Escherichia coli recombinant engineering bacterium for producing citicoline as well as construction method and application of escherichia coli recombinant engineering bacterium

Abstract

The invention discloses escherichia coli recombinant engineering bacteria for producing citicoline as well as a construction method and application of the escherichia coli recombinant engineering bacteria, and belongs to the technical field of biology. A genome cytidine deaminase coding gene cdd is knocked out on the basis of Escherichia coli K-12 MG1655, a temperature control expression vector PBV220 is used for expressing a SpnCCT-SpnCKI-pRpLpyrG mutant gene cluster, the expression of choline transporter protein BetT is enhanced, the gene locus of Escherichia coli ptsH-ptsI-crr is destroyed, meanwhile, a glucose permease gene glf and a glucokinase gene glk from Zymomonas mobilis are introduced, and the mutant gene cluster of the Escherichia coli K-12 MG1655 is obtained. The escherichia coli recombinant engineering bacterium ZMCB02NCL capable of efficiently biosynthesizing citicoline is obtained, and the preservation number of the escherichia coli recombinant engineering bacterium ZMCB02NCL is CCTCC NO: M 2025483.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a recombinant Escherichia coli engineering bacterium for producing cytidine diphosphate choline, and a construction method and application thereof. Background Art

[0002] Cytidine diphosphate choline (CDPC), also known as cytidine-5'-diphosphocholine and cytidine diphosphate choline, is a nucleotide derivative formed by connecting cytidine diphosphate (CDP) and choline through a phosphodiester bond. Its molecular structure contains a hydrophilic choline group and a hydrophobic cytidine part, enabling it to directly participate in the repair and regeneration of nerve cell membranes and regulate the synthesis and release of neurotransmitters such as acetylcholine and dopamine. It is an important precursor for the synthesis of lecithin in cells. In addition, cytidine diphosphate choline significantly improves cerebral energy supply by promoting glucose metabolism and mitochondrial function in the brain, and thus plays an irreplaceable role in neuroprotection, cognitive function enhancement, and the treatment of cerebrovascular diseases.

[0003] Cytidine diphosphate choline sodium is the sodium salt form of cytidine diphosphate choline, which can promote the metabolism of nerve cells, the synthesis and release of neurotransmitters, and improve cerebral blood circulation. It is mainly used for the treatment of cerebrovascular diseases, post-operative consciousness disorders of the brain, brain fog, Alzheimer's disease, anxiety and depression, memory decline, stroke, and other neurodegenerative diseases. Due to the safety and effectiveness of cytidine diphosphate choline in treating nerve injuries, it has been widely used as a health food or drug for the cerebrovascular and cardiovascular systems, and has a good effect on improving attention, learning, and memory.

[0004] Currently, the production methods of cytidine diphosphate choline mainly include chemical synthesis method, enzyme catalysis method, and microbial fermentation method. The chemical synthesis method uses cytidylic acid and choline phosphate as raw materials to generate cytidine diphosphate choline under the action of a condensing agent, but there are problems such as complex reaction steps, low yield, many by-products, and large environmental pollution, which are not suitable for large-scale production. The enzyme catalysis method requires a multi-enzyme system to first express different enzymes and then carry out a catalytic reaction. This method has a cumbersome process, a long time-consuming, and relies on cofactors such as ATP, making it difficult to be applied on a large scale. The microbial fermentation method directly synthesizes through genetic engineering to transform the microbial metabolic pathway, which has the advantage of being green and environmentally friendly, but faces problems such as low product concentration and poor stability.

[0005] The development of genetic engineering technology has provided a feasible solution for the efficient biosynthesis of citicoline and broken through the bottleneck of traditional production technology. Using gene editing tools to knockout endogenous competing genes in the host can effectively block the ineffective conversion of cytidine to uridine. At the same time, genetic engineering allows for cross-species screening and the introduction of enzyme genes with high catalytic activity to make up for the deficiencies in the host's natural metabolic capacity. In addition, by designing an inducer-independent expression system, efficient and controllable synthesis of citicoline can also be achieved. In recent years, the development of metabolic engineering and synthetic biology has provided new ideas for CDPC production. For example, Liu et al. (2022) increased the CDPC production to 1.2 g / L by heterologously expressing choline kinase (SpnCKI) from Streptococcus pneumoniae in Escherichia coli, but still needed to add the CTP precursor externally; the Chen team (2023) used CRISPRi technology to inhibit competing pathway genes (such as cdd ) to increase the CDPC production to 8.5 g / L, but the genetic stability of the strain was poor. In addition, the optimization of the glucose transport system has become a research hotspot. The glucose permease (Glf) and glucose kinase (Glk) of Zymomonas mobilis have been introduced into other hosts because they do not rely on the PTS system, but their compatibility with the host metabolic network still needs to be verified (Zhang et al., 2023).

[0006] Although the above research has made certain progress, the existing technology still has problems such as low productivity, insufficient substrate utilization efficiency, and complex processes. Therefore, the development of an industrial production strain of citicoline with low cost, high conversion rate, and environmental friendliness not only provides an efficient and environmentally friendly production plan for the large-scale production of citicoline, but also promotes the in-depth application of citicoline in the field of neurodrug synthesis, which has important strategic significance for promoting green bio-manufacturing and meeting global health needs. Summary of the Invention

[0007] To solve the problems existing in the prior art, the present invention discloses a recombinant engineering bacterium of Escherichia coli for producing citicoline, its construction method and application. The recombinant engineering bacterium of Escherichia coli can efficiently biosynthesize citicoline, has low production cost, is environmentally friendly, and has ideal large-scale production value.

[0008] The technical solution of the present invention is as follows: One of the purposes of the present invention is to provide a recombinant engineering bacterium of Escherichia coli for producing citicoline. The recombinant engineering bacterium of Escherichia coli was deposited at the China Center for Type Culture Collection on March 17, 2025, with the deposit number CCTCC NO: M 2025483 and named ZMCB02NCL.

[0009] Further, the deposit information is as follows: Strain name: Escherichia coli Latin name:Escherichia coli Strain number: ZMCB02NCL Depository institution: China Center for Type Culture Collection Abbreviation of depository institution: CCTCC Address: Wuhan University, China Date of deposit: March 17, 2025.

[0010] Furthermore, the recombinant engineering Escherichia coli strain ZMCB02NCL is based on Escherichia coli with the cytidine deaminase-encoding gene knocked out from its genome cdd Escherichia coli Escherichia coli K-12 MG1655 as the host cell, carrying a temperature-controlled expression vector pBV220 capable of expressing SpnCCT- SpnCKI-pRpLpyrG a mutant gene cluster, and at the same time replacing the promoter of the endogenous choline transporter gene in Escherichia coli BetT with a strong constitutive promoter P J23119 , and introducing a glucose permease gene glf and a glucokinase gene glk , integrated into the Escherichia coli locus ptsH-ptsI-crr . Furthermore, the mutant gene SpnCCT - SpnCKI is derived from Streptococcus pneumoniae Streptococcus pneumoniae , and its nucleotide sequence is as shown in SEQ ID NO. 1; The SpnCCT-SpnCKI mutant genes in SpnCCT the mutant gene and SpnCKI the mutant gene are fused and expressed through a flexible linker (GGGGS)3, and the nucleotide sequence of the flexible linker is as shown in SEQ ID NO. 2. Furthermore, the SpnCCT-SpnCKI A at position 132 of the mutant gene is mutated to T, T at position 403 is mutated to A, T at position 1116 is mutated to C, A at position 1181 is mutated to C, and A at position 1379 is mutated to G.

[0011] Furthermore, the SpnCCT-SpnCKI-pRpLpyrG mutant gene in the mutant gene cluster pyrG is derived from Escherichia coli Escherichia coli K-12 MG1655, and its nucleotide sequence is as shown in SEQ ID NO. 3.

[0012] Furthermore, the mutant gene pyrG T at position 458 is mutated to A, A at position 730 is mutated to T, G at position 948 is mutated to C, and T at position 1418 is mutated to C.

[0013] Furthermore, the SpnCCT-SpnCKI-pRpLpyrG mutant gene cluster contains pRpL a promoter, and the nucleotide sequence is as shown in SEQ ID NO. 4. Furthermore, the nucleotide sequence of the P J23119 strong constitutive promoter is as shown in SEQ ID NO. 5.

[0014] Furthermore, the glf glucose permease gene glk and the Zymomonas mobilis glucose kinase gene glf-glk are derived from glf Zymomonas mobilis. pTac For the glf gene, the nucleotide sequence of the gene is as shown in SEQ ID NO. 6, where positions 1-1422 are the

[0015] Second objective of the present invention is to provide a method for constructing an Escherichia coli recombinant engineering bacterium for producing cytidine 5'-diphosphate choline, comprising the following steps: (1) Using Escherichia coli Escherichia coli K-12 MG1655 as the host cell, knocking out the cdd gene of the Escherichia coli chassis strain, and expressing the SpnCCT-SpnCKI-pRpLpyrG gene cluster through the temperature-controlled expression vector pBV220 to obtain the Escherichia coli engineering bacterium MG-P1; (2) Randomly mutating the SpnCCT-SpnCKI-pRpLpyrG gene cluster, and then connecting the SpnCCT-SpnCKI- pRpLpyrG mutant gene cluster with the temperature-controlled vector plasmid pBV220 and introducing it into the Escherichia coli engineering bacterium MG-P1, and obtaining the Escherichia coli engineering bacterium MG-P2 through high-throughput screening; (3) Replacing the natural promoter of the endogenous BetT gene with the strong constitutive promoter P J23119 to obtain the Escherichia coli engineering bacterium MG-P3; (4) Integrating the glf glucose permease gene glk and the ptsH-ptsI-crr glucose kinase gene into the gene locus of the Escherichia coli engineering bacterium MG-P3 to finally construct the Escherichia coli recombinant engineering bacterium for producing cytidine 5'-diphosphate choline.

[0016] A third object of the present invention is to provide an application of an Escherichia coli recombinant engineering bacterium for producing cytidine 5'-diphosphate choline (CDPC) in the high-efficiency production of CDPC by fermentation culture using choline chloride, cytidine and glucose as substrates.

[0017] Further, the Escherichia coli recombinant engineering bacterium ZMCB02NCL is cultured to obtain a seed solution, the seed solution is inoculated into a fermentation medium, the pH during fermentation is regulated to 6.8 - 7.4, the induction temperature is 40°C ± 2°C, and the fermentation time is 36 - 38 h. CDPC is obtained by fed-batch fermentation using choline chloride, cytidine and glucose as substrates.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention breaks through the limitations of the existing production technology of CDPC and innovatively constructs an Escherichia coli recombinant engineering bacterium by using a systematic multi-dimensional transformation strategy. First, the genomic cdd gene is knocked out to block cytidine degradation, and the endogenous pyrG gene mutation is combined to enhance CTP synthesis, providing sufficient precursors for the accumulation of CDPC; secondly, the SpnCCT and SpnCKI genes derived from Streptococcus pneumoniae are introduced, and fused and expressed through a flexible linker (GGGGS)³ to reduce the steric hindrance between enzymes and improve the catalytic efficiency; then, the expression levels and enzyme activity ratios of the exogenously introduced key gene SpnCCT-SpnCKI and the endogenous pathway gene pyrG are optimized, and the SpnCCT-SpnCKI-pRpLpyrG gene cluster is used as a whole for directed mutagenesis by in vitro random mutagenesis technology, and the temperature-controlled expression vector pBV220 is used to drive the key gene cluster SpnCCT-SpnCKI-pRpLpyrG , and the temporal regulation of gene expression is achieved through temperature segmentation control, avoiding inducer dependence and simplifying the production process of CDPC.

[0019] 2. The present invention constructs the Escherichia coli recombinant engineering bacterium ZMCB02NCL by integrating frontier biotechnology. The ptsH-ptsI-crr is knocked out to disrupt the natural PTS system in Escherichia coli, and the glucose permease gene glf and the glucose kinase gene glk from Zymomonas mobilis are introduced and integrated into the locus ptsH-ptsI-crr . A new glucose transport system independent of phosphoenolpyruvate (PEP) is used to replace the bacterial phosphotransferase system (PTS) system. By reducing PEP consumption, the carbon flux of the CDPC biosynthetic pathway is increased, the carbon metabolic burden is reduced, the glucose utilization rate is improved, and the efficiency of CDPC synthesis is increased. At the same time, the strong constitutive promoter P J23119 is used to replace the choline transporter BetTThe natural promoter of the gene enhances the choline transporter BetT expression of the gene, promotes the efficient entry of the substrate choline chloride into the cell, and further effectively improves the synthesis efficiency of cytidine diphosphate choline.

[0020] 3. The recombinant Escherichia coli engineering strain ZMCB02NCL constructed by the present invention can simultaneously and efficiently utilize glucose, cytidine and choline chloride, with a high substrate conversion rate. Among them, the endogenous pyrG gene mutation enhances CTP synthesis, providing sufficient precursors for the accumulation of cytidine diphosphate choline, glf and glk the stable integration and co-expression of the genes enhance the glucose uptake efficiency, reduce the carbon metabolic burden, and the strong promoter strengthens the expression of the choline transporter BetT, improving the uptake efficiency of the substrate choline chloride. Using glucose, cytidine, and choline chloride as substrates, the recombinant Escherichia coli engineering strain ZMCB02NCL is used for shake flask fermentation to produce cytidine diphosphate choline, and its fermentation yield can reach 35.2 g / L in a 10 L tank. Moreover, the product separation and purification are simple, the process is simple, green and safe. In addition, during the passage of the recombinant Escherichia coli engineering strain ZMCB02NCL, there are no obvious changes in the biomass of the strain and the content of cytidine diphosphate choline, showing good genetic stability and having ideal industrial application value. Brief Description of the Drawings

[0021] Figure 1 is the construction route of the recombinant Escherichia coli engineering strain ZMCB02NCL of the present invention; Figure 2 is the structural schematic diagram of the recombinant plasmid pBV220-SpnCCT-SpnCKI-pRpLpyrG carried by the recombinant Escherichia coli engineering strain ZMCB02NCL of the present invention; Figure 3 is the fermentation curve of the recombinant Escherichia coli engineering strain ZMCB02NCL for producing CDPC in Example 5 of the present invention. Detailed Embodiments

[0022] The following further describes the present invention in conjunction with preferred embodiments. In the present invention, the endpoints and any values within the disclosed ranges are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values; for numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0023] In the following examples, the experimental methods are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0024] In the following examples, "codon optimization" refers to the redesign of genes by using preferred codons and avoiding codons with low or rare utilization rates. Each organism exhibits a certain degree of codon usage bias or preference, and those most frequently used are the preferred codons.

[0025] The molecular biology experiments not specifically described in the following examples include plasmid construction, restriction digestion, ligation, preparation of competent cells, transformation, medium preparation, etc., and are mainly carried out with reference to "Molecular Cloning: A Laboratory Manual" (Third Edition); the PCR amplification experiment is carried out according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions.

[0026] The whole gene synthesis, primer synthesis and sequencing in the following examples were completed by Shanghai Sangon Biotech Co., Ltd.; the host strain Escherichia coli K-12 MG1655 was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0027] The sequences of the primers used in the following examples are shown in Table 1: Table 1 Primer sequences in the following examples

[0028] The steps of the shake flask fermentation method described in the following examples are as follows: LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 20 g / L agar powder is added to the solid medium.

[0029] Shake flask fermentation medium: 10 g / L glucose, 15 g / L peptone, 20 g / L yeast extract, 2.5 g / L potassium dihydrogen phosphate, 15 g / L dipotassium hydrogen phosphate trihydrate, 0.5 g / L sodium chloride, 1 g / L glycerol, 50 mg / L ampicillin, pH 7.0.

[0030] Shake flask fermentation method: First, the recombinant strain is streaked from the -80°C glycerol stock to the LB solid medium containing ampicillin and cultured at 30°C for activation. A single colony is picked and inoculated into 50 mL of LB liquid medium (containing ampicillin), and cultured with shaking at 30°C and 200 rpm for 14-16 hours until the OD 600 reaches 2-3. It is transferred to the shake flask fermentation medium at an inoculation amount of 1%-2% and cultured at 30°C and 200 rpm until the OD 600Reach 0.6 - 0.8. Heat up to 42°C, and simultaneously add choline chloride to a final concentration of 8 mM and cytidine to a final concentration of 5 mM. Continue culturing at 42°C and 200 rpm for 12 - 24 hours, and regularly take samples to detect OD 600 and the concentration of the product CDPC.

[0031] Fermentation broth detection method: Pipette 12000 rpm centrifuge the fermentation broth for 3 min, dilute the supernatant by a certain multiple, centrifuge through a 0.22 μm filter membrane, and detect with high performance liquid chromatography (HPLC). The parameters of HPLC are as follows: The chromatographic column is Shim-pack GISSC18 column (4.6mm×150mm, 5 μm), the mobile phase is methanol and 10 mM phosphate buffer PBS (pH 4.0), the mobile phase ratio is methanol: phosphate buffer = 3:97, the flow rate is 1.0 mL / min, the injection volume is 5 μL, and the detection wavelength λ = 270 nm.

[0032] The steps of the plasmid elimination method described in the following examples are as follows: Elimination of pTarget F series plasmids: Pick a monoclonal colony and inoculate it into LB medium containing kanamycin Kana and 0.5 mM IPTG antibiotics, culture overnight at 30°C, dilute by a certain multiple and spread on the Kana plate, culture at 30°C, pick a monoclonal colony and streak it on the Kana plate and the spectinomycin Spec plate. When the monoclonal colony grows on the Kana plate but not on the Spec plate, it indicates that the pTarget F plasmid has been successfully eliminated.

[0033] Elimination of pCas9 series plasmids: Pick a monoclonal colony into LB medium, culture overnight at 37°C, dilute by a certain multiple and spread on the LB plate, culture at 37°C, pick a monoclonal colony and streak it on the Kana plate and the LB plate. When the monoclonal colony grows on the LB plate but not on the Kana plate, it indicates that the pCas9 plasmid has been successfully eliminated.

[0034] Example 1 This example provides a method for constructing recombinant Escherichia coli MG-P1, including the following steps: (1) cdd Gene knockout 1)Design cdd Specific target points of 20 bp for the gene, use primers cdd-N20-F and cdd-N20-F-R to perform inverse PCR amplification with the pTarget F plasmid as the template. The amplified product is digested with the restriction enzyme DpnI, and transformed into DH5α competent cells to construct the knockout plasmid pTF-Δcdd; 2)Using the genome of Escherichia coli MG1655 as the template, use primers cdd-LH-F / cdd-LH-R to amplify cddThe upstream homologous arm, the downstream homologous arm of cdd was amplified using primers cdd-RH-F / cdd-RH-R, and the upstream and downstream homologous arms were ligated using primers cdd-LH-F / cdd-RH-R to obtain the targeting DNA homologous recombination fragment; 3) Transform the pCas9 plasmid into competent Escherichia coli MG1655 to obtain Escherichia coli MG1655 / pCas9 containing the pCas9 plasmid. Electrotransform the plasmid pTF-Δcdd into competent cells of MG1655 / pCas9. Perform colony PCR verification on the single colonies grown on the plate, screen positive transformants and perform gene sequencing to obtain the recombinant Escherichia coli strain MG1655 (Δ cdd of the cdd gene); (2) Construction of the pBV220-SpnCCT-SpnCKI-pRpLpyrG plasmid 1) Codon optimization was performed on the genes of phosphocholine cytidylyltransferase SpnCCT and choline kinase SpnCKI from Streptococcus pneumoniae, and they were ligated using the flexible linker (GGGGS)3 to synthesize the sequence SpnCCT-SpnCKI , and the gene sequence is shown in SEQ ID NO.1; 2) Use primers CCT-CKI-F / CCT-CKI-R to perform PCR recovery on the artificially synthesized double-stranded DNA molecule SEQ ID NO.1 to obtain the fragment SpnCCT-SpnCKI ; The vector backbone pBV220 was digested with restriction enzymes EcoR Ⅰ and BamH Ⅰ; 3) Ligate the fragment SpnCCT-SpnCKI and the vector backbone digested with EcoR Ⅰ and BamH Ⅰ to obtain the recombinant plasmid pBV220-SpnCCT-SpnCKI; 4) Use primers pyrG-F / pyrG-R to amplify the pyrG gene with the MG1655 genome as the template, use primers pRpL-F / pRpL-R to obtain the promoter sub-pRpL fragment, and obtain the fragment pRpLpyrG through Overlapping PCR; 5) Use restriction enzymes BamH Ⅰ and Sla Ⅰ to digest the vector backbone pBV220-SpnCCT-SpnCKI; Ligate the fragment pRpLpyrG and the one digested with BamH Ⅰ and SlaThe vector backbone obtained by digestion with enzyme Ⅰ was ligated to construct the recombinant plasmid pBV220-SpnCCT-SpnCKI-pRpLpyrG; (3)Construction of recombinant Escherichia coli engineering strain MG-P1 1) Prepare the competent cells of Escherichia coli MG1655 (Δcdd). Take 1 μL of the constructed recombinant plasmid pBV220-SpnCCT-SpnCKI-pRpLpyrG and add it to the prepared competent cells of Escherichia coli MG1655 (Δ cdd ) and place it at the bottom of a pre-chilled electroporation cuvette. Then perform transformation by electroporation (2200 V, 5 ms); 2) After the transformation is completed, quickly add 1 mL of LB medium to the electroporation cuvette to resuspend the cells, and then transfer them to a centrifuge tube. Incubate at 37 °C and 220 rpm for 1 h to obtain the transformation product; 3) Spread the transformation product on an LB solid plate containing 100 μg / mL of ampicillin and culture at 37 °C. The recombinant Escherichia coli MG-P1 containing the recombinant plasmid pBV220-SpnCCT-SpnCKI-pRpLpyrG was obtained through colony identification.

[0035] (4)Detection of CDPC content The recombinant Escherichia coli MG-P1 was cultured in a shake flask for 24 h, and the fermentation broth was taken for CDPC content detection. The results showed that the CDPC concentration in the supernatant of the fermentation broth of Escherichia coli MG-P1 was 0.09 g / L.

[0036] Example 2 This example provides a method for constructing a recombinant Escherichia coli MG-P2, which includes the following steps: (1)Error-prone PCR mutagenesis Using the QuickMutation™ Gene Random Mutation Kit, with the plasmid pBV220-SpnCCT-SpnCKI-pRpLpyrG as the template, error-prone primers F-SpnCCT / R-pyrG were used for error-prone PCR to mutate the gene SpnCCT-SpnCKI- pRpLpyrG to generate a mutant library of the gene cluster SpnCCT-SpnCKI-pRpLpyrG ; Error-prone PCR system (50 μL): ddH2O 32.5 μL, RandomMut buffer (10X) 5 μL, Mutation enhancer (10X) 5 μL, dNTP (2.5 mM each) 5 μL, template DNA 0.5 μL, primer mixture (10 μM each) 1 μL, RandomMut DNA polymerase 1 μL.

[0037] Error-prone PCR procedure: Pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 4 min, 30 cycles of reaction; finally, extension at 72°C for 10 min, and store the product at 4°C.

[0038] To increase the mutation rate, the amplified product of the above error-prone PCR was recovered and used as a template for continuous error-prone PCR, and the procedure was the same as the above steps; (2)Construction of mutant expression library 1) Use a DNA recovery kit to recover the error-prone PCR product, double-digest it with EcoRⅠ and SalⅠ, and then ligate it to the pBV220 vector treated with the same restriction enzymes. Transform the ligation product E. coli into JM109 to obtain a JM109 bacterial library containing mutants of pBV220-SpnCCT-SpnCKI-pRpLpyrG; E. coli 2) Extract the plasmid from the JM109 bacterial library, and transform the plasmid into Escherichia coli MG1655 (Δcdd) to obtain an Escherichia coli MG1655 (Δcdd) bacterial library containing mutants of pBV220-SpnCCT-SpnCKI-pRpLpyrG; E.coli (3)Screen for high-yield strains of cytidine diphosphate choline CDPC 1) Pick monoclonal colonies from the Escherichia coli MG1655 (Δcdd) bacterial library containing mutants of pBV220-SpnCCT-SpnCKI-pRpLpyrG obtained in step (2) above into a 96-well plate with 150 μL of fermentation medium containing 50 μg / mL of ampicillin per well, culture at 37°C and 150 rpm for 48 h, centrifuge at 5000 rpm, take the supernatant for testing. 2)

[0039] 2) Ten strains with CDPC production greater than 20 mg / 100 mL were screened on a 96-well plate. The CDPC production of the other strains in the wells was generally between 10 mg / 100 mL and 17 mg / 100 mL. The ten strains were respectively renamed as MG2-1, MG2-2, MG2-3, MG2-4, MG2-5, MG2-6, MG2-7, MG2-8, MG2-9, and MG2-10.

[0040] 3) The ten strains were rescreened by shake-flask fermentation. Among them, the strain MG2-9 had the highest CDPC production, reaching 0.19 g / L, which was 2.1 times higher than that of the control strain MG-P1. It was named Escherichia coli MG-P2. The results of the shake-flask rescreening are shown in Table 2.

[0041] Table 2 Content of cytidine diphosphate choline after 24 h of shake-flask fermentation

[0042] Example 3 This example provides a method for constructing recombinant Escherichia coli MG-P3, which includes the following steps: (1) Design a 20-bp specific target for the BetT promoter, and use primers P BetT -N20-F / P BetT -N20-R to perform inverse PCR amplification using the pTarget F plasmid as a template to construct the knockout plasmid pTF-ΔP BetT ; (2) Use primers P BetT -LH-F / P BetT -LH-R to amplify the upstream homologous fragment of P BetT Use primers P BetT -RH-F / P BetT -RH-R to amplify the downstream homologous fragment of P BetT and purify the products; (3) Use primers P BetT -LH-F and P BetT -RH-R to fuse the upstream and downstream homologous fragments to obtain the fusion fragment P BetT LH- P J23119 -P BetT RH ; (4) Electroporate pTarget and the homologous fragments into the competent cells of MG-P2 carrying pCas9, coat them on LB agar containing kanamycin and streptomycin, and then incubate at 37 °C for 16 h; (5) Use primers PBetT -CX-F and P BetT -CX-R verification colonies, successfully obtained strains with the promoter replaced, for the plasmid pTF-ΔP of the strain BetT and the pCas9 plasmid were eliminated to obtain recombinant Escherichia coli MG-P3. BetT

[0043] Example 4 This example provides a method for constructing a recombinant engineering bacterium ZMCB02NCL of Escherichia coli for producing cytidine diphosphate choline, including the following steps: (1) Construction of the knockout plasmid pTF-ΔptsH-ptsI-crr Design specific targets of 20 bp for the ptsH-ptsI-crr gene, use the primers ptsH-ptsI-crr-N20-F / ptsH-ptsI-crr-N20-R to perform inverse PCR amplification with the pTarget F plasmid as the template, the amplified product was digested with the restriction enzyme DpnI, and transformed into DH5α competent cells to construct the knockout plasmid pTF-ΔptsH-ptsI-crr; (2) DNA homologous recombination insertion fragment 1) The glucose permease gene glf and the glucose kinase gene glk derived from Zymomonas mobilis were codon-optimized and artificially synthesized as SEQ ID NO. 6, which was ligated with the vector pTrc99a to obtain the recombinant plasmid pTrc99a-glf-glk; 2) Use the primers pT-F / pT-R to amplify the gene fragment expression cassette glf-glk , using the genome of Escherichia coli MG1655 as the template, use the primers ptsH-ptsI-crr-LH-F / ptsH-ptsI-crr-LH-R to amplify ptsH-ptsI-crr the upstream homologous arm, use the primers ptsH-ptsI-crr-RH-F / ptsH-ptsI-crr-RH-F-R to amplify ptsH-ptsI-crr the downstream homologous arm, and use Overlapping PCR to ligate the upstream and downstream homologous arms with the gene fragment expression cassette glf-glk to obtain the gene insertion expression cassette LH ptsH-ptsI-crr -glf-glk-RH ptsH-ptsI-crr ; (3) Knockout of the locus ptsH-ptsI-crr and glf-glk gene integration 1) Transform the pCas9 plasmid into Escherichia coli MG-P3 competent cells to obtain Escherichia coli MG-P3 / pCas9 containing the pCas9 plasmid; ​2) Insert the plasmid pTF-ΔptsH-ptsI-crr and the gene into the expression cassette LH ptsH-ptsI-crr -glf-glk-RH ptsH-ptsI-crr Electroporate into the competent cells of MG-P3 / pCas9, and perform colony PCR verification on the single colonies grown on the plate; 3) Screen positive transformants and perform gene sequencing. After eliminating the strain plasmids pTF-ΔptsH-ptsI-crr and the pCas9 plasmid, the antibacterial-free strain MG-P4 is obtained; (4)Flask fermentation of strain MG-P4 Cultivate strain MG-P4 in a flask in a fermentation medium containing 10 g / L glucose for 24 h, detect the residual sugar content and the CDPC yield in the supernatant of the fermentation broth and screen. Finally, the recombinant Escherichia coli engineering strain ZMCB02NCL is constructed; The detection results are shown in Table 3. After 24 h of flask fermentation, the CDPC yield of strain MG-P4 reaches 0.32 g / L, and the glucose consumption is 9.8 g / L. Compared with the control strain MG-P3, the CDPC yield of strain MG-P4 and the conversion rate of glucose to CDPC are significantly improved.

[0044] Table 3 Content of cytidine 5'-diphosphate choline and glucose after 24 h of flask fermentation

[0045] Example 5 This example provides a method for applying the recombinant Escherichia coli engineering strain ZMCB02NCL to produce cytidine 5'-diphosphate choline, including the following steps: (1)Prepare the culture medium as follows: Seed flask culture medium: glucose 10 g / L, peptone 15 g / L, yeast extract 20 g / L, potassium dihydrogen phosphate 2.5 g / L, dipotassium hydrogen phosphate trihydrate 15 g / L, sodium chloride 0.5 g / L, glycerol 1 g / L, ampicillin 50 mg / L, pH 7.0; Fermentation medium: glucose 20 g / L, yeast extract 15 g / L, peptone 10 g / L, sodium chloride 3.0 g / L, ammonium sulfate 3.0 g / L, dipotassium hydrogen phosphate trihydrate 3.0 g / L, potassium dihydrogen phosphate 1.5 g / L, ferric ammonium citrate 0.5 g / L, citric acid 1.5 g / L, glycerol 3 g / L, magnesium sulfate heptahydrate 0.5 g / L, antifoaming agent 0.1 mL / L, pH 7.0; (2) Inoculate the Escherichia coli ZMCB02NCL monoclonal into an LB slant medium and culture it at 30 °C for 24 h. Resuspend the cells with 10 mL of sterile water, and then inoculate them into a seed flask containing the primary seed flask medium at an inoculation amount of 2%. Culture at 30 °C for 16 h to obtain the primary seed liquid. (3) Transfer the primary seed liquid into a fermenter containing the secondary seed medium at a transfer amount of 5% and culture at 30 °C for 12 h to obtain the secondary seed liquid. (4) Transfer the secondary seed liquid into a 10 L fermenter (liquid loading volume 7 L) containing the fermentation medium at a transfer amount of 10% and culture at 30 °C. The dissolved oxygen (DO) is maintained above 30% by adjusting the stirring speed to 300 - 600 rpm and the aeration rate to 1 - 2 vvm. Continuously monitor the OD 600 , until the bacteria grow to the mid-logarithmic phase (OD 600 ≈ 35), then raise the culture temperature from 30 °C to 40 °C ± 2 °C. During this process, synchronously increase the stirring speed to 600 - 800 rpm to maintain DO ≥ 20%, and synchronously feed choline chloride solution to make the final concentration in the fermentation broth 8 mM and cytidine to the final concentration of 5 mM. (5) When the residual sugar concentration is lower than 2 g / L, feed glucose (400 g / L) at a rate of 0.5 - 1.5 mL / (L·h) to maintain the carbon source supply. At the same time, according to the OD 600 growth, add additional choline chloride to the final concentration of 5 mM and cytidine to the final concentration of 2 mM. Control the pH of the fermentation process at 6.8 - 7.4. (6) Take the fermentation broth for CDPC content detection. The results show that the recombinant Escherichia coli engineering strain ZMCB02NCL can reach 35.2 g / L after 36 h of fermentation in a 10 L fermenter.

[0046] Performance Test 1. Sequencing of the SpnCCT - SpnCKI - pRpLpyrG mutant gene cluster of Escherichia coli MG - P2 Extract the mutant plasmid pBV220 - SpnCCT - SpnCKI - pRpLpyrG from the Escherichia coli MG - P2 constructed in Example 2, perform PCR using the primers F - SpnCCT / R - pyrG, and send it to Shanghai Sangon Biological Engineering Co., Ltd. for sequencing.

[0047] The results showed that a total of 9 gene mutations occurred in the SpnCCT-SpnCKI-pRpLpyrG gene cluster. Among them, the A at the 132nd position of the SpnCCT-SpnCKI mutant gene on the sequence SpnCCT-SpnCKI-pRpLpyrG mutated to T, the T at the 403rd position mutated to A, the T at the 1116th position mutated to C, the A at the 1181st position mutated to C, and the A at the 1379th position mutated to G. The T at the 458th position of the pyrG mutant gene mutated to A, the A at the 730th position mutated to T, the G at the 948th position mutated to C, and the T at the 1418th position mutated to C.

[0048] 2. Transport efficiency test of choline chloride The Escherichia coli non-antibacterial strain MG-P3 constructed in Example 3 was fermented in a shake flask for 24 h, and its fermentation broth was taken for CDPC content detection.

[0049] The results showed that the CDPC concentration in the supernatant of the fermentation broth of the Escherichia coli non-antibacterial strain MG-P3 was 0.25 g / L, which was 31.8% higher than that of the control strain MG-P2, indicating that using a strong promoter P J23119 to replace the natural BetT promoter improved the yield of choline chloride to cytidine diphosphate choline.

[0050] 3. Genetic stability test The Escherichia coli engineered strain ZMCB02NCL was subcultured to investigate its genetic stability. It was subcultured once every 2 days for 10 generations, and the biomass and cytidine diphosphate choline content of the strain were measured by shake flask fermentation every other generation.

[0051] The results showed that there were no obvious changes in the biomass and cytidine diphosphate choline content of the Escherichia coli engineered strain ZMCB02NCL during the subculture process, indicating good genetic stability.

[0052] The Escherichia coli engineered strain ZMCB02NCL, which was genetically stable and could efficiently accumulate cytidine diphosphate choline simultaneously, was deposited at the China Center for Type Culture Collection (CCTCC) on March 17, 2025. The deposit number is CCTCC NO: M2025483, and the deposit address is Wuhan University, Wuhan, China, Zip Code: 430072.

[0053] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structure or equivalent process transformation made using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, shall be similarly included in the patent protection scope of the present invention.

Claims

1. A recombinant Escherichia coli engineering bacterium for producing cytidine diphosphate choline, characterized in that, The recombinant Escherichia coli engineering strain was deposited at the China Center for Type Culture Collection on March 17, 2025, with the deposit number CCTCC NO: M 2025483 and named ZMCB02NCL.

2. The recombinant Escherichia coli engineering bacteria for producing cytidine-5'-diphosphate choline according to claim 1, characterized in that, The recombinant engineering Escherichia coli strain ZMCB02NCL is Escherichia coli with the cytidine deaminase-encoding gene knocked out from its genome cdd and uses Escherichia coli Escherichia coli K-12 MG1655 as the host cell, carrying the temperature-controlled expression vector pBV220 capable of expressing SpnCCT-SpnCKI-pRpLpyrG the mutant gene cluster. At the same time, the promoter of the endogenous choline transporter gene in Escherichia coli BetT is replaced with a strong constitutive promoter P J23119 , and the glucose permease gene glf and the glucose kinase gene glk are introduced and integrated into the Escherichia coli locus ptsH-ptsI-crr .

3. The recombinant Escherichia coli engineering bacterium for producing cytidine 5'-diphosphate choline according to claim 2, wherein, The mutant gene SpnCCT - SpnCKI is derived from Streptococcus pneumoniae Streptococcus pneumoniae , and its nucleotide sequence is as shown in SEQ ID NO. 1; The said SpnCCT-SpnCKI in the mutant gene SpnCCT the mutant gene and SpnCKI the mutant genes are connected by a flexible linker (GGGGS)3 for fusion expression, and the nucleotide sequence of the flexible linker is as shown in SEQ ID NO.

2.

4. The recombinant engineering Escherichia coli for producing cytidine-5'-diphosphate choline according to claim 2, characterized in that, The SpnCCT-SpnCKI-pRpLpyrG mutant genes in the mutant gene cluster pyrG are derived from Escherichia coli Escherichia coli K-12 MG1655, and the nucleotide sequence is as shown in SEQ ID NO.

3.

5. The recombinant Escherichia coli engineering bacterium for producing cytidine diphosphate choline according to claim 2, wherein The said SpnCCT-SpnCKI-pRpLpyrG The mutant gene cluster contains pRpL a promoter, the nucleotide sequence of which is as shown in SEQ ID NO.

4.

6. The recombinant Escherichia coli engineering bacterium for producing cytidine 5'-diphosphate choline according to claim 2, characterized in that, The strong constitutive promoter P J23119 has the nucleotide sequence shown in SEQ ID NO.

5.

7. The recombinant engineering Escherichia coli for producing cytidine 5'-diphosphate choline according to claim 2, wherein, The glucose permease gene glf and the glucokinase gene glk are derived from Zymomonas mobilis Zymomonas mobilis , glf-glk The nucleotide sequence of the gene is shown in SEQ ID NO. 6, where positions 1-1422 are glf the gene, positions 1423-1451 are pTac the promoter, and positions 1452-2438 are glf the gene.

8. A method for constructing an Escherichia coli recombinant engineering bacterium for producing cytidine 5'-diphosphate choline according to any one of claims 1 to 7, characterized in that, including the following steps: (1) Using Escherichia coli Escherichia coli K-12 MG1655 as the host cell, knock out the cdd gene of the Escherichia coli chassis strain, and express the SpnCCT-SpnCKI-pRpLpyrG gene cluster through the temperature-controlled expression vector pBV220 to obtain the engineered Escherichia coli strain MG-P1; (2) Randomly mutate the SpnCCT-SpnCKI-pRpLpyrG gene cluster, and then SpnCCT-SpnCKI- pRpLpyrG ligate the gene mutation cluster with the temperature-controlled vector plasmid pBV220 and introduce it into the Escherichia coli engineering bacterium MG-P1. After high-throughput screening, the Escherichia coli engineering bacterium MG-P2 is obtained; (3) Replace the natural promoter of the endogenous BetT gene with a strong constitutive promoter P J23119 to obtain the engineered Escherichia coli strain MG-P3; (4) Integrate the glucose permease gene glf and the glucokinase gene glk into the MG-P3 locus of the Escherichia coli engineering bacteria ptsH-ptsI-crr to finally construct the recombinant Escherichia coli engineering bacteria for producing cytidine 5'-diphosphate choline.

9. Use of the recombinant Escherichia coli engineering strain for producing cytidine 5'-diphosphate choline according to any one of claims 1 to 7 in the high-efficiency production of cytidine 5'-diphosphate choline by fermentation culture using choline chloride, cytidine and glucose as substrates.

10. The application according to claim 9, wherein, Cultivate the recombinant Escherichia coli engineering strain ZMCB02NCL to obtain a seed solution, inoculate the seed solution into a fermentation medium, regulate the pH during fermentation to 6.8 - 7.4, the induction temperature to 40°C ± 2°C, and the fermentation time to 36 - 38 h, and produce cytidine 5'-diphosphate choline by fed-batch fermentation using choline chloride, cytidine and glucose as substrates.

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