A recombinant Escherichia coli strain producing citicoline and its construction method and application

Abstract

The present invention discloses a recombinant engineering bacterium of Escherichia coli for producing citicoline and a construction method and application thereof, belonging to the field of biotechnology; Escherichia coli K‑12 MG1655-based knockout of the genomic cytidine deaminase gene cdd The temperature-controlled expression vector PBV220 was used to express SpnCCT‑SpnCKI‑pRpLpyrG Mutating the gene cluster enhances the expression of the choline transporter BetT and disrupts Escherichia coli ptsH‑ptsI‑ crr locus, and simultaneously introduced from Zymomonas mobilis ( Zymomonas mobilis ) glucose permease gene glf and glucokinase genes glk , and obtained the recombinant Escherichia coli ZMCB02NCL capable of efficiently biosynthesizing citicoline, with the deposit number being CCTCC NO: M 2025483.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a recombinant engineering Escherichia coli for producing citicoline, a construction method thereof and an application thereof. Background Art

[0002] Cytidine 5'-diphosphocholine (CDPC), also known as cytidine-5'-diphosphocholine or cytidine diphosphocholine, is a nucleotide derivative formed by cytidine diphosphate (CDP) and choline linked by a phosphodiester bond. Its molecular structure consists of a hydrophilic choline group and a hydrophobic cytidine moiety, 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 also an important precursor for phosphatidylcholine synthesis in cells. Furthermore, cytidine choline significantly improves brain energy supply by promoting glucose metabolism and mitochondrial function in the brain, thus playing an irreplaceable role in neuroprotection, cognitive enhancement, and the treatment of cerebrovascular diseases.

[0003] Citicoline sodium is the sodium salt form of citicoline. It promotes nerve cell metabolism, the synthesis and release of neurotransmitters, and improves cerebral blood circulation. It is primarily used to treat cerebrovascular disease, impaired consciousness after brain surgery, brain fog, Alzheimer's disease, anxiety and depression, memory loss, stroke, and other neurodegenerative diseases. Due to its safety and effectiveness in treating nerve damage, citicoline has been widely used as a drug for the cerebrovascular and cardiovascular systems, and has a beneficial effect on improving attention, learning, and memory.

[0004] Currently, the main methods for producing citicoline include chemical synthesis, enzymatic catalysis, and microbial fermentation. The chemical synthesis method uses cytidylic acid and choline phosphate as raw materials to produce citicoline under the action of a condensing agent. However, it has problems such as complex reaction steps, low yield, many by-products, and severe environmental pollution, making it unsuitable for mass production. The enzymatic catalysis method requires the use of a multi-enzyme system to express different enzymes before catalyzing the reaction. This method is cumbersome and time-consuming, and relies on auxiliary factors such as ATP, making it difficult to apply on a large scale. The microbial fermentation method directly synthesizes by genetically engineering the metabolic pathways of microorganisms, 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 has broken through the bottleneck of traditional production technology. The use of gene editing tools to knock out the host's endogenous competitive genes can effectively block the ineffective conversion of cytidine to uridine. At the same time, genetic engineering allows cross-species screening and introduction of enzyme genes with high catalytic activity to make up for the lack of natural metabolic capacity of the host. In addition, by designing an inducer-independent expression system, efficient and controllable citicoline synthesis 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 Streptococcus pneumoniae choline kinase (SpnCKI) in Escherichia coli, but still required exogenous addition of CTP precursors; Chen's team (2023) used CRISPRi technology to inhibit competitive pathway genes (such as cdd ), increasing CDPC production to 8.5 g / L, but the strain's genetic stability was poor. Furthermore, optimizing glucose transport systems has become a research hotspot. Glucose permease (Glf) and glucokinase (Glk) from Zymomonas mobilis have been introduced into other hosts because they are independent of the PTS system, but their compatibility with host metabolic networks remains to be verified (Zhang et al., 2023).

[0006] Despite the progress made in these studies, existing technologies still face challenges such as low yields, inefficient substrate utilization, and complex processes. Therefore, developing a low-cost, high-conversion, and environmentally friendly strain for the industrial production of citicoline not only provides an efficient and environmentally friendly solution for large-scale production of citicoline, but also promotes its in-depth application in the synthesis of neuropharmaceuticals. This is of strategic importance for promoting green biomanufacturing and addressing global health needs. Summary of the Invention

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

[0008] The technical solutions of the present invention are as follows:

[0009] One of the objects of the present invention is to provide a recombinant engineered Escherichia coli for producing citicoline. The recombinant engineered Escherichia coli was deposited in the China Center for Type Culture Collection on March 17, 2025, with a deposit number of CCTCCNO: M 2025483 and named ZMCB02NCL.

[0010] Furthermore, the deposit information is as follows:

[0011] Bacteria species: Escherichia coli

[0012] Latin name: Escherichia coli

[0013] Strain ID: ZMCB02NCL

[0014] Depository: China Center for Type Culture Collection

[0015] Abbreviation of depository institution: CCTCC

[0016] Address: Wuhan University, China

[0017] Date of preservation: March 17, 2025.

[0018] Furthermore, the recombinant E. coli ZMCB02NCL is used to knock out the cytidine deaminase encoding gene on the genome. cdd Escherichia coli Escherichia coli K-12 MG1655 is a host cell that carries the gene that can express SpnCCT- SpnCKI-pRpLpyrG The temperature-controlled expression vector pBV220 of the mutant gene cluster was used to express the endogenous choline transporter of Escherichia coli. BetT Replace the gene promoter with a strong constitutive promoter P J23119 , and introduced the glucose permease gene glf and glucokinase genes glk , integrated into the E. coli locus ptsH-ptsI-crr middle.

[0019] Furthermore, the mutant gene SpnCCT - SpnCKI From Streptococcus pneumoniae Streptococcus pneumoniae , the nucleotide sequence is shown in SEQ ID NO. 1;

[0020] described SpnCCT-SpnCKI Mutated genes SpnCCT Mutated genes and SpnCKI The mutant gene was fused and expressed via a flexible linker (GGGGS)3, and the nucleotide sequence of the flexible linker was shown in SEQ ID NO. 2.

[0021] Furthermore, the SpnCCT-SpnCKI The A at position 132 of the mutant gene mutated to T, the T at position 403 mutated to A, the T at position 1116 mutated to C, the A at position 1181 mutated to C, and the A at position 1379 mutated to G.

[0022] Furthermore, the SpnCCT-SpnCKI-pRpLpyrG Mutant genes in mutant gene clusters pyrG Derived from Escherichia coliEscherichia coli K-12 MG1655, the nucleotide sequence is shown in SEQ ID NO. 3.

[0023] Furthermore, the mutant gene pyrG The T at position 458 mutated to A, the A at position 730 mutated to T, the G at position 948 mutated to C, and the T at position 1418 mutated to C.

[0024] Furthermore, the SpnCCT-SpnCKI-pRpLpyrG The mutant gene cluster contains pRpL Promoter, the nucleotide sequence is shown in SEQ ID NO. 4.

[0025] Furthermore, the strong constitutive promoter P J23119 The nucleotide sequence is shown in SEQ ID NO. 5.

[0026] Furthermore, the glucose permease gene glf and glucokinase genes glk Derived from Zymomonas mobilis Zymomonas mobilis , glf-glk The nucleotide sequence of the gene is shown in SEQ ID NO. 6, wherein positions 1-1422 are glf Gene, positions 1423-1451 are pTac Promoter, positions 1452-2438 are glf Gene.

[0027] A second object of the present invention is to provide a method for constructing a recombinant engineered Escherichia coli for producing citicoline, comprising the following steps:

[0028] (1) Escherichia coli Escherichia coli K-12 MG1655 was used as the host cell, and the knockout E. coli base strain cdd Gene, expressed through the temperature-controlled expression vector pBV220 SpnCCT-SpnCKI-pRpLpyrG Gene cluster, resulting in the engineered Escherichia coli MG-P1;

[0029] (2) Yes SpnCCT-SpnCKI-pRpLpyrG The gene cluster is randomly mutated and then SpnCCT-SpnCKI- pRpLpyrG The gene mutation cluster was connected with the temperature-controlled vector plasmid pBV220 and introduced into the Escherichia coli engineered strain MG-P1. After high-throughput screening, the Escherichia coli engineered strain MG-P2 was obtained.

[0030] (3) Endogenous BetT Replace the gene's native promoter with a strong constitutive promoter P J23119 Obtained Escherichia coli engineered bacteria MG-P3;

[0031] (4) Transform the glucose permease gene glf and glucokinase genes glk Integrated into the MG-P3 locus of Escherichia coli engineered bacteria ptsH-ptsI-crr Finally, the citicoline-producing Escherichia coli recombinant engineering bacteria were constructed.

[0032] The third object of the present invention is to provide an application of a recombinant engineered Escherichia coli for producing citicoline in the efficient production of citicoline by fermentation using choline chloride, cytidine and glucose as substrates.

[0033] Furthermore, the recombinant engineered Escherichia coli ZMCB02NCL was cultured to obtain a seed solution, which was inoculated into a fermentation medium. During the fermentation process, the pH was regulated to 6.8-7.4, the induction temperature was 40°C ± 2°C, and the fermentation time was 36-38 h. Citicoline was produced by feedback fed-batch fermentation using choline chloride, cytidine, and glucose as substrates.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. This invention breaks through the limitations of existing citicoline production technology and innovatively adopts a systematic multi-dimensional transformation strategy to construct E. coli recombinant engineering bacteria. First, the genome is knocked out. cdd Gene to block cytidine degradation, combined with endogenous pyrG Gene mutations enhance CTP synthesis, providing sufficient precursors for citicoline (CDPC) accumulation; secondly, the introduction of Streptococcus pneumoniae-derived SpnCCT and SpnCKI Genes are fused and expressed through flexible linker (GGGGS)³ to reduce steric hindrance between enzymes and improve catalytic efficiency; then the exogenous introduction of key genes is optimized. SpnCCT-SpnCKI and endogenous pathway genes pyrG The expression level and enzyme activity ratio of SpnCCT-SpnCKI-pRpLpyrG The gene cluster was treated as a whole and subjected to directed mutation at the same time, and the temperature-controlled expression vector pBV220 was used to drive the key gene cluster. SpnCCT-SpnCKI-pRpLpyrG , through segmented temperature control, temporal regulation of gene expression is achieved, dependence on inducers is avoided, and the production process of citicoline is simplified.

[0036] 2. This invention integrates cutting-edge biotechnology to construct the recombinant E. coli ZMCB02NCL and knock out ptsH-ptsI-crr Disruption of the native PTS system in Escherichia coli and introduction of the glucose permease gene from Zymomonas mobilis glf and glucokinase genes glk and integrate it into the locus ptsH-ptsI-crrIn this study, a novel glucose transport system that does not rely on phosphoenolpyruvate (PEP) was used to replace the bacterial phosphotransferase system (PTS). By reducing PEP consumption, the carbon flux of the CDPC biosynthesis pathway was increased, the carbon metabolic burden was reduced, glucose utilization was improved, and the efficiency of CDPC synthesis was improved. At the same time, a strong constitutive promoter was used to P J23119 Replacement of choline transporter BetT Gene native promoter, enhances choline transporter BetT The expression of the gene promotes the efficient entry of the substrate choline chloride into the cell, further effectively improving the synthesis efficiency of citicoline.

[0037] 3. The recombinant E. coli ZMCB02NCL constructed by the present invention can efficiently utilize glucose, cytidine and choline chloride simultaneously, with high substrate conversion rate. pyrG Gene mutations enhance CTP synthesis, providing sufficient precursors for citicoline accumulation. glf and glk The stable integration and co-expression of the genes enhance glucose uptake efficiency and reduce the burden of carbon metabolism. The strong promoter enhances 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 E. coli ZMCB02NCL was used to produce citicoline in shake flask fermentation, with a fermentation yield of 35.2 g / L in a 10 L tank. The product separation and purification were simple, the process was simple, and it was environmentally friendly and safe. Furthermore, during the passage of the recombinant E. coli ZMCB02NCL, there was no significant change in the strain's biomass or citicoline content, demonstrating good genetic stability and ideal value for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the construction route of the Escherichia coli recombinant engineered bacteria ZMCB02NCL of the present invention;

[0039] Figure 2 Schematic diagram of the structure of the recombinant plasmid pBV220-SpnCCT-SpnCKI-pRpLpyrG carried by the recombinant engineered Escherichia coli ZMCB02NCL of the present invention;

[0040] Figure 3 This is a fermentation curve diagram of the production of CDPC by the recombinant engineered E. coli ZMCB02NCL described in Example 5 of the present invention. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with preferred embodiments. The endpoints of the ranges and any values ​​disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and individual point values, and the 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.

[0042] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0043] In the following examples, "codon optimization" refers to the redesign of genes by utilizing preferred codons and avoiding codons that are underutilized or rare. Every organism exhibits some degree of codon usage difference or preference, with those codons that are most frequently used being the preferred codons.

[0044] The molecular biology experiments not specifically described in the following examples, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium configuration, etc., were mainly performed with reference to the Molecular Cloning Laboratory Manual (3rd edition); PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions.

[0045] The whole gene synthesis, primer synthesis and sequencing in the following examples were completed by Shanghai Sangon Biotechnology Co., Ltd.; the host strain Escherichia coli K-12 MG1655 was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0046] The sequences of the primers used in the following examples are shown in Table 1:

[0047] Table 1 Primer sequences in the following examples

[0048]

[0049] The shake flask fermentation method described in the following examples has the following steps:

[0050] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 20 g / L agar powder for solid culture medium.

[0051] Shake flask fermentation medium: glucose 10 g / L, peptone 15 g / L, yeast extract 20 g / L, potassium dihydrogen phosphate 2.5 g / L, potassium dihydrogen phosphate trihydrate 15 g / L, sodium chloride 0.5 g / L, glycerol 1 g / L, ampicillin 50 mg / L, pH 7.0.

[0052] Shake flask fermentation method: First, streak the recombinant strain from -80℃ glycerol stock onto LB solid medium containing ampicillin and activate it at 30℃. Pick a single colony and inoculate it into 50 mL LB liquid medium (containing ampicillin) and shake culture at 30℃ and 200rpm for 14-16 hours until the OD 600 Reach 2-3. Transfer 1%-2% inoculum to shake flask fermentation medium and culture at 30℃ and 200rpm until OD 600 The OD value reaches 0.6-0.8. Raise the temperature 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 to culture at 42°C and 200 rpm for 12-24 hours, and regularly sample and check the OD value. 600 and product CDPC concentration.

[0053] Fermentation broth detection method: The fermentation broth was aspirated and centrifuged at 12000rpm for 3min. The supernatant was diluted a certain number of times, centrifuged through a 0.22 μm filter membrane, and detected by high-performance liquid chromatography (HPLC). The HPLC parameters were as follows: the chromatographic column was a Shim-pack GISSC18 column (4.6mm×150mm, 5 μm), the mobile phase was methanol and 10mM phosphate buffer PBS (pH4.0), the mobile phase ratio was methanol: phosphate buffer = 3:97, the flow rate was 1.0mL / min, the injection volume was 5 μL, and the detection wavelength λ = 270 nm.

[0054] The steps of the plasmid elimination method described in the following examples are as follows:

[0055] Elimination of pTarget F series plasmids: Pick a single clone and inoculate it into LB medium containing kanamycin Kana and 0.5 mM IPTG antibiotics, culture it at 30℃ overnight, dilute it a certain multiple and spread it on Kana plate, culture it at 30℃, pick a single clone and streak it on Kana plate and spectinomycin Spec plate. If the single clone grows on Kana plate but not on Spec plate, it means that the pTarget F plasmid has been successfully eliminated.

[0056] Elimination of pCas9 series plasmids: Pick a single clone in LB medium, culture at 37℃ overnight, dilute it a certain multiple and spread it on LB plate, culture it at 37℃, pick a single clone and streak it on Kana plate and LB plate. If the single clone grows on LB plate but not on Kana plate, it means that the pCas9 plasmid has been successfully eliminated.

[0057] Example 1

[0058] This embodiment provides a method for constructing recombinant Escherichia coli MG-P1, comprising the following steps:

[0059] (1) cdd gene knockout

[0060] 1) Design cdd The specific target site of the gene was 20 bp. The primers cdd-N20-F and cdd-N20-FR were used to perform inverse PCR amplification with the pTarget F plasmid as the template. The amplified product was digested with the restriction endonuclease DpnI and transformed into DH5α competent cells to construct the knockout plasmid pTF-Δcdd.

[0061] 2) The genome of Escherichia coli MG1655 was used as a template and primers cdd-LH-F / cdd-LH-R were used to amplify cdd For the upstream homology arm, primers cdd-RH-F / cdd-RH-R were used to amplify the downstream homology arm of cdd, and primers cdd-LH-F / cdd-RH-R were used to connect the upstream and downstream homology arms to obtain the target DNA homologous recombination fragment;

[0062] 3) The pCas9 plasmid was transformed into Escherichia coli MG1655 competent cells to obtain Escherichia coli MG1655 / pCas9 containing the pCas9 plasmid. The plasmid pTF-Δcdd was electroporated into MG1655 / pCas9 competent cells. The single colonies grown on the plate were verified by colony PCR. The positive transformants were screened and sequenced to obtain the knockout cdd The recombinant Escherichia coli strain MG1655 (Δ cdd );

[0063] (2) Construction of pBV220-SpnCCT-SpnCKI-pRpLpyrG plasmid

[0064] 1) Based on phosphocholine cytidylyltransferase from Streptococcus pneumoniae SpnCCT and choline kinase SpnCKI The genes were codon optimized and connected using flexible linker (GGGGS)3 to synthesize the sequence SpnCCT-SpnCKI , the gene sequence is shown in SEQ ID NO. 1;

[0065] 2) Use primers CCT-CKI-F / CCT-CKI-R to perform PCR on the artificially synthesized double-stranded DNA molecule SEQ ID NO.1 to recover the product and obtain the fragment SpnCCT-SpnCKI ; The vector backbone pBV220 was cleaved with restriction enzymes EcoR Ⅰ and BamHⅠ enzyme digestion;

[0066] 3) The fragment SpnCCT-SpnCKI Japanese Sutra EcoR Ⅰ and BamH The vector backbone obtained by enzyme digestion was connected to obtain the recombinant plasmid pBV220-SpnCCT-SpnCKI;

[0067] 4) Amplification using primers pyrG-F / pyrG-R using the MG1655 genome as a template pyrG Gene was promoted using primers pRpL-F / pRpL-R sub-pRpL Fragments were obtained by Overlapping PCR pRpLpyrG ;

[0068] 5) Use restriction endonucleases BamH Ⅰ and Sla Ⅰ enzyme digestion vector backbone pBV220-SpnCCT-SpnCKI; fragment pRpLpyrG and BamH Ⅰ and Sla The vector backbones obtained by enzyme digestion were connected to construct the recombinant plasmid pBV220-SpnCCT-SpnCKI-pRpLpyrG;

[0069] (3) Construction of recombinant Escherichia coli MG-P1

[0070] 1) Prepare 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 Escherichia coli MG1655 (Δ cdd ) were placed in the bottom of a pre-cooled electrode cup and then transformed using electroporation (2200 V, 5 ms);

[0071] 2) After transformation is complete, quickly add 1 mL of LB medium to the electrode cup to resuspend the cells, then transfer them to a centrifuge tube and resuscitate at 37°C and 220 rpm for 1 hour to obtain the transformation product.

[0072] 3) Spread the transformation product onto a solid LB plate containing 100 μg / mL ampicillin and culture at 37°C. Colony identification revealed recombinant E. coli MG-P1 containing the recombinant plasmid pBV220-SpnCCT-SpnCKI-pRpLpyrG.

[0073] (4) CDPC content detection

[0074] The recombinant Escherichia coli MG-P1 was fermented 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.

[0075] Example 2

[0076] This embodiment provides a method for constructing recombinant Escherichia coli MG-P2, comprising the following steps:

[0077] (1) Error-prone PCR mutations

[0078] QuickMutation™ random gene mutation kit was used to perform error-prone PCR with plasmid pBV220-SpnCCT-SpnCKI-pRpLpyrG as template and error-prone primers F-SpnCCT / R-pyrG to make the gene SpnCCT-SpnCKI- pRpLpyrG Mutation occurs, and the PCR product is the gene cluster SpnCCT-SpnCKI-pRpLpyrG Mutation library;

[0079] Error-prone PCR system (50 μL):

[0080] ddH2O 32.5 μL, RandomMut buffer (10X) 5 μL, Mutation enhancer (10X) 5 μL, dNTP (2.5mM each) 5 μL, template DNA 0.5 μL, primer mix (10μM each) 1 μL, RandomMutDNA polymerase 1 μL.

[0081] Error-prone PCR procedures:

[0082] Pre-denaturation at 94°C for 3 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 4 min; finally, extension at 72°C for 10 min, and storage of the product at 4°C.

[0083] To increase the mutation rate, the amplified products of the above error-prone PCR were recovered and used as templates for continuous error-prone PCR, and the procedure was the same as the above steps;

[0084] (2) Construction of mutant expression library

[0085] 1) Use DNA recovery kit to recover error-prone PCR products, and after double digestion with EcoRI and SalI, ligate to pBV220 vector treated with the same enzymes. E. coli JM109, obtained a mutant containing pBV220-SpnCCT-SpnCKI-pRpLpyrGE. coli JM109 bacterial library;

[0086] 2) From E.coli Plasmids were extracted from the JM109 bacterial library and transformed into Escherichia coli MG1655 (Δcdd) to obtain an Escherichia coli MG1655 (Δcdd) bacterial library containing the pBV220-SpnCCT-SpnCKI-pRpLpyrG mutant.

[0087] (3) Screening of high-yield strains of citicoline (CDPC)

[0088] 1) Pick a single clone of the Escherichia coli MG1655 (Δcdd) bacterial library containing the pBV220-SpnCCT-SpnCKI-pRpLpyrG mutant obtained in step (2) above and place it into a 96-well plate with 150 μL of fermentation medium containing 50 μg / mL ampicillin per well. Incubate at 37°C, 150 rpm for 48 h, centrifuge at 5000 rpm, and collect the supernatant for testing.

[0089] 2) Ten strains were screened on 96-well plates with CDPC production greater than 20 mg / 100 mL. The remaining strains generally had CDPC production between 10 mg / 100 mL and 17 mg / 100 mL. The 10 strains were renamed MG2-1, MG2-2, MG2-3, MG2-4, MG2-5, MG2-6, MG2-7, MG2-8, MG2-9, and MG2-10.

[0090] 3) Ten strains were screened by shake flask fermentation. Strain MG2-9 had the highest CDPC production, reaching 0.19 g / L, a 2.1-fold increase compared to the control strain MG-P1. It was named Escherichia coli MG-P2. The results of the shake flask screening are shown in Table 2.

[0091] Table 2 Citicoline content after 24 h of shake flask fermentation

[0092]

[0093] Example 3

[0094] This embodiment provides a method for constructing recombinant Escherichia coli MG-P3, comprising the following steps:

[0095] (1) Design a 20 bp specific target site for the BetT promoter using primer P BetT -N20-F / P BetT -N20-R was amplified by reverse PCR using the pTarget F plasmid as a template to construct the knockout plasmid pTF-ΔP BetT ;

[0096] (2) Use primer P BetT -LH-F / P BetT -LH-R amplification P BetT The upstream homologous fragment was amplified with primer P BetT -RH-F / P BetT -RH-R amplification P BetT 1. Detect downstream homologous fragments of α-aminobutyric acid and purify the products;

[0097] (3) Use primer P BetT -LH-F and P BetT -RH-R fuses the upstream and downstream homologous fragments to obtain a fusion fragment P BetT LH- P J23119 -P BetT RH ;

[0098] (4) pTarget and homologous fragments were electroporated into MG-P2 competent cells carrying pCas9, plated on LB agar containing kanamycin and streptomycin, and then incubated at 37 °C for 16 h;

[0099] (5) Using primer P BetT -CX-F and P BetT -CX-R verified colony and successfully replaced BetT promoter strains, plasmid pTF-ΔP BetT After eliminating the pCas9 plasmid, recombinant Escherichia coli MG-P3 was obtained.

[0100] Example 4

[0101] This embodiment provides a method for constructing a recombinant engineered Escherichia coli strain ZMCB02NCL that produces citicoline, comprising the following steps:

[0102] (1) Construction of knockout plasmid pTF-ΔptsH-ptsI-crr

[0103] A 20-bp specific target site of the ptsH-ptsI-crr gene was designed and inverse PCR amplified using primers ptsH-ptsI-crr-N20-F / ptsH-ptsI-crr-N20-R with the pTarget F plasmid as a template. The amplified product was digested with the restriction endonuclease DpnI and transformed into DH5α competent cells to construct the knockout plasmid pTF-ΔptsH-ptsI-crr.

[0104] (2) DNA homologous recombination insertion fragment

[0105] 1) The glucose permease gene from Zymomonas mobilis was glf and glucokinase genes glk After codon optimization, SEQ ID NO. 6 was artificially synthesized and ligated with the vector pTrc99a to obtain the recombinant plasmid pTrc99a-glf-glk;

[0106] 2) Use primers pT-F / pT-R to amplify the gene fragment expression cassette glf-glk The genome of Escherichia coli MG1655 was used as a template and primers ptsH-ptsI-crr-LH-F / ptsH-ptsI-crr-LH-R were used to amplify ptsH-ptsI-crr Upstream homology arm, amplified using primers ptsH-ptsI-crr-RH-F / ptsH-ptsI-crr-RH-FR ptsH-ptsI-crr Downstream homology arms, using Overlapping PCR to connect the upstream and downstream homology arms with the gene fragment expression cassette glf-glk After ligation, the gene insertion expression cassette LH was obtained ptsH-ptsI-crr -glf-glk-RH ptsH-ptsI-crr ;

[0107] (3) Locus ptsH-ptsI-crr Knockout and glf-glk Gene integration

[0108] 1) Transform the pCas9 plasmid into competent E. coli MG-P3 to obtain E. coli MG-P3 / pCas9 containing the pCas9 plasmid;

[0109] 2) Insert the plasmid pTF-ΔptsH-ptsI-crr and gene into the expression cassette LH ptsH-ptsI-crr -glf-glk-RH ptsH-ptsI-crr Electroporate into MG-P3 / pCas9 competent cells, and perform colony PCR verification on single colonies grown on the plate;

[0110] 3) Screening positive transformants and performing gene sequencing to eliminate the strain plasmids pTF-ΔptsH-ptsI-crr and pCas9 plasmid to obtain the resistance-free strain MG-P4;

[0111] (4) Shake flask fermentation of strain MG-P4

[0112] The strain MG-P4 was cultured in a shake flask in a fermentation medium containing 10 g / L glucose for 24 hours, and the residual sugar content and CDPC yield in the fermentation supernatant were detected and screened, ultimately constructing the recombinant engineered E. coli strain ZMCB02NCL.

[0113] The test results are shown in Table 3. After 24 h of shake flask fermentation, the CDPC production of strain MG-P4 reached 0.32 g / L, and the glucose consumption was 9.8 g / L. Compared with the control strain MG-P3, the CDPC production and the conversion rate of glucose to CDPC of strain MG-P4 were significantly improved.

[0114] Table 3 Citicoline and glucose contents after 24 h of shake flask fermentation

[0115]

[0116] Example 5

[0117] This embodiment provides an application method of producing citicoline using the recombinant engineered Escherichia coli ZMCB02NCL, comprising the following steps:

[0118] (1) Prepare the culture medium as follows:

[0119] Seed shake flask medium: glucose 10 g / L, peptone 15 g / L, yeast extract 20 g / L, potassium dihydrogen phosphate 2.5 g / L, potassium dihydrogen phosphate trihydrate 15 g / L, sodium chloride 0.5 g / L, glycerol 1 g / L, ampicillin 50 mg / L, pH 7.0;

[0120] 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, potassium phosphate dibasic trihydrate 3.0 g / L, potassium dihydrogen phosphate 1.5 g / L, ammonium ferric citrate 0.5 g / L, citric acid 1.5 g / L, glycerol 3 g / L, magnesium sulfate heptahydrate 0.5 g / L, defoamer 0.1 mL / L, pH 7.0;

[0121] (2) Escherichia coli ZMCB02NCL monoclonal was inoculated into LB slant medium and cultured at 30°C for 24 h. The cells were resuspended in 10 mL of sterile water and then inoculated into a seed shake flask containing primary seed shake flask medium at a 2% inoculum volume. The cells were cultured at 30°C for 16 h to obtain the primary seed solution.

[0122] (3) Inoculate the first-level seed solution into a fermentation tank containing the second-level seed culture medium at a transplant volume of 5%, and culture at 30°C for 12 hours to obtain the second-level seed solution;

[0123] (4) Transfer 10% of the secondary seed solution into a 10 L (7 L liquid volume) fermentation tank filled with fermentation medium and culture at 30°C. Maintain the dissolved oxygen (DO) above 30% by stirring at 300-600 rpm and aeration at 1-2 vvm. Continuously monitor OD 600 , until the bacteria grow to the mid-logarithmic phase (OD600 When the fermentation temperature reaches 35 °C, the culture temperature is raised from 30 °C to 40 °C ± 2 °C. During this process, the stirring speed needs to be increased to 600-800 rpm to maintain DO ≥ 20%. Choline chloride solution is added simultaneously to make the final concentration in the fermentation broth 8 mM and cytidine to make the final concentration 5 mM.

[0124] (5) When the residual sugar concentration is lower than 2 g / L, glucose (400 g / L) is added at a rate of 0.5-1.5 mL / (Lh) to maintain the carbon source supply. 600 Choline chloride was added to a final concentration of 5 mM and cytidine to a final concentration of 2 mM. The pH of the fermentation process was controlled at 6.8-7.4.

[0125] (6) The fermentation broth was taken for CDPC content testing. The results showed that the recombinant engineered E. coli ZMCB02NCL could reach 35.2 g / L after fermentation for 36 h in a 10 L fermenter.

[0126] Performance Testing

[0127] 1. Sequencing of the SpnCCT-SpnCKI-pRpLpyrG mutant gene cluster of Escherichia coli MG-P2

[0128] The mutant plasmid pBV220-SpnCCT-SpnCKI-pRpLpyrG was extracted from the E. coli MG-P2 constructed in Example 2, and PCR was performed using primers F-SpnCCT / R-pyrG. The resultant plasmid was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.

[0129] The results showed that a total of 9 gene mutations occurred in the SpnCCT-SpnCKI-pRpLpyrG gene cluster. Among them, the A at position 132 of the SpnCCT-SpnCKI mutant gene mutated to T, the T at position 403 mutated to A, the T at position 1116 mutated to C, the A at position 1181 mutated to C, and the A at position 1379 mutated to G. The T at position 458 of the pyrG mutant gene mutated to A, the A at position 730 mutated to T, the G at position 948 mutated to C, and the T at position 1418 mutated to C.

[0130] 2. Choline chloride transport efficiency test

[0131] The antibiotic-free E. coli strain MG-P3 constructed in Example 3 was used for shake flask fermentation for 24 h, and the fermentation broth was collected for CDPC content detection.

[0132] The results showed that the CDPC concentration in the fermentation supernatant of the E. coli non-resistant strain MG-P3 was 0.25 g / L, which was 31.8% higher than that of the control strain MG-P2, indicating that the use of a strong promoterP J23119 Replace natural BetT The promoter increases the yield of citicoline from choline chloride.

[0133] 3. Genetic stability test

[0134] The engineered Escherichia coli ZMCB02NCL was subcultured to investigate its genetic stability. The cells were subcultured every 2 days for 10 generations. Shake flask fermentation was performed every other generation to determine the biomass and citicoline content of the strain.

[0135] The results showed that the biomass and citicoline content of the engineered E. coli strain ZMCB02NCL did not change significantly during the subculture process, indicating good genetic stability.

[0136] The genetically stable engineered Escherichia coli strain ZMCB02NCL capable of simultaneously and efficiently accumulating citicoline was deposited in the China Center for Type Culture Collection (CCTCC) on March 17, 2025, with the deposit number CCTCC NO: M2025483. The deposit address is Wuhan University, Wuhan, China, Postal Code: 430072.

[0137] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An Escherichia coli producing citicoline ( Escherichia coli The recombinant engineered bacteria is characterized in that The deposit number of the recombinant Escherichia coli strain is CCTCC NO: M 2025483, and it is named the recombinant Escherichia coli strain ZMCB02NCL.

2. The citicoline-producing recombinant Escherichia coli according to claim 1, characterized in that: The E. coli recombinant engineering bacteria ZMCB02NCL is used to knock out the cytidine deaminase encoding gene on the genome cdd Escherichia coli K-12MG1655 was used as the host cell, carrying the SpnCCT-SpnCKI-pRpLpyrG The temperature-controlled expression vector pBV220 of the mutant gene cluster was used to express the endogenous choline transporter of Escherichia coli. BetT Replace the gene promoter with a strong constitutive promoter P J23119 , and introduced the glucose permease encoding gene glf and glucokinase encoding genes glk , and destroy the phosphotransferase system in Escherichia coli; among them, SpnCCT and SpnCKI They are respectively the genes encoding phosphocholine cytidylyltransferase and choline kinase from Streptococcus pneumoniae; pRpLpyrG For promoter pL and cytidine triphosphate synthase encoding genes.

3. Use of the citicoline-producing recombinant Escherichia coli according to claim 1 or 2 in the efficient production of citicoline by fermentation using choline chloride, cytidine and glucose as substrates.

4. The use according to claim 3, characterized in that The recombinant engineered Escherichia coli ZMCB02NCL was cultured to obtain a seed solution, which was inoculated into a fermentation medium. During the fermentation process, the pH was regulated to 6.8-7.4, the induction temperature was 40°C ± 2°C, and the fermentation time was 36-38 h. Citicoline was produced by feedback fed-batch fermentation using choline chloride, cytidine, and glucose as substrates.

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