A genetically engineered yeast strain of citicoline and a construction method and application thereof

Abstract

The application provides a genetically engineered yeast of citicoline, a construction method and application thereof. The genome of the genetically engineered yeast comprises three expression cassettes: the first expression cassette is a fusion gene of an ERMES complex subunit protein and a CCT enzyme, the second expression cassette is a mitochondrial ADP / ATP carrier protein or a function-enhanced mutant thereof, and the third expression cassette is a vacuole membrane transport protein gene. The application also provides a construction method of the genetically engineered yeast and application thereof in preparing citicoline. The application realizes the synergistic production of synthesis-energy supply-storage trinity by anchoring the synthetic enzyme system to the membrane contact site, combining ATP supply strengthening and vacuole compartmentalized storage, and effectively improves the yield of citicoline.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a genetically engineered yeast containing cytidine diphosphate choline, its construction method, and its application. Background Technology

[0002] Citicoline (CDP-choline) is a key precursor in the phosphatidylcholine biosynthesis pathway. It plays a role in improving phospholipid metabolism in nerve cell membranes and repairing damaged nerve function, and is widely used in the clinical treatment of ischemic stroke, traumatic brain injury, and neurodegenerative diseases. Currently, the industrial production of CDP-choline mainly relies on chemical synthesis and biotransformation. Chemical synthesis involves the use of toxic reagents and has a low substrate conversion rate, and has been gradually replaced by biological methods. Regarding biotransformation, the main technical route is the synthesis of CDP-choline from phosphocholine cytidine transferase (CCT) derived from yeast. In recent years, with the intervention of genetic engineering technology, researchers have begun to overexpress key enzyme genes such as CCT, CKI (choline kinase), HNM1 (choline transporter), and sATP6 (ATP synthase subunit) in yeast chassis strains such as Saccharomyces cerevisiae, Pichia pastoris, and Isaac's orientalis, while downregulating the expression or activity of negative regulatory genes such as His4, Ku70, 5'-NT, and Cda, in order to increase the fermentation yield of cytidine diphosphate choline. Furthermore, the use of whole-cell yeast catalysis combined with an ATP regeneration system has also been explored in the production of cytidine diphosphate choline.

[0003] However, current technologies still face several key technological bottlenecks. First, existing genetic engineering strategies mainly focus on the overexpression and knockout of single enzymes or genes, lacking a systematic design for the synergistic coordination of metabolic flows between different organelles within the cell. The synthesis of cytidine diphosphate choline (CDP-choline) requires the endoplasmic reticulum (ER) to provide precursors, mitochondria to provide ATP, and vacuoles to provide storage space; these three steps are spatially separate, and current technologies have failed to achieve effective spatial coupling among them. Second, current strategies primarily enhance ATP supply by overexpressing wild-type ATP synthase subunits, lacking targeted optimization of ATP carriers for the specific metabolic background of CDP-choline synthesis, resulting in insufficient adaptability between energy supply and synthesis requirements. Furthermore, the accumulation of the product in the cytoplasm easily triggers feedback inhibition and enzymatic degradation, and current technologies have not yet established effective product compartmentalization storage strategies to overcome this constraint. In addition, the role of the ER-mitochondrial membrane contact site in metabolic regulation has not been addressed or utilized in current technologies, and the efficiency of material exchange between organelles remains a significant factor limiting yield. These problems restrict further increases in CDP-choline fermentation yield and the promotion of its industrial application. Summary of the Invention

[0004] To overcome the above-mentioned defects of the prior art, the present invention provides a genetically engineered yeast strain containing cytidine diphosphate choline, its construction method and application, which solves the problems of lack of intercellular metabolic flow co-design, poor adaptability of ATP energy supply and synthesis requirements, and failure to eliminate product feedback inhibition and degradation in the prior art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A genetically engineered yeast strain, wherein the genome of the genetically engineered yeast strain contains: A first expression cassette includes a first gene and a second gene, wherein the first gene encodes a subunit protein of the ERMES complex and the second gene encodes a protein with phosphocholine cytidine transferase activity, and the first gene and the second gene are fused together via a linker peptide encoding sequence. The second expression cassette contains a gene encoding the mitochondrial ADP / ATP carrier protein or a functionally enhanced mutant thereof; The third expression cassette contains a gene encoding a vacuolar membrane transporter that has the activity of transporting cytidine diphosphate choline.

[0006] Preferably, the subunit protein of the ERMES complex is Mdm34 protein; the protein with phosphocholine cytyltransferase activity is CCT protein; the mitochondrial ADP / ATP carrier protein is AAC2 protein; and the vacuolar membrane transport protein is Ycf1 protein.

[0007] Preferably, the linker peptide coding sequence encodes a flexible linker peptide, and the flexible linker peptide is (GGGGS)3; the coding sequence of the first gene is located upstream or downstream of the coding sequence of the second gene.

[0008] Preferably, the functionally enhanced mutant is a mutant whose ATP output rate is increased by at least 20% in a culture medium containing phosphocholine and cytidine, with wild-type AAC2 protein as a control; the mutant has an amino acid residue mutation in the C1 loop region or M2 loop region of the AAC2 protein.

[0009] Preferably, the amino acid sequence of the functionally enhanced mutant is shown in SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25.

[0010] Preferably, the 3' end of the gene encoding the vacuolar membrane transporter is fused with a vacuolar membrane localization signal coding sequence, wherein the vacuolar membrane localization signal is a transmembrane domain of a vacuolar membrane integrative protein or a GPI anchoring signal sequence.

[0011] Preferably, the genome of the genetically engineered yeast further includes a fourth expression cassette, which contains a gene encoding choline kinase, and the gene encoding choline kinase is fused with a vacuolar membrane anchoring signal coding sequence.

[0012] Preferably, the promoters in the first expression box, the second expression box, and the third expression box are each independently constitutive promoters, inductive promoters, or bidirectional promoters; the first expression box and the second expression box are regulated by the same bidirectional promoter.

[0013] Preferably, a method for constructing a genetically engineered yeast strain containing cytidine diphosphate choline includes the step of introducing the expression cassettes into a yeast chassis strain.

[0014] Preferably, the application of a genetically engineered yeast containing cytidine diphosphate choline includes the preparation of cytidine diphosphate choline, cytidine diphosphate choline-containing pharmaceutical compositions, feed, or cosmetics.

[0015] The technical effects and advantages of this invention, including a genetically engineered yeast strain containing cytidine diphosphate choline, its construction method, and its application, are as follows: 1. This invention is the first to use membrane contact sites as engineered targets for cytidine diphosphate choline (CDP-choline) synthesis, achieving cross-organelle synergistic optimization of metabolic flux. Existing technologies focus only on the overexpression and knockout of single enzymes or genes, resulting in fragmented strategies that do not address inter-organelle communication. This invention, by fusing the CCT enzyme with an ERMES complex subunit protein, physically anchors the CDP-choline synthase system to the endoplasmic reticulum-mitochondrial contact site for the first time. This allows the synthesis reaction to occur in a microenvironment with abundant ATP supply, shortening the diffusion distance between substrates and energy carriers and enhancing substrate channel effects.

[0016] 2. The integrated "synthesis-energy supply-storage" design of this invention exhibits significant synergistic effects. This invention spatially rearranges three functional modules dispersed in different cellular regions—synthesis (ERMES-CCT fusion), energy supply (AAC2 functionally enhanced mutant), and storage (Ycf1 vacuolar compartmentalization)—through membrane contact site engineering, forming a closed-loop metabolic flow of "produced and used immediately, energy supplied on-site, and products stored instantly." Experimental evidence shows that the combined effect of the three modules far exceeds the arithmetic sum of the effects of the individual modules, demonstrating a significant synergistic effect.

[0017] 3. This invention obtains a functionally enhanced AAC2 mutant, achieving targeted enhancement of mitochondrial ATP output. Existing technologies mainly improve ATP supply through overexpression of wild-type AAC2, a relatively singular strategy. This invention, through error-prone PCR-directed evolution, screened and obtained a functionally enhanced AAC2 mutant that significantly increases ATP output rate under high concentrations of phosphocholine and cytidine monophosphate, achieving a match between ATP supply and cytidine monophosphate choline synthesis load, avoiding energy metabolism imbalances caused by indiscriminate overexpression.

[0018] 4. This invention constructs a vacuolar compartmentalized storage system, achieving both increased and stable production through immediate product isolation. This invention fuses the Ycf1 vacuolar membrane transporter with a vacuolar membrane localization signal, causing cytidine diphosphate choline to be pumped into the vacuolar for storage immediately after synthesis. This effectively reduces intracellular product concentration, relieves feedback inhibition, and utilizes the acidic environment of the vacuolar to reduce chemical and enzymatic degradation of the product, thus stabilizing the product and increasing yield.

[0019] 5. This invention forms a multi-layered protection gradient, organically unifying the core invention with auxiliary enhancements. Based on the three-in-one core modification, this invention further integrates degradation pathway blocking (knockout of the 5'-nucleotidase-encoding gene) and ATP supply enhancement (overexpression of the mitochondrial ATP synthase-encoding gene). The above-mentioned auxiliary modifications, together with the three core expression cassettes, form a complete closed loop from synthesis enhancement, energy supply optimization, product storage to degradation blocking, further amplifying the synergistic effect of the core modification.

[0020] 6. The construction method of this invention is highly systematic and has good industrial applicability. The multi-gene co-transformation construction method provided by this invention integrates multiple expression cassettes into the same chassis strain, with clear operation steps, high transformation efficiency, and is easy to scale up for industrial production. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the genetic structure of the genetically engineered yeast strain of this invention; Figure 2 This is a structural diagram of the gene elements of the first expression cassette of the present invention; Figure 3 This is a schematic diagram of the synergistic mechanism of "synthesis-energy supply-storage" in this invention; Figure 4 This is a flowchart of the method for constructing the genetically engineered yeast strain of the present invention; Figure 5 This is a flow chart of the fermentation production process of cytidine diphosphate choline according to the present invention. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include," "contain," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "includes..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0024] refer to Figure 1-5 This invention provides a genetically engineered yeast strain containing cytidine diphosphate choline (CDP-choline), its construction method, and its applications. The genome of the genetically engineered yeast strain includes a first expression cassette, a second expression cassette, and a third expression cassette. The first expression cassette contains a first gene encoding an ERMES complex subunit protein and a second gene encoding cytidine diphosphate choline transferase (CDP-choline) activity, the first and second genes being fused via a linker peptide coding sequence. The second expression cassette contains a gene encoding a mitochondrial ADP / ATP carrier protein or a functionally enhanced mutant thereof. The third expression cassette contains a gene encoding a vacuolar membrane transporter protein, the vacuolar membrane transporter protein having CDP-choline transporting activity. This invention also provides a method for constructing the genetically engineered yeast strain, comprising the step of introducing the first, second, and third expression cassettes into a yeast chassis strain. This invention further provides the application of the genetically engineered yeast strain in the preparation of CDP-choline, CDP-choline-containing pharmaceutical compositions, feed, or cosmetics. This invention anchors the cytidine diphosphate choline synthase system to the endoplasmic reticulum-mitochondrial contact site, introduces an AAC2 functional enhancement mutant to enhance ATP supply, and combines it with a vacuolar compartmentalized storage system. For the first time, it has achieved the synergistic and efficient production of cytidine diphosphate choline in yeast cells through a three-in-one process of "synthesis-energy supply-storage", which effectively improves the yield of cytidine diphosphate choline and substrate conversion rate.

[0025] Example 1: Purpose of implementation: The purpose of this embodiment is to provide a genetically engineered yeast strain that simultaneously contains a first expression cassette, a second expression cassette, and a third expression cassette. In the first expression cassette, a subunit protein of the ERMES complex is fused to a protein with phosphocholine cytidine transferase activity via a linker peptide coding sequence. The second expression cassette contains a functionally enhanced mutant encoding a mitochondrial ADP / ATP carrier protein. The third expression cassette contains a gene encoding a vacuolar membrane transporter protein with cytidine diphosphate choline transport activity. This embodiment is used to verify that the above expression cassettes can be successfully constructed and correctly expressed in a yeast chassis strain.

[0026] Implementation System: The genetically engineered yeast strain constructed in this embodiment contains the following in its genome: (a) A first expression cassette comprising a first gene and a second gene, the first gene encoding a subunit protein of the ERMES complex and the second gene encoding a protein having phosphocholine cytidine transferase activity, the first gene and the second gene being fused together via a linker peptide encoding sequence; (b) A second expression cassette containing a functionally enhanced mutant of the gene encoding the mitochondrial ADP / ATP carrier protein; (c) A third expression cassette containing a gene encoding a vacuolar membrane transporter that has the activity of transporting cytidine diphosphate choline.

[0027] The subunit protein of the ERMES complex is Mdm34 protein; the protein with phosphocholine cytyltransferase activity is CCT protein; the mitochondrial ADP / ATP carrier protein is AAC2 protein; and the vacuolar membrane transporter protein is Ycf1 protein. The linker peptide coding sequence encodes the flexible linker peptide (GGGGS)3. The amino acid sequence of the functionally enhanced mutant is shown in SEQ ID NO:23. The vacuolar membrane transporter protein coding gene has a vacuolar membrane localization signal coding sequence fused to its 3' end, and the vacuolar membrane localization signal is the transmembrane domain of the Vam3p protein.

[0028] Implementation steps: Step 1: Preparation of the substrate strain. Saccharomyces cerevisiae BY4741 (MATahis3Δ1leu2Δ0met15Δ0ura3Δ0) was used as the substrate strain. This strain carries histidine, leucine, methionine, and uracil auxotrophic markers. BY4741 strain was inoculated into 5ml LYPD liquid medium (1% yeast extract, 2% peptone, 2% glucose) and cultured overnight at 30℃ and 250rpm for later use.

[0029] Step 2: Construction of the first expression cassette. Using Saccharomyces cerevisiae BY4741 genomic DNA as a template, the coding sequence of the MDM34 gene was amplified by polymerase chain reaction (PCR). The MDM34 gene encodes the Mdm34 protein, and its amino acid sequence is shown in SEQ ID NO:1. Using a plasmid containing the CCT gene as a template, the coding sequence of the CCT gene was amplified by PCR. The CCT gene encodes the CCT protein, and its amino acid sequence is shown in SEQ ID NO:3. The coding sequences of the MDM34 gene and the CCT gene were fused by overlap extension PCR using a linker peptide coding sequence. The linker peptide coding sequence encodes the (GGGGS)3 flexible linker peptide, and its nucleotide sequence is shown in SEQ ID NO:13. The 5' to 3' ends of the fusion gene are sequentially: MDM34 coding sequence — (GGGGS)3 coding sequence — CCT coding sequence. The fusion gene was cloned into the BamHI and XhoI sites of the pYES2 plasmid and linked downstream of the GAL1 promoter to obtain the first expression cassette vector pYES2-MDM34-CCT.

[0030] Step 3: Construction of the second expression cassette. Using the wild-type AAC2 gene as a template, the amino acid sequence of the wild-type AAC2 protein encoded by the wild-type AAC2 gene is shown in SEQ ID NO:22. Random mutations were performed using error-prone polymerase chain reaction. The AAC2 mutant library was transformed into Saccharomyces cerevisiae BY4741, and the fastest-growing transformants were screened on SD solid medium containing a final concentration of 8 g / L phosphocholine and 8 g / L cytidine monophosphate. Genomic DNA was extracted from the fastest-growing transformants, the AAC2 gene was amplified and sequenced, and the AAC2 functionally enhanced mutant was obtained, the amino acid sequence of which is shown in SEQ ID NO:23. The AAC2 functionally enhanced mutant gene shown in SEQ ID NO:23 was cloned into the pRS425 plasmid between the SalI and SacI sites and ligated downstream of the GAL10 promoter to obtain the second expression cassette vector pRS425-AAC2-M1.

[0031] Step 4: Construction of the third expression cassette. Using *Saccharomyces cerevisiae* BY4741 genomic DNA as a template, the coding sequence of the YCF1 gene was amplified by polymerase chain reaction (PCR). The YCF1 gene encodes the Ycf1 protein, and its amino acid sequence is shown in SEQ ID NO: 5. Using *Saccharomyces cerevisiae* BY4741 genomic DNA as a template, the transmembrane domain coding sequence of the VAM3 gene was amplified by PCR. The amino acid sequence of the transmembrane domain of the Vam3p protein encoded by the VAM3 gene is shown in SEQ ID NO: 8. The coding sequence of the YCF1 gene and the coding sequence of the VAM3 transmembrane domain were fused by overlap extension PCR, so that the 3' end of the YCF1 gene is linked to the coding sequence of the VAM3 transmembrane domain. The fusion gene was cloned into the pRS415 plasmid between the EcoRI and SalI sites and ligated downstream of the TEF1 promoter to obtain the third expression cassette vector pRS415-YCF1-VAM3.

[0032] Step 5: Co-transformation of multiple genes and screening of positive transformants. The first expression cassette vector pYES2-MDM34-CCT obtained in Step 2, the second expression cassette vector pRS425-AAC2-M1 obtained in Step 3, and the third expression cassette vector pRS415-YCF1-VAM3 obtained in Step 4 were co-transformed into the *Saccharomyces cerevisiae* BY4741 chassis strain using the lithium acetate transformation method. The specific procedure is as follows: Take 100 μL of competent cells, add 1 μg of each of the above three expression cassette vectors, 240 μL of PEG3350 (50% w / v), 36 μL of 1M lithium acetate, and 50 μL of single-stranded vector DNA (2 mg / mL), and vortex to mix. Incubate at 30°C for 30 minutes, heat shock at 42°C for 15 minutes, and incubate on ice for 2 minutes. Spread on SD selective plates that do not contain uracil, histidine, or leucine, and incubate at 30°C for 3-5 days to screen for positive transformants.

[0033] Step Six: Verification of Positive Transformants. Genomic DNA was extracted from positive transformants, and the expression of each target gene was verified using real-time quantitative polymerase chain reaction (qPCR). The primer pairs used were as follows: forward primer sequence for MDM34 gene (SEQ ID NO: 14), reverse primer sequence (SEQ ID NO: 15); forward primer sequence for AAC2 gene (SEQ ID NO: 16), reverse primer sequence (SEQ ID NO: 17); forward primer sequence for YCF1 gene (SEQ ID NO: 18), reverse primer sequence (SEQ ID NO: 19); forward primer sequence for internal reference gene ACT1 (SEQ ID NO: 20), reverse primer sequence (SEQ ID NO: 21). Transformants expressing all target genes were selected and named engineered strain YE001.

[0034] Implementation results: In this embodiment, the genetically engineered yeast strain YE001 was successfully constructed. Real-time quantitative polymerase chain reaction (qPCR) results showed that the transcriptional signals of the MDM34-CCT fusion gene, the AAC2 functionally enhanced mutant gene, and the YCF1-VAM3 fusion gene in the engineered strain YE001 were significantly higher than those in the wild-type control strain, indicating that the first, second, and third expression cassettes were correctly integrated and transcribed in the yeast chassis strain. The acquisition of the engineered strain YE001 provides stable biological material for subsequent fermentation experiments in this invention.

[0035] Example 2: Purpose of implementation: The purpose of this embodiment is to verify the application effect of the genetically engineered yeast YE001 constructed in Example 1 in the production of cytidine diphosphate choline, and to measure its cytidine diphosphate choline yield under shake-flask fermentation conditions, so as to prove that the genetically engineered yeast of the present invention can effectively synthesize cytidine diphosphate choline.

[0036] Implementation System: This embodiment uses the genetically engineered yeast YE001 constructed in Example 1 as the production strain. The genome of the genetically engineered yeast YE001 contains a first expression cassette (MDM34-CCT fusion gene, regulated by the GAL1 promoter), a second expression cassette (the AAC2 functionally enhanced mutant gene shown in SEQ ID NO:23, regulated by the GAL10 promoter), and a third expression cassette (YCF1-VAM3 fusion gene, regulated by the TEF1 promoter). The fermentation medium composition is: glucose 30 g / L, yeast extract 10 g / L, peptone 20 g / L, phosphocholine 10 g / L, cytidine 10 g / L, magnesium sulfate 1.0 g / L, manganese sulfate 0.05 g / L, potassium dihydrogen phosphate 2 g / L, pH 6.0. The fermentation conditions are: temperature 30℃, stirring speed 250 rpm. The induction conditions are: when OD... 600 When the concentration reaches 0.8, add galactose to a final concentration of 2% (w / v).

[0037] Implementation steps: Step 1: Preparation of seed culture. The engineered bacteria YE001 obtained in Example 1 was inoculated into 5ml LYPD liquid medium and cultured overnight at 30℃ and 250rpm to obtain the seed culture.

[0038] Step 2: Shake-flask fermentation. The seed culture from Step 1 is fermented according to the initial OD... 600=0.5g was inoculated into a 250mL Erlenmeyer flask containing 50mL of fermentation medium. The fermentation medium consisted of: glucose 30g / L, yeast extract 10g / L, peptone 20g / L, phosphocholine 10g / L, cytidine monophosphate 10g / L, magnesium sulfate 1.0g / L, manganese sulfate 0.05g / L, potassium dihydrogen phosphate 2g / L, and pH 6.0. The fermentation temperature was 30℃, and the stirring speed was 250rpm.

[0039] Step 3: Induction of expression, when the fermentation broth OD 600 When the value reached 0.8, galactose was added to a final concentration of 2% (w / v) to induce the expression of the MDM34-CCT fusion gene controlled by the GAL1 promoter and the AAC2 functionally enhanced mutant gene controlled by the GAL10 promoter. The total fermentation time was 72 hours.

[0040] Step 4: Sample Processing. After fermentation, take 1 mL of fermentation broth and centrifuge at 4℃ and 10000 rpm for 10 minutes, collecting the supernatant and bacterial cells separately. The bacterial cells are homogenized using a high-pressure homogenizer at 1000 bar pressure, and then centrifuged at 12000 rpm for 20 minutes to collect the supernatant. Combine the fermentation supernatant and the homogenized supernatant for analysis.

[0041] Step 5: Determination of cytidine diphosphate choline (CDP-choline) content. The CDP-choline content in the supernatant collected in Step 4 was determined using high-performance liquid chromatography (HPLC). Chromatographic conditions were as follows: column: TechMate C18ST (4.6 × 250 mm, 5 μm); mobile phase: methanol:phosphate buffer (5:95, v / v); flow rate: 1.0 mL / min; detection wavelength: 265 nm; column temperature: 30 °C; injection volume: 20 μL. A standard curve was plotted using CDP-choline standards, and quantification was performed using the external standard method.

[0042] Implementation results: Under shake-flask fermentation conditions, the engineered yeast YE001 achieved a cytidine diphosphate choline (CDP-choline) yield of 6.8 g / L. High-performance liquid chromatography (HPLC) analysis showed a distinct absorption peak at a position consistent with the retention time of the CDP-choline standard, confirming the fermentation product as CDP-choline. These results demonstrate that the genetically engineered yeast provided by this invention can efficiently synthesize CDP-choline and has good potential for industrial application.

[0043] Example 3: Purpose of implementation: The purpose of this embodiment is to verify the contributions of the first expression cassette (ERMES-CCT fusion), the second expression cassette (AAC2 functionally enhanced mutant), and the third expression cassette (Ycf1 vacuolar membrane transporter) of the present invention to the production of cytidine diphosphate choline, and to demonstrate that the combination of the three has a synergistic effect that transcends the simple superposition of the effects of each module, thereby supporting the technical effect of the synergistic action of the three expression cassettes in the genetically engineered yeast of the present invention.

[0044] Implementation System: This embodiment constructed a series of control strains, each containing a different combination of expression cassettes in its genome: Strain A (first expression cassette only): The genome contains the first expression cassette (MDM34-CCT fusion gene, regulated by the GAL1 promoter), but does not contain the second or third expression cassette.

[0045] Strain B (second expression cassette only): The genome contains the second expression cassette (the AAC2 functionally enhanced mutant gene shown in SEQ ID NO:23, regulated by the GAL10 promoter), but does not contain the first or third expression cassette.

[0046] Strain C (third expression cassette only): The genome contains the third expression cassette (YCF1-VAM3 fusion gene, regulated by the TEF1 promoter), but does not contain the first or second expression cassette.

[0047] Strain D (first expression cassette + second expression cassette): The genome contains the first and second expression cassettes, but not the third expression cassette.

[0048] Strain E (first expression cassette + third expression cassette): The genome contains the first and third expression cassettes, but not the second expression cassette.

[0049] Strain F (second expression cassette + third expression cassette): The genome contains the second and third expression cassettes, but not the first expression cassette.

[0050] Engineered strain YE001 (first expression cassette + second expression cassette + third expression cassette): The genome contains the first expression cassette, the second expression cassette, and the third expression cassette simultaneously.

[0051] The construction methods for each strain were the same as steps one through six of Example 1, except for the combination of transformation plasmids. The fermentation medium, fermentation conditions, induction conditions, sample processing methods, and detection methods were all exactly the same as steps one through five of Example 2.

[0052] Implementation steps: Step 1: Construction of control strains. Following steps one through six of Example 1, strains A (containing only the first expression cassette), B (containing only the second expression cassette), C (containing only the third expression cassette), D (containing both the first and second expression cassettes), E (containing both the first and third expression cassettes), and F (containing both the second and third expression cassettes) were constructed. The expression of the target gene in each strain was verified using real-time quantitative polymerase chain reaction.

[0053] Step 2: Shake-flask fermentation. The strains A to F constructed in Step 1, as well as the engineered strain YE001 constructed in Example 1, were subjected to shake-flask fermentation according to the methods described in Steps 1 to 3 of Example 2. The fermentation medium, fermentation conditions, and induction conditions for each strain were exactly the same as in Example 2.

[0054] Step 3: Sample processing and determination of cytidine diphosphate choline content. Following the methods in steps four and five of Example 2, the fermentation broth of each strain was processed and subjected to high-performance liquid chromatography (HPLC) to determine the cytidine diphosphate choline yield of each strain.

[0055] Implementation results: The results of cytidine diphosphate choline (CDP-choline) production in each strain are as follows: Wild-type BY4741 (Comparative Example 1) had no exogenous expression cassette and a production of 0.5 g / L; Strain A contained only the first expression cassette and a production of 2.1 g / L; Strain B contained only the second expression cassette and a production of 1.8 g / L; Strain C contained only the third expression cassette and a production of 1.5 g / L; Strain D contained both the first and second expression cassettes and a production of 4.2 g / L; Strain E contained both the first and third expression cassettes and a production of 3.6 g / L; Strain F contained both the second and third expression cassettes and a production of 3.3 g / L; and engineered strain YE001 contained the first, second, and third expression cassettes and a production of 6.8 g / L.

[0056] When the three expression cassettes existed individually, the yields of cytidine diphosphate choline were 2.1 g / L, 1.8 g / L, and 1.5 g / L, respectively, with an arithmetic sum of their individual effects of 5.4 g / L. The engineered strain YE001 (combination of the three cassettes) achieved a yield of 6.8 g / L, a 25.9% increase compared to the arithmetic sum (6.8 vs 5.4), and a 223.8% increase compared to the optimal single module (strain A, 2.1 g / L). These results indicate a significant synergistic effect among the first expression cassette (ERMES-CCT fusion, enabling synthase anchoring at membrane contact sites), the second expression cassette (AAC2 functionally enhanced mutant, enhancing mitochondrial ATP output), and the third expression cassette (Ycf1 vacuolar membrane transporter, enabling vacuolar compartmentalized storage of the product). The yield of the combination of the three cassettes is far higher than the simple sum of the effects of each module, verifying the scientific validity and effectiveness of the "synthesis-energy supply-storage" integrated design in the genetically engineered yeast described in this invention.

[0057] Example 4: Purpose of implementation: The purpose of this embodiment is to verify the effect of different AAC2 functional enhancement mutants (amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25) in the second expression cassette of the present invention on the production of cytidine diphosphate choline, and to verify the effect of downregulation of VMA1 and VMA2 gene expression (i.e., vacuolar pH optimization) on the further enhancement of cytidine diphosphate choline production.

[0058] Implementation System: This embodiment uses the engineered strains YE001-M1 (containing the AAC2 functionally enhanced mutant shown in SEQ ID NO:23), YE001-M2 (containing the AAC2 functionally enhanced mutant shown in SEQ ID NO:24), and YE001-M3 (containing the AAC2 functionally enhanced mutant shown in SEQ ID NO:25) constructed in Example 1, as well as a control strain containing wild-type AAC2. The genomes of all the above strains simultaneously contain both the first expression cassette (MDM34-CCT fusion gene) and the third expression cassette (YCF1-VAM3 fusion gene).

[0059] In the vacuolar pH optimization experiment, the expression of the engineered strain YE001-M1 was downregulated in its genome, specifically the expression of the VMA1 gene (encoding the amino acid sequence shown in SEQ ID NO: 11) and the VMA2 gene (encoding the amino acid sequence shown in SEQ ID NO: 12).

[0060] Implementation steps: Step 1: Fermentation of engineered bacteria containing different AAC2 functionally enhanced mutants. Engineered bacteria YE001-M1 (containing SEQ ID NO: 23), YE001-M2 (containing SEQ ID NO: 24), YE001-M3 (containing SEQ ID NO: 25), and a control strain containing wild-type AAC2 were subjected to shake-flask fermentation according to steps one through three of Example 2. The fermentation medium, fermentation conditions, and induction conditions for each strain were exactly the same as in Example 2. Sample processing and high-performance liquid chromatography (HPLC) detection were performed according to steps four through five of Example 2 to determine the cytidine diphosphate choline (CDP-choline) production of each strain. Simultaneously, the intracellular ATP content of each strain after fermentation was determined using a luciferase method. Specifically, 1 mL of fermentation broth was collected by centrifugation, ATP extract was added, and the luminescence value was measured on a fluorescence detector using an ATP detection kit. The relative fold increase was calculated using the ATP content of the wild-type AAC2 control strain as a benchmark.

[0061] Step 2: Construction of strains with downregulated VMA1 and VMA2 gene expression. CRISPR-Cas9 technology was used to downregulate the expression of the VMA1 and VMA2 genes in the engineered strain YE001-M1. sgRNAs targeting the promoter regions of the VMA1 and VMA2 genes were designed and cloned into Cas9 expression vectors, respectively. Simultaneously, a repair template containing the CYC1 weak promoter sequence and flanking homologous arms was constructed. The Cas9 expression vector and repair template were co-transformed into the engineered strain YE001-M1. Transformants with successful promoter replacement were selected and named engineered strain YE001-M1-V. The downregulation of the transcriptional levels of the VMA1 and VMA2 genes was verified using real-time quantitative polymerase chain reaction.

[0062] Step 3: Determination of pH within the vacuoles. Engineered bacteria YE001-M1 and YE001-M1-V were inoculated into liquid culture medium and cultured to the logarithmic growth phase. The cells were collected by centrifugation, and BCECF-AM fluorescent dye (final concentration 10 μM) was added. The cells were incubated at 30°C in the dark for 30 minutes. After washing, the cells were resuspended in buffer solution, and the fluorescence intensity was detected by flow cytometry. The pH value within the vacuoles was calculated based on the standard curve.

[0063] Step 4: Fermentation and detection of pH-optimized strains in vacuoles. The engineered strain YE001-M1-V was fermented in shake flasks according to the methods in steps one to three of Example 2. The samples were processed and detected by high performance liquid chromatography according to the methods in steps four to five of Example 2. The production of cytidine diphosphate choline was determined and compared with that of engineered strain YE001-M1 without VMA1 and VMA2 downregulation.

[0064] Implementation results: The fermentation results of different AAC2 functionally enhanced mutants are as follows: the control strain containing wild-type AAC2 produced 4.5 g / L of cytidine diphosphate choline and had an intracellular ATP content 1.0 times that of the wild-type control; the YE001-M1 strain containing the mutant shown in SEQ ID NO:23 produced 6.8 g / L of ATP and had an ATP content 2.1 times that of the wild-type control; the YE001-M2 strain containing the mutant shown in SEQ ID NO:24 produced 6.5 g / L of ATP and had an ATP content 2.0 times that of the wild-type control; and the YE001-M3 strain containing the mutant shown in SEQ ID NO:25 produced 6.2 g / L of ATP and had an ATP content 1.8 times that of the wild-type control. All three AAC2 functionally enhanced mutants significantly increased intracellular ATP content (1.8-2.1 times that of the wild-type) and cytidine diphosphate choline production (1.38-1.51 times that of the wild-type AAC2 control strain), with the mutant shown in SEQ ID NO:23 showing the best effect.

[0065] The results of vacuolar pH optimization are as follows: In engineered strain YE001-M1, VMA1 and VMA2 were normally expressed, and the vacuolar pH was 5.0, with a cytidine diphosphate choline (CDP-choline) yield of 6.8 g / L. In engineered strain YE001-M1-V, VMA1 and VMA2 expression was downregulated, the vacuolar pH was 5.8, and the yield was 7.5 g / L. Real-time quantitative polymerase chain reaction (qPCR) verification results showed that the transcription levels of VMA1 and VMA2 in engineered strain YE001-M1-V were reduced by more than 60% compared to YE001-M1. After adjusting the vacuolar pH from 5.0 to 5.8, the CDP-choline yield increased from 6.8 g / L to 7.5 g / L, an increase of 10.3%. The results indicate that downregulating the expression of VMA1 and VMA2 genes and adjusting the vacuolar pH to a more stable range for CDP-choline can effectively reduce product degradation during vacuolar storage and further increase yield.

[0066] Example 5: Purpose of implementation: The purpose of this embodiment is to verify the effect of knocking out the 5'-nucleotidase encoding gene and / or overexpressing the mitochondrial ATP synthase encoding gene on the production of cytidine diphosphate choline in the genome of the genetically engineered yeast described in this invention. This embodiment serves to support the technical feature of this invention: "knockout of the 5'-nucleotidase encoding gene and / or overexpression of the mitochondrial ATP synthase encoding gene."

[0067] Implementation System: This embodiment uses the engineered bacterium YE001-M1 obtained in Example 4 as the starting strain. The genome of the engineered bacterium YE001-M1 contains a first expression cassette (MDM34-CCT fusion gene), a second expression cassette (AAC2 functionally enhanced mutant gene shown in SEQ ID NO:23), and a third expression cassette (YCF1-VAM3 fusion gene).

[0068] Based on this, the following strains were constructed: Strain YE001-M1-ΔN: In the YE001-M1 genome, the 5'-nucleotidase encoding gene (SDT1 gene) is knocked out.

[0069] Strain YE001-M1-ATP: In the YE001-M1 genome, the gene encoding mitochondrial ATP synthase (ATP2 gene, encoding the β subunit of F1F0-ATP synthase) is overexpressed.

[0070] Strain YE001-M1-ΔN-ATP: In the YE001-M1 genome, the SDT1 gene is knocked out and the ATP2 gene is overexpressed.

[0071] Implementation steps: Step 1: Construction of the 5'-nucleotidase-encoding gene knockout strain. The SDT1 gene in the engineered strain YE001-M1 was knocked out using CRISPR-Cas9 technology. An sgRNA targeting the SDT1 gene was designed and cloned into a Cas9 expression vector. Simultaneously, a knockout fragment containing the KanMX resistance marker and flanking homologous arms (500 bp upstream and downstream of the SDT1 gene) was constructed. The Cas9 expression vector and the knockout fragment were co-transformed into the engineered strain YE001-M1. Positive transformants were screened on YPD plates containing G418 (200 μg / mL). Genomic DNA was extracted from the positive transformants, and SDT1 gene knockout was verified using polymerase chain reaction (PCR). Primers were designed to include the internal sequence of the SDT1 gene and the sequences outside the upstream and downstream homologous arms. The correctly verified transformant was named YE001-M1-ΔN.

[0072] Step 2: Construction of the strain overexpressing the mitochondrial ATP synthase encoding gene. Using the genomic DNA of *Saccharomyces cerevisiae* BY4741 as a template, the coding sequence of the ATP2 gene was amplified by polymerase chain reaction. The ATP2 gene was cloned into the pRS426 plasmid (containing a URA3 marker) between the BamHI and XhoI sites, and ligated downstream of the GAL1 promoter to obtain the ATP2 overexpression plasmid. The ATP2 overexpression plasmid was transformed into the engineered strain YE001-M1, and positive transformants were screened on SD selective plates without uracil, named YE001-M1-ATP.

[0073] Step 3: Construction of the combined strain of 5'-nucleotidase gene knockout and mitochondrial ATP synthase gene overexpression. The ATP2 overexpression plasmid constructed in Step 2 was transformed into YE001-M1-ΔN constructed in Step 1. Positive transformants were screened on SD selective plates without uracil. The overexpression of the ATP2 gene was verified by real-time quantitative polymerase chain reaction and named YE001-M1-ΔN-ATP.

[0074] Step 4: Fermentation and detection of each strain. The engineered strains YE001-M1, YE001-M1-ΔN, YE001-M1-ATP and YE001-M1-ΔN-ATP were fermented in shake flasks according to the methods in steps one to three of Example 2. The samples were processed and detected by high performance liquid chromatography according to the methods in steps four to five of Example 2 to determine the cytidine diphosphate choline production of each strain.

[0075] Implementation results: The results of cytidine diphosphate choline (CDP-choline) production in each strain are as follows: The starting strain YE001-M1, without any additional gene modification, produced 6.8 g / L; YE001-M1-ΔN, with SDT1 gene knocked out, produced 7.8 g / L, a 14.7% increase compared to YE001-M1; YE001-M1-ATP, with ATP2 gene overexpression, produced 7.3 g / L, a 7.4% increase compared to YE001-M1; and YE001-M1-ΔN-ATP, with both SDT1 knocked out and ATP2 overexpressed, produced 8.5 g / L, a 25.0% increase compared to YE001-M1.

[0076] Polymerase chain reaction (PCR) results showed that the internal sequence of the SDT1 gene in YE001-M1-ΔN and YE001-M1-ΔN-ATP could not be amplified, while the outer sequences of the upstream and downstream homologous arms could be amplified normally, confirming that the SDT1 gene had been successfully knocked out. Real-time quantitative PCR results showed that the transcription level of the ATP2 gene in YE001-M1-ATP and YE001-M1-ΔN-ATP was more than 8-fold higher than that in YE001-M1, confirming successful overexpression of the ATP2 gene.

[0077] SDT1 gene knockout (strain YE001-M1-ΔN) increased cytidine diphosphate choline (CDP-choline) production from 6.8 g / L to 7.8 g / L, indicating that reducing the CDP-choline degradation pathway can effectively increase product accumulation. ATP2 gene overexpression (strain YE001-M1-ATP) increased production to 7.3 g / L, indicating that enhanced ATP supply can further support CDP-choline synthesis. The combination of SDT1 gene knockout and ATP2 gene overexpression (strain YE001-M1-ΔN-ATP) achieved a production of 8.5 g / L, a 25.0% increase compared to YE001-M1, indicating a synergistic effect between knocking out the degradation enzyme encoding gene and overexpressing the ATP synthase encoding gene.

[0078] Comparative Example 1 Purpose of implementation: The purpose of this comparative example is to provide wild-type Saccharomyces cerevisiae without any genetic modification as a control to verify the significant improvement in cytidine diphosphate choline production of the genetically engineered yeast strain described in this invention compared to the wild-type strain.

[0079] Implementation System: This comparative example uses a wild-type Saccharomyces cerevisiae strain BY4741 without any genetic modification as a control strain. This strain's genome does not contain any of the first, second, or third expression cassettes. The fermentation medium and conditions are exactly the same as in Example 2, but since this strain does not contain the exogenous gene expression cassette controlled by the GAL1 promoter, galactose was not added for induction.

[0080] Implementation steps: Step 1: Cultivation of wild-type strains. Wild-type strain of Saccharomyces cerevisiae BY4741 was inoculated into 5ml LYPD liquid medium and cultured overnight at 30℃ and 250rpm to obtain seed culture.

[0081] Step 2: Shake-flask fermentation. The seed culture from Step 1 is fermented according to the initial OD... 600 =0.5g was inoculated into a 250mL Erlenmeyer flask containing 50mL of fermentation medium. The composition of the fermentation medium, fermentation temperature, and stirring speed were exactly the same as in step two of Example 2. Since this strain does not contain the exogenous gene controlled by the GAL1 promoter, no galactose was added for induction. The total fermentation time was 72 hours.

[0082] Step 3: Sample processing and determination of cytidine diphosphate choline content. Following the methods in steps four and five of Example 2, the fermentation broth was processed and analyzed by high performance liquid chromatography to determine the yield of cytidine diphosphate choline.

[0083] Implementation results: The wild-type *Saccharomyces cerevisiae* strain BY4741 produced 0.5 g / L of cytidine diphosphate choline under shake-flask fermentation conditions. Compared with the engineered strains obtained in the various embodiments of this invention: the wild-type BY4741 produced 0.5 g / L; the engineered strain YE001 (containing the first, second, and third expression cassettes) produced 6.8 g / L, a 12.6-fold increase compared to the wild-type; and the engineered strain YE001-M1-ΔN-ATP (further knocking out SDT1 and overexpressing ATP2 based on the first, second, and third expression cassettes) produced 8.5 g / L, a 16.0-fold increase compared to the wild-type.

[0084] The above results indicate that although unmodified wild-type Saccharomyces cerevisiae possesses a weak ability to synthesize cytidine diphosphate choline (0.5 g / L), the yield is extremely low. In wild-type yeast, the ERMES complex is at a normal physiological level, the mitochondrial ADP / ATP carrier is wild-type AAC2, and although Ycf1 protein is present on the vacuolar membrane, it is not expressed. The cytidine diphosphate choline synthase system is also not anchored at the membrane contact site. Therefore, it does not possess the ability to efficiently synthesize and accumulate cytidine diphosphate choline. This invention, by introducing a first expression cassette (anchoring CCT enzyme to the ERMES complex to optimize the synthesis module), a second expression cassette (introducing an AAC2 functionally enhanced mutant to optimize the ATP energy supply module), and a third expression cassette (overexpressing the Ycf1 vacuolar membrane transporter to optimize the product storage module), combined with SDT1 gene knockout (reducing product degradation) and ATP2 gene overexpression (further enhancing ATP supply), increased the production of cytidine diphosphate choline from 0.5 g / L to 8.5 g / L, a 16-fold increase, fully demonstrating the effectiveness and industrial application value of the technical solution of this invention.

[0085] Comparing Examples 1-5 with Comparative Example 1, the progressive effects and industrial value of the present invention are clearly demonstrated. In Comparative Example 1, the wild-type Saccharomyces cerevisiae BY4741 strain without any genetic modification produced only 0.5 g / L of cytidine diphosphate choline under the same shake-flask fermentation conditions, indicating that the natural synthesis capacity of wild-type yeast is extremely low and has no potential for production application. Example 1 successfully constructed the engineered strain YE001, which integrates a first expression cassette (MDM34-CCT fusion, anchoring the synthase to the ERMES complex), a second expression cassette (AAC2 functionally enhanced mutant, enhancing mitochondrial ATP output), and a third expression cassette (YCF1-VAM3 fusion, achieving vacuolar compartmentalized storage) in its genome. The fermentation results of Example 2 showed that the cytidine diphosphate choline production of this engineered strain jumped to 6.8 g / L, 12.6 times higher than that of the wild type, preliminarily verifying the effectiveness of the "synthesis-energy supply-storage" three-in-one modification strategy. To further analyze the contribution of each module, Example 3 constructed a series of control strains containing only a single expression cassette or two expression cassettes. The results showed that the yields of the three modules individually were 2.1, 1.8, and 1.5 g / L, respectively, with an arithmetic sum of their individual effects of 5.4 g / L. However, the yield of YE001, with the combination of the three modules, reached 6.8 g / L, representing a 25.9% increase compared to the arithmetic sum and a 223.8% increase compared to the optimal single module. This comparison fully demonstrates that the three modules are not simply additive, but exhibit a significant synergistic effect—the coupling of membrane contact site anchoring of CCT enzymes, mitochondrial ATP-directed enhanced supply, and active vacuole storage forms a positive cycle, enabling the yield to exceed the upper limit of the isolated effects of each module.

[0086] In further optimization experiments, Example 4 compared the effects of different AAC2 functionally enhanced mutants. It was found that the mutant shown in SEQ ID NO:23 increased intracellular ATP content to 2.1 times that of the wild-type control, and the cytidine diphosphate choline (CDP-choline) production reached 6.8 g / L, while the wild-type AAC2 control strain only produced 4.5 g / L, confirming the key role of the mutant in enhancing energy supply. Simultaneously, by downregulating the expression of VMA1 and VMA2 genes, adjusting the vacuolar pH from 5.0 to 5.8, the production of the engineered strain YE001-M1-V was further increased to 7.5 g / L, a 10.3% increase compared to the unoptimized state, indicating that moderate regulation of the acidic environment within the vacuolar reduced product storage degradation. Example 5 further introduced auxiliary modifications based on YE001-M1, comparing the individual and combined effects of 5'-nucleotidase-encoding gene (SDT1) knockout and mitochondrial ATP synthase-encoding gene (ATP2) overexpression. The results showed that SDT1 knockout increased yield by 14.7% (to 7.8 g / L), and ATP2 overexpression increased yield by 7.4% (to 7.3 g / L). The combined strain YE001-M1-ΔN-ATP yield reached 8.5 g / L, a 25.0% increase compared to the starting strain and a 16.0-fold increase compared to the wild type. In summary, from 0.5 g / L in the wild type, to 6.8 g / L in the engineered strain, then to 7.5 g / L after vacuolar pH optimization, and finally reaching 8.5 g / L after degradative enzyme knockout and ATP synthase overexpression enhancement, this invention, through modular design, synergistic integration, and auxiliary enhancement, achieved a stepwise leap in cytidine diphosphate choline (CDP-choline) yield, fully demonstrating the enormous application potential of this genetically engineered yeast and its construction strategy in industrial production.

[0087] The above are merely specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of protection of the claims.

[0088] In conclusion, the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A genetically engineered yeast, characterized in that, The genome of the genetically engineered yeast contains: A first expression cassette includes a first gene and a second gene, wherein the first gene encodes a subunit protein of the ERMES complex and the second gene encodes a protein with phosphocholine cytidine transferase activity, and the first gene and the second gene are fused together via a linker peptide encoding sequence. The second expression cassette contains a gene encoding the mitochondrial ADP / ATP carrier protein or a functionally enhanced mutant thereof; The third expression cassette contains a gene encoding a vacuolar membrane transporter that has the activity of transporting cytidine diphosphate choline.

2. The genetically engineered yeast according to claim 1, characterized in that, The subunit protein of the ERMES complex is Mdm34 protein; the protein with phosphocholine cytyltransferase activity is CCT protein; the mitochondrial ADP / ATP carrier protein is AAC2 protein; and the vacuolar membrane transport protein is Ycf1 protein.

3. The genetically engineered yeast according to claim 1, characterized in that, The linker peptide coding sequence encodes a flexible linker peptide, which is (GGGGS)3; the coding sequence of the first gene is located upstream or downstream of the coding sequence of the second gene.

4. The genetically engineered yeast according to claim 1, characterized in that, The enhanced mutant is defined as a mutant whose ATP export rate is increased by at least 20% in a medium containing phosphocholine and cytidine, compared to wild-type AAC2 protein; the mutant has an amino acid residue mutation in the C1 or M2 loop region of the AAC2 protein.

5. The genetically engineered yeast according to claim 1, characterized in that, The amino acid sequences of the enhanced mutants are shown in SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:

25.

6. The genetically engineered yeast according to claim 1, characterized in that, The 3' end of the gene encoding the vacuolar membrane transporter is fused with a vacuolar membrane localization signal coding sequence, wherein the vacuolar membrane localization signal is a transmembrane domain of the vacuolar membrane integrative protein or a GPI anchoring signal sequence.

7. The genetically engineered yeast according to claim 1, characterized in that, The genome of the genetically engineered yeast also contains a fourth expression cassette, which contains a gene encoding choline kinase, and the gene encoding choline kinase is fused with a vacuolar membrane anchoring signal coding sequence.

8. The genetically engineered yeast according to claim 1, characterized in that, The promoters in the first expression box, the second expression box, and the third expression box are each independently constitutive promoters, inductive promoters, or bidirectional promoters; the first expression box and the second expression box are regulated by the same bidirectional promoter.

9. A method as claimed in claim 1 The method for constructing the genetically engineered yeast according to any one of the eight claims is characterized in that, Including claim 1 8. The steps for introducing each expression cassette into a yeast chassis strain as described in any one of the above.

10. As claimed in claim 1 The use of any one of the genetically engineered yeast strains in the preparation of cytidine diphosphate choline, cytidine diphosphate choline-containing pharmaceutical compositions, feed, or cosmetics.

Images