Method for producing phosphatidylethanolamine using a carbon source

Abstract

To provide a method for producing phosphatidylethanolamine using a carbon source.SOLUTION: A production method includes a step of culturing modified cyanobacteria, a step of fermentation light treatment, a step of filtration treatment, and a step of oil-water separation treatment. In the step of culturing modified cyanobacteria, modified cyanobacteria are cultured in an environment containing a carbon source, and the carbon source is converted into serine. In the step of fermentation light treatment, serine, phosphatidylcholine, and phospholipase are mixed in a light reaction tank to obtain a phosphatidylethanolamine mixture. In the step of filtration treatment, the phosphatidylethanolamine mixture is filtered through a filtration membrane to obtain a filtrate. In the step of oil-water separation treatment, the filtrate is placed in an oil-water separator, left to stand for a predetermined time, and phosphatidylethanolamine is obtained from the upper layer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing phosphatidylethanolamine, and in particular to a method for producing phosphatidylethanolamine using a carbon source. [Background technology]

[0002] Cyanobacteria are self-sufficient organisms that synthesize the nutrients they need through photosynthesis. To mitigate the greenhouse effect and combat environmental destruction, existing technologies utilize the ability of cyanobacteria to fix carbon dioxide as a metabolic product and produce alcohols and organic acids such as ethanol, butanol, 2,3-butanediol, succinic acid, lactic acid, and isoprene. However, because cyanobacteria lack the ability to convert carbon sources into serine, current technology does not allow for the production of serine using cyanobacteria.

[0003] Phospholipid ethanolamine (PE), also known as phosphatidylethanolamine, is the second most common phospholipid after soybean phosphatidylcholine (PC). It is a phospholipid found in the brain and a component of the cell membranes of nerve cells. Phosphatidylethanolamine is generally believed to aid in the conduction of electrical pulses and promote the activation of neurotransmitters involved in learning, memory, and emotion, and is therefore used in health products to regulate physiological functions. Existing technology requires extraction and purification from soybean phosphatidylcholine, which contains approximately 0.5-3% phosphatidylethanolamine, making it a rare source of phosphatidylethanolamine.

[0004] Therefore, one of the important issues that we want to solve in this project is to develop a new method for producing phosphatidylethanolamine and increase the supply source of phosphatidylethanolamine. Summary of the Invention [Problem to be solved by the invention]

[0005] The technical problem that the present invention aims to solve is to provide a method for producing phosphatidylethanolamine using a carbon source in order to make up for the shortcomings of existing techniques. [Means for solving the problem]

[0006] To solve the above technical problems, the present invention provides a method for producing phosphatidylethanolamine using a carbon source. The method for producing phosphatidylethanolamine using a carbon source includes: culturing modified cyanobacteria in a culture medium; providing the modified cyanobacteria with an environment containing a carbon source so that the modified cyanobacteria convert the carbon source into serine; filtering the culture medium to separate the modified cyanobacteria and obtain a filtered culture medium; synthesizing the phosphatidylethanolamine by mixing the filtered culture medium, serine, phosphatidylcholine, and phospholipase in a photoreactor to obtain a phosphatidylethanolamine mixture; and separating the phosphatidylethanolamine mixture from an oil-water separator, allowing it to stand for a predetermined period of time, and obtaining the phosphatidylethanolamine from the upper layer. The modified cyanobacteria contain the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0007] In an embodiment of the present invention, a method for obtaining a modified cyanobacterium includes synthesizing a DNA sequence, inserting the DNA sequence into a plasmid, and inserting the plasmid into a cyanobacterium by electroporation to obtain the modified cyanobacterium.

[0008] In a specific embodiment of the present invention, the electroporation treatment is performed at a voltage of 0.5 to 1.5 kV for 2 to 10 msec.

[0009] In certain embodiments of the invention, the electroporation treatment further comprises adding 0.5 to 2% polyethylene glycol.

[0010] In a particular embodiment of the invention, the environment comprises 25-50 mM sodium bicarbonate.

[0011] In a particular embodiment of the invention, said cyanobacteria is an elongated, spherical cyanobacterium.

[0012] In certain embodiments of the invention, the phospholipase is a magnetic catalyst.

[0013] In a specific embodiment of the present invention, the temperature of the fermentation light treatment is 30 to 41°C.

[0014] In a particular embodiment of the invention, the modified cyanobacterium has the ability to produce 3-phosphate glycerate dehydrogenase, phosphoserine phosphorylase, and phosphoserine transferase.

[0015] In certain embodiments of the invention, the carbon source is carbon dioxide, glucose, sucrose, fructose, or lactose.

[0016] In order to solve the above technical problems, another technical solution adopted by the present invention is to provide a method for producing phosphatidylethanolamine using a carbon source, which method includes: a fermentation phototreatment step of producing serine using modified cyanobacteria; a phosphatidylethanolamine synthesis step of mixing serine, phosphatidylcholine, and phospholipase in a photoreactor to obtain a phosphatidylethanolamine mixed solution; a filtration step of filtering the phosphatidylethanolamine mixed solution through a membrane filter to obtain a filtrate; and an oil-water separation step of putting the filtrate into an oil-water separator, leaving it to stand for a predetermined time, and obtaining the phosphatidylethanolamine from the upper layer.

[0017] In a particular embodiment of the invention, the modified cyanobacterium comprises the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0018] In a particular embodiment of the invention, the weight ratio of said phosphatidylcholine to said serine is 1:5.

[0019] In a particular embodiment of the invention, the pore size of the filtration membrane is 0.45 μm.

[0020] In certain embodiments of the invention, the predetermined period of time is 10 minutes or more.

[0021] One advantage of the present invention is that the method for producing phosphatidylethanolamine using a carbon source provided by the present invention involves culturing modified cyanobacteria in an environment containing a carbon source, and allowing the modified cyanobacteria to convert the carbon source into serine, and the modified cyanobacteria comprises the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. This allows phosphatidylethanolamine to be produced using L-serine produced from the carbon source as a reaction precursor, thereby improving the economic efficiency of waste gas treatment.

[0022] In order to better understand the features and technical contents of the present invention, please refer to the following detailed description of the present invention and the accompanying drawings, which are provided for reference and explanation only and are not intended to limit the scope of the present invention. [Brief explanation of the drawings]

[0023] [Figure 1] 1 is a flowchart showing a method for producing phosphatidylethanolamine using a carbon source of the present invention. [Figure 2] 1 is a schematic diagram of the metabolic pathway of the modified cyanobacteria of the present invention. [Figure 3] FIG. 1 is a schematic diagram of the construction of the plasmid of the present invention. [Figure 4] 1 is a line graph showing the relationship between NaHCO3 concentration and cyanobacterial growth. [Figure 5]1 is a line graph showing the relationship between temperature and cyanobacterial growth in a fermentation photoreactor. DETAILED DESCRIPTION OF THE INVENTION

[0024] The following describes the embodiments of the present invention. Those skilled in the art can understand the advantages and effects of the present invention from the disclosure of this specification. The present invention can be implemented or applied in other different embodiments. Each clause in this specification can also be modified and changed equivalently based on various aspects or applications without departing from the spirit of the present invention. In addition, the drawings of the present invention are for simple and schematic illustration only and are not drawn to actual scale. The following embodiments will further explain the technical matters related to the present invention, but the disclosed contents do not limit the present invention. In addition, the term "or" used in this specification may include any one or more combinations of related items according to actual circumstances.

[0025] It should be understood that the term "or" used herein may include any one or more combinations of the relevant listed items depending on the actual circumstances. Unless the context requires otherwise, the term "comprises" should be understood as implying a stated integer or step, or set of integers or steps, and not excluding any other integer or step, or any other set of integers or steps. In this description, the terms "comprises," "contains," "including," and "having" can be used interchangeably.

[0026] As used herein, the term "exogenous gene", also known as a heterologous gene, refers to a gene or nucleic acid fragment that is not endogenous to the host or target cell itself, but is derived from another species or cell, or is artificially synthesized, and is introduced into the host or target cell by genetic engineering techniques.

[0027] As shown in FIGS. 1 to 3, a first embodiment of the present invention provides a method for converting a carbon source into serine. The method for converting a carbon source into serine includes the following steps S1 to S4: In step S1, a DNA sequence is synthesized. In step S2, the DNA sequence is inserted into a plasmid so that the plasmid contains the sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In step S3, the plasmid is inserted into a cyanobacterium by electroporation to obtain a modified cyanobacterium. In step S4, a carbon source is provided to the modified cyanobacterium, and the modified cyanobacterium converts the carbon source into serine.

[0028] In Figures 2 and 3, NADP refers to nicotinamide adenine dinucleotide phosphate, and NADPH refers to reduced nicotinamide adenine dinucleotide phosphate. ATP refers to adenosine triphosphate, and ADP refers to adenosine diphosphate. PSII refers to photosynthetic system II, PSI refers to photosynthetic system I, and Cytb6f is cytochrome b6f, which is central to the light-dependent reactions of oxygenic photosynthesis. RuBP refers to ribulose-1,5-bisphosphate, and CA refers to carbonic anhydrase. rbs refers to the ribosome-binding site.

[0029] Step S1 of DNA sequence synthesis involves artificially synthesizing a genetic code suitable for cyanobacteria, allowing them to recognize and produce the corresponding substance. In particular, a genetic code suitable for identifying the elongated, spherical cyanobacterium Synechococcus elongates PCC7942 is synthesized. Furthermore, the artificially synthesized DNA sequence can be mass-produced using polymerase chain reaction (PCR). In one embodiment of the present invention, the PCR conditions are as follows: denaturation at 98°C for 30 seconds, followed by 30 cycles of denaturation at 98°C for 10 seconds, low-temperature annealing at 56°C for 20 seconds, and polymerization at 72°C for 45 seconds, followed by PCR amplification with polymerization at 72°C for 10 minutes.

[0030] Next, we mass-produced and isolated plasmid DNA from the native E. coli DH5α strain, inserted the DNA sequence from step S2 into the plasmid, introduced the designed DNA sequence into the E. coli plasmid, and replicated it in denatured E. coli for mass production to obtain a recombinant plasmid, which we named pSerSyn. However, because E. coli cannot utilize CO2, even if we obtained the designed DNA sequence, it cannot convert the carbon source into serine. Therefore, the mass-produced denatured plasmid DNA must be further extracted and transferred to native cyanobacteria.

[0031] Specifically, the plasmid used in the present invention is constructed using pSyn_1 as a backbone, carrying the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, which are integrated into the cyanobacterial genome by homologous recombination and expressed. Subsequently, modified cyanobacteria of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 that have successfully undergone homologous recombination are selected using antibiotic selection. In other words, the successfully modified cyanobacterial strains can grow on solid media containing antibiotics.

[0032] In step S3 of inserting the plasmid into cyanobacteria, the cyanobacteria were cultured in BG11 medium and OD 730 The growth concentration of the strain can be measured by electroporation. Furthermore, the plasmid is inserted into the cyanobacteria by electroporation, resulting in modified cyanobacteria. Electroporation involves applying an electric current to the cyanobacterial cells for a very short time (microseconds to milliseconds), creating a potential difference across the cell membrane in a high-voltage, low-capacity environment, which changes the structure of the cell membrane. This compresses and thins the cell membrane, creating countless tiny pores that allow the plasmid to pass through the membrane and enter the cyanobacterial cell.

[0033] To achieve optimal plasmid penetration, 0.5% to 2% polyethylene glycol (PEG) can be added in this step. For example, any concentration between 0.5% and 2%, such as 1.0% or 1.5%. Preferably, electroporation is performed at a voltage between 0.5 KV and 1.5 KV, for example, 0.6 KV, 0.7 KV, 0.8 KV, 0.9 KV, 1.0 KV, 1.1 KV, 1.2 KV, 1.3 KV, or 1.4 KV, for 2 msec to 10 msec. Alternatively, electroporation can be performed at any voltage between 2 msec and 10 msec, for example, 3 msec, 4 msec, 5 msec, 6 msec, 7 msec, 8 msec, or 9 msec. In the present invention, 1 x 10 PEG is added during electroporation. 6 A study was conducted to treat protozoan bacteria under different voltage and time conditions to obtain the amount of successfully modified cyanobacterial strains. The results are shown in Table 1 below.

[0034] [Table 1]

[0035] Based on the results in Table 1 above, the preferred conditions for the electroporation treatment of the present invention, with 1% PEG added, are to treat the cyanobacteria at a voltage of 0.5 kV for 10 msec, the more preferred conditions are to treat the cyanobacteria at a voltage of 1.5 kV for 5 msec, and the even more preferred conditions are to treat the cyanobacteria at a voltage of 1.0 kV for 5 msec, which will result in the maximum number of bacteria removed.

[0036] In general, protozoan cyanobacteria are capable of reducing carbon dioxide to glyceraldehyde 3-phosphate (G3P). However, due to a lack of the relevant metabolic enzymes, protozoan cyanobacteria cannot further metabolize G3P to L-serine. The present invention uses cyanobacteria to process carbon sources and produce modified cyanobacteria to convert the carbon source to serine.

[0037] In the present invention, industrial waste gas can be used as a carbon source. This gas is a mixture of hydrogen, acetylene, methane, hydrogen sulfide, and acetaldehyde. Furthermore, this mixture can contain 30 ppm to 50 ppm of hydrogen, 150 ppm to 250 ppm of acetylene, 100 ppm to 200 ppm of methane, 0.1 ppm to 1 ppm of hydrogen sulfide, and 1 ppm to 5 ppm of acetaldehyde. For example, the industrial waste gas is a mixture of 40 ppm of hydrogen (H), 200 ppm of acetylene (C2H2), 150 ppm of methane (CH4), 0.5 ppm of hydrogen sulfide (HS), and 3 ppm of acetaldehyde (CH3CHO).

[0038] In step S4 of providing a carbon source to the modified cyanobacterium, the modified cyanobacterium of the present invention has the ability to produce 3-phosphoglycerate dehydrogenase (SerA), phosphoserine phosphatase (SerB), and phosphoserine aminotransferase (SerC), which can independently carry out the reaction of the following formula 1 to convert G3P to L-serine. [ka] formula 1

[0039] The modified cyanobacteria of the present invention contain multiple exogenous genes, including the nucleic acid sequence of the gene for 3-phosphate glycerol dehydrogenase (SerA), the nucleic acid sequence of the gene for phosphate serine phosphorylase (SerB), and the nucleic acid sequence of the gene for phosphate serine transferase (SerC), and these genes can be expressed or overexpressed in the modified cyanobacteria. In other words, the modified cyanobacteria of the present invention contain expression plasmids of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0040] Specifically, the modified cyanobacteria of the present invention retain the characteristic of protozoan cyanobacteria converting carbon sources into glyceraldehyde 3-phosphate (G3P). The modified cyanobacteria of the present invention also produce 3-phosphate glycerate dehydrogenase (SerA), which can convert G3P into 3-phosphohydroxypyruvate (3P-HP). Furthermore, the modified cyanobacteria of the present invention produce phosphoserine transferase (SerC), which can convert 3-phosphate hydroxypyruvate into 3-phosphoserine (3P-Serine). The modified cyanobacteria of the present invention also produce phosphoserine kinase (SerB), which can convert 3P-Serine into serine, particularly L-serine.

[0041] Therefore, the modified cyanobacteria of the present invention have the ability to convert a carbon source into L-serine. For example, the carbon source can be carbon dioxide, glucose, sucrose, fructose, or lactose. However, the present invention is not limited to these examples. Preferably, the modified cyanobacteria of the present invention can utilize a carbon source from carbon-containing industrial waste gas and convert the carbon source in the industrial waste gas into L-serine. This allows the modified cyanobacteria to treat industrial waste and simultaneously produce economically valuable chemicals.

[0042] Another embodiment of the present invention provides a method for converting a carbon source to serine, comprising converting the carbon source to serine using at least a modified cyanobacterium of the present invention, the modified cyanobacterium comprising the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In other words, the modified cyanobacterium comprises expression plasmids of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0043] Based on this, the modified cyanobacterium can convert a carbon source into glyceraldehyde 3-phosphate (G3P) and release it extracellularly. By expressing the sequence of SEQ ID NO:1, the cyanobacterium produces 3-phosphate glycerate dehydrogenase (SerA), which can further convert G3P into 3-phosphate hydroxypyruvate (3P-HP). By expressing the sequence of SEQ ID NO:3, the cyanobacterium produces phosphoserine transferase (SerC), which can convert 3-phosphate hydroxypyruvate (3P-HP) into phosphate serine (3P-Serine). By expressing the sequence of SEQ ID NO:2, the cyanobacterium produces phosphoserine kinase (SerB), which can convert phosphate serine (3P-Serine) into serine.

[0044] The present invention also includes a method for producing phosphatidylethanolamine using a carbon source. The method includes the following steps: S10: culturing modified cyanobacteria; S20: performing fermentation light treatment; S30: performing filtration treatment; S40: synthesizing phosphatidylethanolamine; and S50: performing oil-water separation treatment. In other words, in step S10, modified cyanobacteria are cultured in BG11 medium.

[0045] Specifically, in the fermentation light treatment in step S20, the modified cyanobacteria are cultured in a flue gas (industrial waste gas) environment containing CO2. In a preferred embodiment, sodium bicarbonate (NaHCO3) is added to the culture medium of the modified cyanobacteria to help dissolve the flue gas into the culture medium of the modified cyanobacteria. Furthermore, as shown in Figure 4, the concentration of NaHCO3 is 25-50 mM (e.g., any positive integer between 25 and 50). After 24 hours of culture with NaHCO3 added, the growth rate of the modified cyanobacteria is OD 1.0 or higher than that without NaHCO3 addition. 730 It has been shown that the addition of 25-50 mM NaHCO3 promotes the growth of modified cyanobacteria and improves L-serine production.

[0046] In the filtration step S30, membrane filtration can be used. For example, the culture medium or reaction solution is introduced into a backwash filter and filtered through a 0.45 μm filter membrane to remove the denatured cyanobacterial cells, yielding a filtrate containing serine or phosphatidylethanolamine. The filtered denatured cyanobacteria is recovered in a photofermentation reactor and reused in the photofermentation treatment, thereby reducing production costs.

[0047] Specifically, in one embodiment, the culture medium is first filtered to remove cyanobacterial cells, and the filtered culture medium is then mixed with phosphatidylcholine and phospholipase to proceed with the phosphatidylethanolamine synthesis step. This embodiment prevents interference between the reaction of the modified cyanobacterium converting a carbon source to serine and the reaction of synthesizing phosphatidylethanolamine. In another embodiment, phosphatidylcholine and phospholipase are added directly to the modified cyanobacterial culture medium to synthesize phosphatidylethanolamine, performing the reaction in a one-pot synthesis to reduce equipment costs.

[0048] In step S40, the phosphatidylethanolamine synthesis step, the modified cyanobacteria and the L-serine produced therefrom are mixed with phosphatidylcholine and phospholipase in a photoreactor, or a filtered culture medium containing L-serine (not containing modified cyanobacterial cells) is mixed with phosphatidylcholine and phospholipase in a photoreactor to obtain a phosphatidylethanolamine mixture. In one embodiment of the present invention, the temperature of the fermentation photoreactor is 30 to 41°C, e.g., any temperature between 30 and 41°C, such as 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or 41°C. It is noteworthy that, as shown in FIG. 5, the optimal temperature for the fermentation photoreactor is 38°C, which allows for optimal strain growth.

[0049] More specifically, the phospholipase of the present invention is added in the form of a magnetic catalyst, and the method for preparing the magnetic catalyst includes the steps of: (A) preparation of magnetic nanoparticles (MNPs); (B) surface modification; and (C) immobilization of the enzyme, as detailed below.

[0050] (A) Fabrication of nanomagnetic beads Add 200 mL of 8.85% (w / v) iron(II) sulfate heptahydrate, 100 mL of 10.11% (w / v) potassium nitrate, and 50 mL of 27.62% (w / v) potassium hydroxide to a 500 mL serum vial and stir in a 90 °C water bath for 120 minutes. After separating and collecting the MNPs using a magnet, wash them three times with the same volume of deionized water and 95% alcohol, alternating between each wash. After each wash, use a magnet to separate and adsorb the MNPs for approximately 5 minutes, then remove the wash solution.

[0051] (B) Surface modification After adjusting the concentration of the prepared MNPs to 10 mg / mL, transfer 1 mL to a microcentrifuge tube and use a magnet to separate and adsorb the MNPs for approximately 5 minutes, then remove the supernatant. Then, wash the MNPs three times with 1 mL of deionized water to completely remove the alcohol. After each wash, use a magnet to separate and adsorb the MNPs for approximately 5 minutes, then remove the supernatant. After removing the alcohol, add 500 μL of deionized water to create an MNP suspension.

[0052] Weigh 2.5 mg of dopamine hydrochloride (DA) into a small glass bottle, add 500 μl of deionized water, and stir to dissolve. The MNP suspension was added to the dopamine hydrochloride solution, and 1 μl of 10 N NaOH was added to adjust the pH to 8.5. The mixture was then stirred for 3 hours. After approximately 2 hours of stirring, the pH dropped to 7, requiring additional NaOH to adjust the pH back to 8.5. The DA-MNPs were separated and collected with a magnet, then washed five times with 1 mL of deionized water. After each wash, the DA-MNPs were separated and collected with a magnet for approximately 5 minutes, and the supernatant was removed. After washing, the DA-MNPs were resuspended in 1 mL of deionized water, sonicated for 15 minutes, and stored at 4°C.

[0053] (C) Immobilization of phospholipase D DA-MNPs and phospholipase D were mixed in different ratios in 5 mM phosphate buffer (pH 6.8) and allowed to stand for 5 minutes. The DA-MNPs were then adsorbed and separated using a magnet for 5 minutes, washed three times with ultrapure water, and then repeatedly adsorbed and separated using a magnet. The phospholipase D-coated DA-MNPs were then redissolved in the same volume of ultrapure water and left to stand.

[0054] In one embodiment of the present invention, 1 mol of phosphatidylcholine and 0.05-5 wt% (relative to phosphatidylcholine) of nanomagnetic beads are added to 1 L of cyanobacteria / L-serine (containing 5 mol of serine), and the trimethylethanolaminium functional group in the phosphatidylcholine is substituted by the catalytic action of phospholipase to produce phosphatidylethanolamine, as shown in Scheme 2 below. [ka] formula 2

[0055] It is noteworthy that in the present invention, the pH adjustment makes the phosphatidylethanolamine obtained after the reaction insoluble in water, and it can be purified by an oil-water separation system. Specifically, the pH of the filtrate obtained by the filtration treatment is 1 to 3.

[0056] Therefore, in step S50 of the oil-water separation process, the filtrate is placed in an oil-water separator for layer separation. The oil-water separator has a separator plate. The upper organic phase spills out the separator's right side, while the lower aqueous phase remains in the oil-water separator, achieving oil-water separation and obtaining phosphatidylethanolamine as the organic phase. The separated aqueous phase is also recovered and reused for disposing of the NaHCO3 aqueous solution, thereby reducing production costs. In one embodiment of the present invention, the layer separation time is 10 minutes or more.

[0057] In one embodiment of the present invention, the modified cyanobacteria of the present invention can produce up to 2.78 g / L of L-serine by culturing them for 60 hours at 38°C in an environment of 3% CO2 and 25 mM NaHCO3. Furthermore, soybean-derived phosphatidylcholine and L-serine can be mixed in a 1:5 ratio in the presence of 10 mM Ca. 2+ After 12 hours of reaction at 45°C, 1.86 g / L of phosphatidylethanolamine was produced, a conversion rate of approximately 83.2%. In other words, by increasing the weight ratio of soybean-derived phosphatidylcholine to L-serine to 1:5, the efficiency of converting serine obtained from modified cyanobacteria to phosphatidylethanolamine could be further improved, increasing economic benefits.

[0058] [Beneficial Effects of the Embodiments] One beneficial effect of the present invention is to provide a method for producing phosphatidylethanolamine using a carbon source, which involves culturing modified cyanobacteria in an environment containing a carbon source, and allowing the modified cyanobacteria to convert the carbon source into serine, and the modified cyanobacteria comprising the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. This allows phosphatidylethanolamine to be produced using L-serine obtained from the carbon source as a reaction precursor, thereby improving the economic efficiency of waste gas treatment.

[0059] More specifically, the present invention uses a modified cyanobacterial strain containing the sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, thereby expressing the ability to convert a carbon source into serine. Specifically, in an environment rich in carbon sources, the modified cyanobacterium of the present invention can produce 35.2 mg / L to 46.3 mg / L of serine per hour. Furthermore, phosphatidylcholine and L-serine are blended in a ratio of 1:5, and 10 mM Ca is added. 2+ A high conversion rate of 83.2% was achieved when the reaction was carried out for 12 hours at 45°C. Furthermore, in this invention, phospholipase was modified into a magnetic catalyst and directly added to a fermentation photoreactor to perform a one-pot reaction, which is simple to operate, reusable, and reduces production costs.

[0060] The above disclosure is merely a preferred embodiment of the present invention, and does not limit the scope of the claims of the present invention. Therefore, all equivalent technical modifications made based on the contents of the specification and accompanying drawings of the present invention shall be included in the scope of the claims of the present invention. [Explanation of symbols]

[0061] S10~S50: Step

Claims

1. a modified cyanobacteria culturing step of culturing the modified cyanobacteria in a culture medium; a fermentation light treatment step, providing the modified cyanobacteria with an environment containing a carbon source so that the modified cyanobacteria convert the carbon source into serine; a filtration step of filtering the culture solution to separate the modified cyanobacteria and obtain a filtered culture solution; a phosphatidylethanolamine synthesis step of mixing the filtered culture medium containing serine, phosphatidylcholine, and phospholipase in a photoreactor to obtain a phosphatidylethanolamine mixture; an oil-water separation step in which the phosphatidylethanolamine mixture is left to stand in an oil-water separator for a predetermined time, and then phosphatidylethanolamine is obtained from the upper layer; Including, The modified cyanobacteria comprises the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

3.

1. A method for producing phosphatidylethanolamine using a carbon source, comprising:

2. The modified cyanobacteria is synthesizing a DNA sequence; inserting the DNA sequence into a plasmid; inserting the plasmid into a cyanobacterium by electroporation to obtain the modified cyanobacterium; obtained by a method comprising: A method for producing phosphatidylethanolamine using the carbon source according to claim 1.

3. 3. The method for producing phosphatidylethanolamine using a carbon source according to claim 2, wherein the electroporation treatment is carried out at a voltage of 0.5 to 1.5 kV for 2 to 10 mSec.

4. 3. The method for producing phosphatidylethanolamine using a carbon source according to claim 2, wherein the electroporation treatment further comprises adding polyethylene glycol at a concentration of 0.5 to 2%.

5. The method for producing phosphatidylethanolamine using a carbon source according to claim 1, wherein the environment contains 25 to 50 mM sodium bicarbonate.

6. The method for producing phosphatidylethanolamine using a carbon source according to claim 1, wherein the cyanobacteria is an elongated, spherical cyanobacterium.

7. The method for producing phosphatidylethanolamine using a carbon source according to claim 1 , wherein the phospholipase is a magnetic catalyst.

8. The method for producing phosphatidylethanolamine using a carbon source according to claim 1, wherein the temperature of the fermentation light treatment is 30 to 41°C.

9. 2. The method for producing phosphatidylethanolamine using a carbon source according to claim 1, wherein the modified cyanobacterium has the ability to produce 3-phosphate glycerate dehydrogenase, phosphoserine phosphorylase, and phosphoserine transferase.

10. 2. The method for producing phosphatidylethanolamine using a carbon source according to claim 1, wherein the carbon source is carbon dioxide, glucose, sucrose, fructose, or lactose.

11. a fermentation phototreatment step in which modified cyanobacteria are used to treat a carbon source and produce serine; a phosphatidylethanolamine synthesis step of mixing the serine, phosphatidylcholine, and phospholipase in a photoreactor to obtain a phosphatidylethanolamine mixture; a filtration step of passing the phosphatidylethanolamine mixture through a filtration membrane to obtain a filtrate; an oil-water separation step in which the filtrate is left to stand in an oil-water separator for a predetermined time, and phosphatidylethanolamine is obtained from the upper layer; Including, The modified cyanobacteria comprises the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

3.

1. A method for producing phosphatidylethanolamine using a carbon source, comprising:

12. The method for producing phosphatidylethanolamine using a carbon source according to claim 11, wherein the weight ratio of the phosphatidylcholine to the serine is 1:

5.

13. The method for producing phosphatidylethanolamine using a carbon source according to claim 11, wherein the pore size of the filtration membrane is 0.45 μm.

14. The method for producing phosphatidylethanolamine using a carbon source according to claim 11, wherein the predetermined time is 10 minutes or more.

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