Method for producing phosphatidylethanolamine using 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 particularly to a method for producing phosphatidylethanolamine using a carbon source.

Background Art

[0002] Cyanobacteria are self-sufficient organisms that synthesize their own necessary nutrients through photosynthesis. As measures against global warming and environmental destruction, existing technologies utilize the ability of cyanobacteria to fix carbon dioxide as a metabolite and apply it to the production of alcohols such as ethanol, butanol, 2,3-butanediol, and organic acids such as succinic acid, lactic acid, and isoprene. However, since cyanobacteria do not have the ability to convert a carbon source into serine, the production of serine using cyanobacteria is impossible with current technologies.

[0003] Phosphatidylethanolamine (PE) is the main phospholipid after phosphatidylcholine (PC) in soybeans, also known as phosphatidylethanolamine, is a phospholipid present in the brain, and is also a component of the cell membrane of nerve cells. Generally, phosphatidylethanolamine is thought to assist in the conduction of electrical pulses and promote the activation of neurotransmitters such as learning, memory, and emotions, and is used as a health product for regulating physiological functions. In existing technologies, it is obtained by extraction and purification from soy phosphatidylcholine, and soy phosphatidylcholine contains about 0.5 to 3% of phosphatidylethanolamine, and the supply source of phosphatidylethanolamine is scarce.

[0004] Therefore, developing a new method for producing phosphatidylethanolamine and increasing the supply source of phosphatidylethanolamine is one of the important issues to be solved in this industry.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for producing phosphatidylethanolamine using a carbon source in order to compensate for the deficiencies of the existing technology.

Means for Solving the Problem

[0006] To solve the above technical problem, the present invention provides a method for producing phosphatidylethanolamine using a carbon source. The method for producing phosphatidylethanolamine using a carbon source includes a step of culturing modified cyanobacteria in a culture solution, a step of providing an environment containing a carbon source to the modified cyanobacteria 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 obtaining a filtered culture solution, a step of synthesizing phosphatidylethanolamine by mixing the filtered culture solution, serine, phosphatidylcholine and phospholipase in a photoreaction tank to obtain a phosphatidylethanolamine mixture, and an oil-water separation step of putting the phosphatidylethanolamine mixture into an oil-water separator, standing for a predetermined 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, the following method is included to obtain modified cyanobacteria. Synthesizing a DNA sequence, inserting the DNA sequence into a plasmid, and inserting the plasmid into cyanobacteria by electroporation to obtain the modified cyanobacteria.

[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 a specific embodiment of the present invention, the electroporation treatment further includes adding 0.5 to 2% polyethylene glycol.

[0010] In a specific embodiment of the present invention, the environment contains 25 to 50 mM of sodium hydrogen carbonate.

[0011] In a specific embodiment of the present invention, the cyanobacteria are elongated spherical cyanobacteria.

[0012] In a specific embodiment of the present 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 specific embodiment of the present invention, the modified cyanobacteria have the production ability of 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase and phosphoserine transferase.

[0015] In a specific embodiment of the present 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, and the method includes a fermentation light treatment step of producing serine using modified cyanobacteria, and a phosphatidylethanolamine synthesis step of mixing serine, phosphatidylcholine and phospholipase in a photoreaction tank to obtain a phosphatidylethanolamine mixture, a filtration treatment step of filtering the phosphatidylethanolamine mixture through a filtration membrane to obtain a filtrate, and an oil-water separation treatment step of putting the filtrate into an oil-water separator, standing for a predetermined time, and obtaining the phosphatidylethanolamine from the upper layer.

[0017] In a specific embodiment of the present invention, the modified cyanobacteria contain the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

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

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

[0020] In a specific embodiment of the present invention, the predetermined time is 10 minutes or more.

[0021] One advantage of the present invention is that in the method for producing phosphatidylethanolamine using the carbon source provided by the present invention, through the technical solutions of "culturing modified cyanobacteria in an environment containing a carbon source, and the modified cyanobacteria converting the carbon source into serine" and "the modified cyanobacteria containing the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3", L-serine produced from the carbon source is used as a reaction precursor to produce phosphatidylethanolamine, and the economic effect of waste gas treatment can be improved.

[0022] To further understand the features and technical content of the invention, the following detailed description of the present invention and the accompanying drawings are referred to. However, the provided accompanying drawings are only for reference and explanation, and are not for limiting the scope of the claims of the present invention.

Brief Description of the Drawings

[0023]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Embodiments for Carrying Out the Invention

[0024] The embodiments disclosed by the present invention will be described below. Those skilled in the art can understand the advantages and effects of the present invention based on the disclosure of this specification. The present invention can be implemented or applied by other different embodiments. Each detail in this specification can also be equivalently modified and changed without departing from the spirit of the present invention based on various viewpoints or applications. Also, the drawings of the present invention are for simple and schematic explanation and do not show actual dimensions. In the following embodiments, technical matters related to the present invention will be further described, but the disclosed content does not limit the present invention. Also, the term "or" used in this specification can include any one or a combination of more than one of the related listed items according to the actual situation.

[0025] It should be understood that the term "or" used in the text may include any one or a combination of more than one of the related listed items according to the actual situation. Unless there are special requirements in the context, the term "comprising" should be understood as implying the stated integer or step, or a set of integers or steps, and does not exclude any other arbitrary integer or step, or any other arbitrary set of integers or steps. In this specification, the terms "comprising", "containing", "including", and "having" can be used interchangeably.

[0026] The term "exogenous gene" used in the text, also called a heterologous gene, refers to a gene or nucleic acid fragment that is not the endogenous gene composition of the host cell or target cell itself, but is from another species or cell, or is artificially synthesized, and is introduced into the host cell or target cell by genetic engineering techniques.

[0027] As shown in FIGS. 1 to 3, the 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 cyanobacteria by electroporation to obtain denatured cyanobacteria. In step S4, a carbon source is provided to the denatured cyanobacteria, and the denatured cyanobacteria convert the carbon source into serine.

[0028] In FIGS. 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 photosystem II, PSI refers to photosystem I, Cytb6f is cytochrome b6f, which is the center of the light-dependent reaction of photosynthesis containing oxygen. RuBP refers to ribulose-1,5-bisphosphate, and CA refers to carbonic anhydrase. Rbs refers to the ribosome-binding site.

[0029] In step S1 of synthesizing the DNA sequence, a gene code suitable for cyanobacteria is artificially synthesized, which means that cyanobacteria can identify and produce corresponding substances. In particular, a gene code suitable for identifying the elongated spherical cyanobacterium Synechococcus elongates PCC7942 is synthesized. Furthermore, the artificially synthesized DNA sequence can be mass-produced using the polymerase chain reaction (PCR). In the embodiment of the present invention, the PCR conditions are as follows: After denaturation at 98°C for 30 seconds, denaturation at 98°C for 10 seconds, low-temperature annealing at 56°C for 20 seconds, and polymerization at 72°C for 45 seconds are performed for 30 cycles, and then PCR amplification is performed by polymerization at 72°C for 10 minutes.

[0030] Furthermore, plasmid DNA is mass-produced and isolated from the original Escherichia coli DH5α strain, the DNA sequence in step S2 is inserted into the plasmid, the designed DNA sequence is introduced into the plasmid of Escherichia coli, replicated in the denatured Escherichia coli and mass-produced to obtain a recombinant plasmid. This is named pSerSyn. However, since Escherichia coli cannot utilize CO2, even if the designed DNA sequence is obtained, the carbon source cannot be converted to serine. Therefore, it is necessary to further extract the mass-produced denatured plasmid DNA and transfer it to the original cyanobacteria.

[0031] Specifically, the plasmid construction used in the present invention uses pSyn_1 as the backbone, carries the gene sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and is integrated into the genome of cyanobacteria by homologous recombination for expression. Then, homologous recombination is successfully carried out using antibiotic selection, and the denatured cyanobacteria of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 are selected and obtained. In other words, the cyanobacterial strain that has successfully undergone denaturation can grow on a solid medium containing antibiotics.

[0032] In step S3 of inserting the plasmid into cyanobacteria, the cyanobacteria are cultured in BG11 medium, and OD 730 can be used to measure the growth concentration of the strain. Furthermore, the plasmid is inserted into cyanobacteria by electroporation to obtain denatured cyanobacteria. Electroporation applies an electric current to the cells of cyanobacteria in an extremely short time (microseconds to milliseconds), generates a potential difference across the cell membrane in an environment of high voltage and low volume, and changes the structure of the cell membrane. As a result, the cell membrane is compressed and thinned, countless tiny pores are generated, and the plasmid can pass through the cell membrane and enter the cells of cyanobacteria.

[0033] To achieve the optimal plasmid permeation effect, an additional 0.5% - 2% polyethylene glycol (PEG) can be added in this step. For example, it can be any concentration between 0.5% and 2%, such as 1.0%, 1.5%, etc. Preferably, the electroporation treatment is performed on cyanobacteria at a voltage of 0.5 KV - 1.5 KV, for example, any voltage between 0.5 KV and 1.5 KV such as 0.6 KV, 0.7 KV, 0.8 KV, 0.9 KV, 1.0 KV, 1.1 KV, 1.2 KV, 1.3 KV, 1.4 KV, etc., for 2 mSec - 10 mSec. For example, any millisecond between 2 mSec and 10 mSec such as 3 mSec, 4 mSec, 5 mSec, 6 mSec, 7 mSec, 8 mSec, 9 mSec, etc. In the present invention, furthermore, during the electroporation treatment, 1×10 6 of native cyanobacteria were treated under different voltage and time conditions to obtain the amount of strains of successfully modified cyanobacteria, as shown in Table 1 below.

[0034]

Table 1

[0035] Based on the results in Table 1 above, with 1% PEG added, the preferred condition for the electroporation treatment of the present invention is to treat cyanobacteria at a voltage of 0.5 kV for 10 mSec. A more preferred condition is to treat cyanobacteria at a voltage of 1.5 kV for 5 mSec. An even more preferred condition is to treat cyanobacteria at a voltage of 1.0 kV for 5 mSec, which can obtain the maximum number of colonies.

[0036] Generally, native cyanobacteria have the ability to reduce carbon dioxide to glyceraldehyde 3 - phosphate (G3P). However, due to the lack of related metabolic enzymes, native cyanobacteria cannot further metabolize glyceraldehyde 3 - phosphate (G3P) to L - serine. The present invention uses cyanobacteria to process a carbon source and produces 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 (H2), 200 ppm of acetylene (C2H2), 150 ppm of methane (CH4), 0.5 ppm of hydrogen sulfide (H2S), and 3 ppm of acetaldehyde (CH3CHO).

[0038] In step S4 for providing a carbon source to the modified cyanobacteria, the modified cyanobacteria of the present invention have the production ability of 3-phosphoglycerate dehydrogenase (SerA), phosphoserine phosphatase (SerB), and phosphoserine aminotransferase (SerC), and can independently perform the reaction of formula 1 below to convert G3P into L-serine.

Chemical formula

[0039] The modified cyanobacteria of the present invention have a plurality of foreign genes, including the nucleic acid sequences of the genes of 3-phosphoglycerate dehydrogenase (SerA), phosphoserine phosphatase (SerB), and phosphoserine aminotransferase (SerC), and these genes can be expressed or overexpressed in the modified cyanobacteria. In other words, the modified cyanobacteria of the present invention contain the 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 that the native cyanobacteria convert a carbon source into glyceraldehyde 3-phosphate (G3P). Further, the modified cyanobacteria of the present invention produce 3-phosphoglycerate dehydrogenase (SerA), whereby glyceraldehyde 3-phosphate (G3P) can be converted into 3-phosphohydroxypyruvate (3P-HP). Furthermore, the modified cyanobacteria of the present invention produce phosphoserine transferase (SerC), which can convert 3-phosphohydroxypyruvate into phosphoserine (3P-Serine). Additionally, the modified cyanobacteria of the present invention produce phosphoserine phosphatase (SerB), which can convert phosphoserine (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 the above examples. Preferably, the modified cyanobacteria of the present invention can utilize a carbon source from industrial waste gas containing carbon and convert the carbon source in the industrial waste gas into L-serine, and at the same time, treat industrial waste using the modified cyanobacteria and obtain economically valuable chemicals.

[0042] Another embodiment of the present invention provides a method for converting a carbon source into serine, which includes converting the carbon source into serine using at least the modified cyanobacteria of the present invention. The modified cyanobacteria include the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In other words, the modified cyanobacteria include the expression plasmids of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0043] Based on this, the modified cyanobacteria can convert a carbon source into glyceraldehyde 3-phosphate (G3P) and release it extracellularly. The cyanobacteria produce 3-phosphoglycerate dehydrogenase (SerA) by the expression of the sequence of SEQ ID NO:1 and can further convert glyceraldehyde 3-phosphate (G3P) into 3-phosphohydroxypyruvate (3P-HP). The cyanobacteria produce phosphoserine transferase (SerC) by the expression of the sequence of SEQ ID NO:3 and can convert 3-phosphohydroxypyruvate (3P-HP) into phosphoserine (3P-Serine). The cyanobacteria produce phosphoserine phosphatase (SerB) by the expression of the sequence of SEQ ID NO:2 and can convert phosphoserine (3P-Serine) into serine.

[0044] In addition, the present invention can also include 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, the modified cyanobacteria are cultured in BG11 medium.

[0045] Specifically, in the fermentation light treatment of step S20, the modified cyanobacteria are cultured in a flue gas (industrial waste gas) environment containing CO2. In a better embodiment, sodium bicarbonate (NaHCO3) is added to the culture medium of the modified cyanobacteria, which helps the flue gas dissolve into the culture medium of the modified cyanobacteria. Further, as shown in FIG. 4, the concentration of NaHCO3 is 25-50 mM (for example, any positive integer from 25 to 50). After culturing for 24 hours with the addition of NaHCO3, the growth amount of the modified cyanobacteria is higher in terms of OD 730 value than when no NaHCO3 is added, and it has been shown that adding 25-50 mM of NaHCO3 promotes the growth of the modified cyanobacteria and improves the production amount of L-serine.

[0046] In step S30 of the filtration process, the membrane filtration method can be used. For example, the culture solution or reaction solution is introduced into a backwash filter, filtered through a 0.45 μm filter membrane to remove the cells of denatured cyanobacteria, and a filtrate containing serine or phosphatidylethanolamine is obtained. The filtered denatured cyanobacteria are recovered in the fermentation photoreaction tank and reused in the fermentation light treatment, which can save manufacturing costs.

[0047] Specifically, in one embodiment, first, the culture solution is filtered to remove the cells of cyanobacteria, and the filtered culture solution, phosphatidylcholine, and phospholipase are mixed to proceed with the synthesis step of phosphatidylethanolamine. In this embodiment, it is possible to avoid the interference between the reaction of converting the carbon source to serine by denatured cyanobacteria and the synthesis reaction of phosphatidylethanolamine. In another embodiment, phosphatidylcholine and phospholipase are directly added to the culture solution of denatured cyanobacteria to synthesize phosphatidylethanolamine, and the reaction is carried out by one-pot synthesis to save equipment costs.

[0048] In the phosphatidylethanolamine synthesis step of step S40, denatured cyanobacteria and the L-serine produced by it are mixed with phosphatidylcholine and phospholipase in a photoreaction tank, or the filtered culture solution containing L-serine (without the cells of denatured cyanobacteria) is mixed with phosphatidylcholine and phospholipase in a photoreaction tank to obtain a phosphatidylethanolamine mixture. In one embodiment of the present invention, the temperature of the fermentation light treatment is 30 to 41 °C, for example, any temperature from 30 to 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, 41 °C. It should be noted that, as shown in FIG. 5, the optimal temperature of the fermentation light treatment is 38 °C, and the best strain growth amount can be obtained.

[0049] More specifically, the phospholipase of the present invention is added in the form of a magnetic catalyst. The manufacturing method of the magnetic catalyst includes the following steps: (A) manufacturing magnetic nanoparticles (MNP); (B) surface modification; and (C) immobilization of the enzyme, the details of which are as follows.

[0050] (A) Manufacturing of magnetic nanoparticles Add 200 mL of 8.85% (w / v) iron(II) sulphate 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 water bath at 90 °C for 120 minutes. After separating and collecting the MNP generated using a magnet, wash it alternately three times with the same volume of deionized water and 95% alcohol, and after each washing, use a magnet to separate and adsorb the MNP for about 5 minutes, and then remove the washing solution.

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

[0052] Weigh 2.5 mg of dopamine hydrochloride (DA), put it into a small glass bottle, add 500 μl of deionized water, stir and dissolve it to prepare a dopamine hydrochloride solution. Add the MNP suspension to the dopamine hydrochloride solution, add 1 μl of 10 N NaOH to adjust the pH to 8.5, and then stir for 3 hours. After stirring for about 2 hours, since the pH drops to 7, it is necessary to add NaOH again to adjust the pH to 8.5. After separating and collecting DA-MNP with a magnet, wash it 5 times with 1 mL of deionized water. After each washing, separate and collect DA-MNP with a magnet for about 5 minutes and remove the supernatant. After the washing is completed, resuspend the volume of DA-MNP to 1 mL with deionized water, perform ultrasonic treatment for 15 minutes, and then store it at 4 °C.

[0053] (C) Immobilization of Phospholipase D Mix DA-MNP and phospholipase D in 5 mM phosphate buffer (pH 6.8) at different ratios, and then let it stand for 5 minutes. Then, use a magnet to adsorb and separate DA-MNP for 5 minutes, repeat washing 3 times with ultrapure water and adsorbing and separating with a magnet, and then redissolve the DA-MNP coated with phospholipase D in the same volume of ultrapure water and wait.

[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 trimethylethanaminium functional group in phosphatidylcholine is substituted by the catalytic action of phospholipase to generate phosphatidylethanolamine. Specifically, it is shown in the following formula 2.

Chemical formula

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

[0056] Therefore, in the step S50 of the oil-water separation treatment, the filtrate is left standing in an oil-water separator for layer separation. The oil-water separator has a separation plate. The upper organic phase overflows from the separation plate to the right side of the separator, and the lower aqueous phase remains in the oil-water separator, thereby achieving oil-water separation and obtaining phosphatidylethanolamine in the organic phase. In addition, the separated aqueous phase can be recovered and reused for the preparation of the NaHCO3 aqueous solution, thus saving the production cost. In one embodiment of the present invention, the time for standing and layer separation is 10 minutes or more.

[0057] In one embodiment of the present invention, by culturing the modified cyanobacteria of the present invention for 60 hours in an environment of 38 °C, 3% CO2, and 25 mM NaHCO3, up to 2.78 g / L of L-serine can be produced. Furthermore, by reacting soy-derived phosphatidylcholine and L-serine at a ratio of 1:5 in the presence of 10 mM Ca 2+ ions at 45 °C for 12 hours, 1.86 g / L of phosphatidylethanolamine can be generated, and the conversion rate reaches about 83.2%. That is, by setting the weight ratio of soy-derived phosphatidylcholine to L-serine to 1:5, the efficiency of converting serine obtained from modified cyanobacteria into phosphatidylethanolamine can be further improved, and economic benefits can be enhanced.

[0058] [Beneficial effects according to the embodiment] One beneficial effect of the present invention is to provide a method for producing phosphatidylethanolamine using a carbon source. Thus, by culturing modified cyanobacteria in an environment containing a carbon source and having the modified cyanobacteria convert the carbon source into serine, and through the technical solutions of "the modified cyanobacteria contain the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3", phosphatidylethanolamine can be produced using L-serine obtained from the carbon source as a reaction precursor, enhancing the economic effect of waste gas treatment.

[0059] More specifically, in the present invention, a strain of modified cyanobacteria is used and the ability to convert a carbon source into serine is expressed by containing the sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. Specifically, under an environment rich in a carbon source, the modified cyanobacteria of the present invention can produce 35.2 mg / L to 46.3 mg / L of serine per hour. Also, when phosphatidylcholine and L-serine are blended at a ratio of 1:5 and reacted for 12 hours in an environment of 10 mM Ca 2+ ions at 45 °C, a high conversion rate of 83.2% is achieved. Furthermore, in the present invention, phospholipase is modified with a magnetic catalyst and directly added to the fermentation photoreactor for a one-pot reaction, making the operation simple and reusable, and saving the manufacturing cost.

[0060] The content disclosed above is only 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 content of the specification and the accompanying drawings of the present invention shall be included in the scope of the claims of the present invention.

Description of the Reference Numerals

[0061] S10~S50: Step

Claims

1. A modified cyanobacteria culturing step of culturing modified cyanobacteria in a culture solution, A fermentation light treatment step of providing an environment containing a carbon source to the modified cyanobacteria for the modified cyanobacteria to convert the carbon source into serine, A filtration treatment step of filtering the culture solution to separate the modified cyanobacteria and obtaining a filtered culture solution, A phosphatidylethanolamine synthesis step of mixing the filtered culture solution, the serine, phosphatidylcholine and phospholipase in a photoreactor to obtain a phosphatidylethanolamine mixture, An oil-water separation treatment step of allowing the phosphatidylethanolamine mixture to stand in an oil-water separator and obtaining phosphatidylethanolamine from the upper layer after a predetermined time has elapsed, comprising, the modified cyanobacteria contain the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 A method for producing phosphatidylethanolamine using a carbon source, characterized in that.

2. The modified cyanobacteria are obtained by a method comprising: a step of synthesizing a DNA sequence, a step of inserting the DNA sequence into a plasmid, a step of inserting the plasmid into cyanobacteria by electroporation treatment to obtain the modified cyanobacteria, and A method for producing phosphatidylethanolamine using a carbon source according to Claim 1.

3. The electroporation treatment is performed at a voltage of 0.5 to 1.5 kV for 2 to 10 mSec, and the method for producing phosphatidylethanolamine using a carbon source according to Claim 2.

4. The electroporation treatment further includes adding polyethylene glycol at a concentration of 0.5 to 2%, and the method for producing phosphatidylethanolamine using a carbon source according to Claim 2.

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

6. The cyanobacteria are elongated spherical cyanobacteria, and the method for producing phosphatidylethanolamine using a carbon source according to Claim 1.

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

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

9. The method for producing phosphatidylethanolamine using the carbon source according to claim 1, wherein the modified cyanobacteria have the ability to produce 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase, and phosphoserine transferase.

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

11. A fermentation light treatment step of producing serine using modified cyanobacteria, A phosphatidylethanolamine synthesis step of mixing the serine, phosphatidylcholine, and phospholipase in a photoreaction tank to obtain a phosphatidylethanolamine mixture, A filtration treatment step of obtaining a filtrate by passing the phosphatidylethanolamine mixture through a filtration membrane, An oil-water separation treatment step of allowing the filtrate to stand in an oil-water separator for a predetermined time and then obtaining phosphatidylethanolamine from the upper layer, comprising A method for producing phosphatidylethanolamine using a carbon source, characterized in that.

12. The method for producing phosphatidylethanolamine using the carbon source according to claim 11, wherein the modified cyanobacteria contain the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:

3.

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

5.

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

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

Images