 30 s; b. Denaturation at 98℃ for 10 s, annealing at 55℃ for 5 s, extension at 72℃ for 40 s, 32 cycles; c. Extension at 72℃ for 5 min, cooling to 4℃, digesting the template plasmid containing the initial phosphocholine cytyltransferase gene with DpnI enzyme, digesting at 37℃ for 30 min.
[0049] (3) Transformation of DH5α competent cells
[0050] The digestion product of the pET28a(+) plasmid containing the phosphocholine cytyltransferase mutant obtained in (2) was transformed into DH5α competent cells.
[0051] DH5α competent cells were placed on ice and allowed to thaw. 10 μL of plasmid solution was added and the cells were kept on ice for 30 min. They were then heat-shocked at 42°C for 45 s and allowed to stand on ice for 2 min. 700 μL of sterile LB liquid medium was added and the cells were cultured in a shaker at 37°C and 200 rpm for 1 h. After centrifugation at 4000 rpm for 1 min, 600 μL of supernatant was discarded. The precipitated cells were resuspended in the remaining supernatant and spread onto LB agar plates containing Kana resistance (50 μg / mL). The plates were incubated upside down at 37°C overnight.
[0052] (4) Screening of positive clones
[0053] Single colonies were picked from LB plates and inoculated into LB liquid medium containing Kana resistance (50 μg / mL). The culture was carried out overnight at 37°C and 200 rpm. After preservation (with sterile glycerol final concentration of 15%), the culture was sent to Ansonda (Suzhou, China) for sequencing to identify whether the initial phosphocholine cytyltransferase mutation was successful.
[0054] 3. Heterologous expression of phosphocholine cytyltransferase mutants
[0055] (1) Transformation of protein-expressing competent cells
[0056] Multiple BL21(DE3) competent cells were taken from an ultra-low temperature freezer at -80℃ and placed on ice. After the cells thawed, 1 μL of pET28a(+) plasmid containing the phosphocholine cytyltransferase mutant was added to each competent cell. The cells were placed on ice for 30 min, heat-shocked at 42℃ for 45 s, and then placed on ice for 2 min. 700 μL of sterile LB liquid medium was added, and the cells were cultured at 37℃ and 200 rpm for 1 h on a shaker. 200 μL of the culture was then taken and spread on LB agar plates containing Kana resistance (50 μg / mL), and incubated upside down at 37℃ overnight.
[0057] (2) Preservation of strains expressing phosphocholine cytidine transferase mutant
[0058] Single colonies were picked from LB plates and inoculated into test tubes containing LB liquid medium with Kana resistance (50 μg / mL). The culture was carried out overnight at 37°C and 200 rpm. The strains used as expression strains of phosphocholine cytidine transferase mutants were added to the bacterial culture with sterile glycerol at a final volume concentration of 15% and stored in an ultra-low temperature freezer at -80°C for long-term storage.
[0059] (3) Protein expression
[0060] The overnight cultured bacterial culture was inoculated into 50 mL of sterile LB liquid medium at an inoculation rate of 1%, with a final kanamycin concentration of 50 μg / mL. The culture was incubated at 37℃ and 200 rpm for about 2.5 h. When the OD600 reached 0.6~0.8, sterile IPTG with a final concentration of 0.1 mM was added, and the culture was induced at 37℃ and 200 rpm for 4 h.
[0061] (4) Crushing process
[0062] After induction, 50 mL of bacterial suspension was collected and centrifuged at 8000 rpm for 10 min at 4°C. The bacterial pellet was then resuspended in Tris-HCl (50 mM, pH 7.5) buffer and precisely diluted to ensure that the OD600 of all cell suspensions used for comparison was uniformly 10. This step ensured that the cell biomass used in subsequent reactions was completely consistent. The standardized cell suspensions were placed in an ice-water bath and sonicated (40% W power, 3 s sonication, 5 s interval, total processing time 6 min). After sonication, standardized crude enzyme solution was obtained and directly used for subsequent activity assays.
[0063] Example 2: Synthesis of cytidine diphosphate choline using the phosphate choline cytidine transferase mutant provided in Example 1 of this invention.
[0064] The schematic diagram of the reaction principle for the synthesis of cytidine diphosphate choline from phosphate choline and cytidine triphosphate using a phosphate choline cytidine yltransferase mutant in this embodiment is attached. Figure 1 As shown. The specific steps are as follows:
[0065] In a 2 mL EP tube, add 20 μL of crude enzyme solution, 100 μL of 20 mM substrate phosphocholine, 100 μL of 20 mM substrate cytidine triphosphate, and 100 μL of 400 mM magnesium chloride in sequence. Make up the reaction volume to 1 mL with buffer solution and carry out the reaction in a metal bath at 30 °C.
[0066] Enzyme activity assay:
[0067] The assay system for phosphorylated cytidine ditransferase activity is as follows:
[0068] 680 μL Tris-HCl buffer (50 mM, pH 7.5), 100 μL 20 mM phosphocholine, 100 μL 20 mM cytidine triphosphate, and 20 μL of the mutant crude enzyme solution were mixed and added to a 2 mL ep tube. The reaction was carried out in a metal bath at 30 °C.
[0069] The reaction solution after 10 min of reaction was inactivated by boiling water bath and centrifuged at 13000 r / min for 3 min. The supernatant was mixed with an equal volume of 20% acetonitrile water and centrifuged at 13000 r / min for 15 min. The supernatant was then filtered through a 0.22 µm sterile membrane into a liquid chromatography bottle for analysis.
[0070] The content of cytidine diphosphate choline was determined by high performance liquid chromatography (HPLC). The injection volume was 20 µL, the chromatographic column was a Da Cao PC HILIC 5μm 4.6×250 nm, the mobile phase was 0.05% formic acid water, acetonitrile-methanol (8:2), the column temperature was 30℃, the flow rate was 1 mL / min, and the detection wavelength was 280 nm.
[0071] The HPLC results are attached. Figure 2 As shown, the HPLC chromatogram of the cytidine diphosphate choline standard is shown below. Figure 3 ,from Figure 2 The results show that the retention time of cytidine diphosphate choline is 9.25 min.
[0072] The results showed that the phosphate choline cytidine transferase mutants obtained by the present invention produced more cytidine diphosphate choline products than the initial phosphate choline cytidine transferase under the same reaction conditions and within a reaction time of 10 min. The catalytic effects of different phosphate choline cytidine transferase mutants are shown in Table 1 below.
[0073] Table 1 Catalytic effects of different phosphocholine cytidine transferase mutants
[0074]
[0075] The results in Table 1 show that the yield of cytidine diphosphate choline (CDP-choline) catalyzed by wild-type phosphocholine cytidine transferase was only 343.95 mg / L. However, after a single-site mutation, the yield of CDP-choline increased to 500.05–579.08 mg / L within the same time period. When there were 2–4 mutation sites, the catalytic effects of the mutants showed significant differences. In particular, mutations of N194D in wild-type phosphocholine cytidine transferase, followed by mutations of K91E / G98S, C156V / S148A, or S148A, resulted in mutants with relative enzyme activities as high as 241.2%, 202.7%, and 216.2%, respectively. Their highest catalytic efficiency was 2.02–2.41 times that of the initial phosphocholine cytidine transferase. Even the G98S / S148A / N194D mutant had an enzyme activity 1.90 times that of the wild-type enzyme, greatly improving the catalytic efficiency.
[0076] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
[0077] sequence list
[0078] SEQ ID NO:1
[0079] MSNKVSRTDSLKKTLSRKSSLTNLFKKNKRKHARDDDEDDEDENGISSNDKSLTKIDNEDDDTDTSRSHKRRHVEDAAKEKLRLIAFQKNEKLLDGKLPKELRKYRPQGYSLNLPPKDRPIRIYADGVFDLFHLGHMKQLEQCKKSFPNVTLICGVPSDKVTHKLKGLTVLTDKQRCETLTHCRWVDEVIANAPWCVTPEFLKDHKIDYVAHDDIPYVSA DSDDIYKPIKEMGKFLTTQRTNGISTSDIITKIIRDYDKYLLRNFARGATRQELNVSWLKKNELEFKKHIKDFREYFKKNQESNLVNSKDLYFEVREILLRKTLGNKLYKKLLGNNKFNPKNIHSHEIKKLQDRLSDISDSSSQKDNDDEDADEESSEIDAEEREAIIRSKSPPSEFADQYTGEKRVKSKQANSQYHEDEDEKTYKFIEHAQQDVEDE
[0080] SEQ ID NO:2
[0081]
Claims
1. A phosphocholine cytidine transferase mutant, characterized in that, It is obtained by simultaneously mutating phosphocholine cytyltransferase MsCCT001 with the following mutations: K91E, G98S, and N194D. The amino acid sequence of phosphocholine cytyltransferase MsCCT001 is shown in SEQ ID NO:
1.
2. The gene encoding the phosphocholine cytidine transferase mutant of claim 1.
3. A recombinant expression vector, characterized in that, It contains the gene described in claim 2.
4. A recombinant genetically engineered bacterium, characterized in that, The recombinant genetically engineered bacteria are obtained by transforming the recombinant expression vector of claim 3 into a host microorganism.
5. A catalyst, characterized in that, The active ingredient of the catalyst includes the phosphate choline cytidine transferase mutant as described in claim 1.
6. The use of the mutant of claim 1 or the catalyst of claim 5 in the preparation of cytidine diphosphate choline.
7. The application as described in claim 6, characterized in that, Using phosphocholine and cytidine triphosphate as substrates, and adding the mutant described in claim 1 or the catalyst described in claim 5, a catalytic reaction is carried out under the action of magnesium chloride at 25-40°C and pH 5.0-8.0 to synthesize cytidine diphosphate choline; wherein the molar ratio of phosphocholine to cytidine triphosphate is 1:1-2, and the molar ratio of phosphocholine to magnesium chloride is 1:15-30.
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