Method for producing cephalin by using carbon source

Abstract

The invention discloses a method for producing cephalin by using a carbon source. The method comprises the following steps: culturing modified cyanobacteria, performing fermentation light treatment, performing filtration treatment and performing oil-water separation treatment. The step of culturing the modified blue-green bacteria is that the modified blue-green bacteria are cultured in an environment containing a carbon source, so that the carbon source is converted into serine by the modified blue-green bacteria. In the fermentation light treatment, serine, lecithin and phospholipase are mixed in a light reaction tank to obtain cephalin mixed liquor. And the filtering treatment is that the cephalin mixed liquid passes through a filtering membrane to obtain filtrate. The oil-water separation treatment comprises the following steps: placing the filtrate in an oil-water separator, standing for a preset time, and taking the upper layer to obtain cephalin. According to the method disclosed by the invention, L-serine produced from a carbon source is used as a reaction precursor to produce cephalin, so that the economic benefit of waste gas treatment is improved.

Description

Technical Field

[0001] The present invention relates to a method for producing cephalin, and particularly to a method for producing cephalin using a carbon source. Background Art

[0002] Cyanobacteria are autotrophic organisms that can synthesize their own nutrients through photosynthesis. To mitigate the greenhouse effect and environmental damage, in the prior art, due to the ability of cyanobacteria to fix carbon dioxide into metabolites, cyanobacteria are applied to the production of alcohols and organic acids such as ethanol, butanol, 2,3-butanediol, succinic acid, lactic acid, and isopropene. However, since cyanobacteria lack the ability to convert carbon sources into serine, cyanobacteria cannot be applied to the production of serine in the prior art.

[0003] Phosphatidylethanolamine (PE), the main phospholipid second only to phosphatidylcholine (PC) in soybean lecithin, can also be called cephalin. It is a phospholipid present in the brain and is also a component of the cell membrane of nerve cells. Generally, cephalin is considered to be able to help transmit electrical impulses, thereby promoting the activity of neurotransmitters such as learning, memory, and emotions, and is used as a health product for regulating physiological functions. In the prior art, it is mainly extracted and purified from the lecithin of soybeans. Only about 0.5-3% of cephalin is contained in the lecithin of soybeans, making the source of cephalin scarce.

[0004] Therefore, how to increase the source of obtaining cephalin by developing a new method for producing cephalin has become one of the important issues to be solved in this field. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for producing cephalin using a carbon source in view of the deficiencies of the prior art.

[0006] To solve the above technical problems, one of the technical solutions adopted by the present invention is to provide a method for producing cephalin using a carbon source. The method includes: culturing modified cyanobacteria by culturing the modified cyanobacteria in a culture solution; fermentation light treatment by providing a carbon source to the modified cyanobacteria so that the modified cyanobacteria convert the carbon source into serine; filtration treatment by filtering the culture solution to separate the modified cyanobacteria to obtain a filtered culture solution; cephalin synthesis by mixing the filtered culture solution, the serine, lecithin and phospholipase in a photoreaction tank to obtain a cephalin mixture; and oil-water separation treatment by allowing the cephalin mixture to stand in an oil-water separator for a predetermined time and taking the upper layer to obtain the cephalin. Among them, the modified cyanobacteria include the gene sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0007] Further, the modified cyanobacteria are obtained through the following steps: synthesizing a DNA sequence; implanting the DNA sequence into a plasmid; and implanting the plasmid into cyanobacteria through electroporation treatment to obtain the modified cyanobacteria.

[0008] Further, the electroporation treatment is to treat with a voltage of 0.5 to 1.5 kV for 2 to 10 mSec.

[0009] Further, the electroporation treatment further includes adding polyethylene glycol with a concentration of 0.5 to 2%.

[0010] Further, the environment includes 25 to 50 mM of sodium bicarbonate.

[0011] Further, the cyanobacteria are Synechococcus elongatus.

[0012] Further, the phospholipase is a magnetic catalyst.

[0013] Further, the temperature of the fermentation light treatment is 30 to 41 °C.

[0014] Further, the modified cyanobacteria have the ability to produce 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase and phosphoserine aminotransferase.

[0015] Further, the carbon source is carbon dioxide, glucose, sucrose, fructose or galactose.

[0016] To solve the above technical problems, another technical solution adopted by the present invention is to provide a method for producing cephalin using a carbon source. The method includes: fermentation light treatment to produce serine using modified cyanobacteria; cephalin synthesis by mixing the serine with lecithin and phospholipase in a photoreaction tank to obtain a cephalin mixture; filtration treatment by passing the cephalin mixture through a filter membrane to obtain a filtrate; and oil-water separation treatment by placing the filtrate in an oil-water separator and standing for a predetermined time, and taking the upper layer to obtain the cephalin.

[0017] Furthermore, the modified cyanobacteria include the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0018] Furthermore, the weight ratio of the lecithin to the serine is 1:5.

[0019] Furthermore, the pore size of the filter membrane is 0.45 μm.

[0020] Furthermore, the predetermined time is 10 minutes or more.

[0021] One of the beneficial effects of the present invention is that the method for producing cephalin using a carbon source provided by the present invention can use L-serine produced from the carbon source as a reaction precursor to produce cephalin through the technical solutions of "culturing modified cyanobacteria in an environment containing a carbon source to enable the modified cyanobacteria to convert the carbon source into serine" and "the modified cyanobacteria include the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3", thereby improving the economic benefits of waste gas treatment.

[0022] To further understand the features and technical content of the present invention, please refer to the following detailed description and drawings of the present invention. However, the provided drawings are only for reference and illustration, and are not used to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a flowchart of the method for producing cephalin using a carbon source according to the present invention.

[0024] Figure 2 It is a schematic diagram of the metabolic pathway of the modified cyanobacteria according to the present invention.

[0025] Figure 3 It is a schematic diagram of plasmid construction according to the present invention.

[0026] Figure 4 It is a line graph showing the relationship between the concentration of NaHCO3 and the growth of cyanobacteria.

[0027] Figure 5A line graph showing the relationship between the temperature of the fermentation photoreactor and the growth of cyanobacteria.

[0028] Reference numerals in the drawings: S10 to S50: steps Detailed implementation manners

[0029] The following are specific examples to illustrate the implementation manners of the present invention regarding the "method for producing cephalin using a carbon source" disclosed herein. Those skilled in the art can understand the advantages and effects of the present invention from the content disclosed in this specification. The present invention can be implemented or applied through other different specific embodiments, and various details in this specification can also be modified and changed based on different viewpoints and applications without departing from the concept of the present invention. Additionally, the drawings of the present invention are only for simple schematic illustration and are not drawn according to actual sizes, hereby stating in advance. The following implementation manners will further detail the relevant technical content of the present invention, but the disclosed content is not intended to limit the protection scope of the present invention.

[0030] It should be understood that the term "or" used herein may, depending on the actual situation, include any one or a combination of more of the associated listed items. Unless otherwise required by the context, the term "comprising" should be understood to imply including a stated integer or step or a group of integers or steps, but not excluding any other integer or step or any other group of integers or steps. In this specification, the terms "comprising", "containing", "including" or "having" can be used interchangeably.

[0031] The term "exogenous gene" used herein, also known as a heterologous gene, refers to a gene or nucleotide fragment that is not from the endogenous genome originally possessed by the host cell or target cell itself, but is taken from other species or cells, or is artificially synthesized, and is introduced into the host cell or target cell via genetic engineering techniques.

[0032] The first embodiment of the present invention provides a method for converting a carbon source into serine, which includes: Step S1: Synthesize a DNA sequence; Step S2: Implant the DNA sequence into a plasmid so that the plasmid includes the sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; Step S3: Implant the plasmid into cyanobacteria via electroporation treatment to obtain modified cyanobacteria; and Step S4: Provide a carbon source to the modified cyanobacteria so that the modified cyanobacteria convert the carbon source into serine.

[0033] In Figure 2 and Figure 3Among them, NADP refers to nicotinamide adenine dinucleotide phosphate; NADPH refers to reduced nicotinamide adenine dinucleotide phosphate; ATP refers to adenosine triphosphate; ADP refers to adenosine diphosphate; PSII is photosystem II; PSI is photosystem I; Cytb6f refers to cytochrome b6f, which is the core of the light-dependent reaction of oxygenic photosynthesis; RuBP is ribulose-1,5-bisphosphate; CA refers to carbonic anhydrase; rbs refers to ribosome-binding site.

[0034] In step S1 of synthesizing the DNA sequence, it refers to artificially synthesizing a DNA sequence with a gene coding recognition suitable for cyanobacteria, so that cyanobacteria can recognize and produce the corresponding substances. In particular, a gene coding with a recognition suitable for Synechococcus elongates PCC7942 is synthesized. Further, the artificially synthesized DNA sequence can be mass-produced by polymerase chain reaction (PCR). In an embodiment of the present invention, the PCR conditions can be: denaturation at 98°C for 30 seconds, followed by 30 cycles of denaturation at 98°C for 10 seconds, low-temperature adhesion at 56°C for 20 seconds, and polymerization at 72°C for 45 seconds, and then PCR amplification is carried out under the condition of polymerization at 72°C for 10 minutes.

[0035] In addition, plasmid DNA is mass-produced and isolated from the original Escherichia coli DH5α strain, so that in step S2 of implanting the DNA sequence into the plasmid, the designed DNA sequence is sent into the plasmid of Escherichia coli, and the modified Escherichia coli is used for replication and mass production to obtain a recombinant plasmid, which is named pSerSyn. However, Escherichia coli cannot utilize CO2, and even if the designed DNA sequence is obtained, it cannot convert the carbon source into serine. Therefore, the mass-produced modified plasmid DNA needs to be taken out and transferred to the original cyanobacteria.

[0036] Specifically, the plasmid construction used in the present invention can use pSyn_1 as the backbone and carry the gene sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, so as to be integrated into the genome of cyanobacteria for expression through homologous recombination. Subsequently, antibiotics are used to screen out the modified cyanobacteria that have successfully undergone homologous recombination to obtain SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. In other words, the successfully modified cyanobacteria strain can grow on a solid medium containing antibiotics.

[0037] In step S3 of implanting the plasmid into cyanobacteria, the cyanobacteria can be cultured in BG11 medium and with OD730 Measure the growth concentration of the strain. Further, a plasmid is implanted into the cyanobacterium via electroporation to obtain a modified cyanobacterium. Electroporation is to apply an electric current to the cyanobacterium cells within a very short time (microseconds to milliseconds), making them in an environment of high voltage and low capacitance. A potential difference will be generated across the cell membrane, causing a change in the cell membrane structure. The cell membrane will be compressed and thinned, and then numerous tiny pores will be generated, allowing the plasmid to pass through the cell membrane and enter the cells of the cyanobacterium.

[0038] To achieve the best plasmid permeability effect, 0.5 to 2% polyethylene glycol (PEG), such as any concentration between 0.5% and 2% like 1.0%, 1.5%, etc., can be further added in this step. Preferably, the electroporation treatment is carried out at a voltage of 0.5 to 1.5 kV, such as any voltage value between 0.5 and 1.5 kV like 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 kV, and the cyanobacterium is treated for 2 to 10 mSec, such as any millisecond between 2 and 10 like 3, 4, 5, 6, 7, 8, 9 mSec. The present invention further studies that in the electroporation treatment, the native cyanobacteria with a quantitative concentration of 1x10 6 Under the treatment conditions of different voltages and times, the amount of the strain of the modified cyanobacterium successfully obtained from the native cyanobacteria is shown in Table 1 below.

[0039] Table 1 (1% PEG is added to each group)

[0040] Voltage (kV) Time (mSec) Number of colonies (colony) 0.5 2 6 0.5 5 12 0.5 10 21 1.0 2 19 1.0 5 36 1.0 10 11 1.5 2 17 1.5 5 32 1.5 10 8

[0041] Based on the results of Table 1 above, in the case of adding 1% PEG, the electroporation treatment in the present invention is preferably to treat the cyanobacterium at a voltage of 0.5 for 10 mSec, more preferably at a voltage of 1.5 kV for 5 mSec, and even more preferably at a voltage of 1.0 kV for 5 mSec to obtain the maximum number of colonies.

[0042] Generally speaking, native cyanobacteria have the ability to reduce carbon dioxide to glyceraldehyde 3 - phosphate (G3P). However, due to the lack of relevant metabolic enzymes, native cyanobacteria cannot further metabolize glyceraldehyde 3 - phosphate (G3P) into L - serine. The present invention prepares a modified cyanobacterium in order to utilize cyanobacteria to process carbon sources and convert the carbon sources into serine.

[0043] In the present invention, the carbon source may be industrial waste gas, namely a mixture of hydrogen, acetylene, methane, hydrogen sulfide and acetaldehyde. Further, the mixture may contain 30 to 50 ppm of hydrogen, 150 to 250 ppm of acetylene, 100 to 200 ppm of methane, 0.1 to 1 ppm of hydrogen sulfide and 1 to 5 ppm of acetaldehyde. For example, the industrial waste gas may be 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).

[0044] In step S4 of providing a carbon source to the modified cyanobacteria, the modified cyanobacteria of the present invention have the ability to produce 3-phosphoglycerate dehydrogenase (3-phosphoglycerate dehydrogenase, SerA), phosphoserine phosphatase (phosphoserine phosphatase, SerB) and phosphoserine aminotransferase (phosphoserine aminotransferase, SerC), and can independently carry out the reaction of formula 1 below to convert G3P into L-serine.

[0045]

[0046] The modified cyanobacteria of the present invention have multiple exogenous genes, which include the nucleotide sequence of the gene encoding 3-phosphoglycerate dehydrogenase (SerA), the nucleotide sequence of the gene encoding phosphoserine phosphatase (SerB) and the nucleotide sequence of the gene encoding 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 have expression plasmids including SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0047] Specifically, the modified cyanobacteria of the present invention still retain the characteristics of the native cyanobacteria to convert the carbon source into glyceraldehyde 3-phosphate (G3P). And the modified cyanobacteria of the present invention can also produce 3-phosphoglycerate dehydrogenase (SerA), and then can convert glyceraldehyde 3-phosphate (G3P) into 3-phosphohydroxypyruvate (3-phosphohydroxypyruvate, 3P-HP). Furthermore, the modified cyanobacteria of the present invention can also produce phosphoserine aminotransferase (SerC), and then can convert 3-phosphohydroxypyruvate into phosphoserine (3-phosphoserine, 3P-Serine). In addition, the modified cyanobacteria of the present invention can also produce phosphoserine phosphorylase (SerB), and then can convert phosphoserine (3P-Serine) into serine, especially L-serine (L-Serine).

[0048] 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 galactose. However, the present invention is not limited to the examples listed above. Preferably, the modified cyanobacteria of the present invention can utilize the carbon source in the carbon-containing industrial waste gas to convert the carbon source in the industrial waste gas into L-serine, so that while treating industrial waste with the modified cyanobacteria, chemicals with economic benefits can also be obtained.

[0049] Another embodiment of the present invention also provides a method for converting a carbon source into serine, which at least includes using the modified cyanobacteria of the present invention to convert the carbon source into serine. 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 have an expression plasmid including SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0050] Accordingly, the modified cyanobacteria can convert the carbon source into glyceraldehyde 3-phosphate (G3P). The cyanobacteria can also utilize the expression of the SEQ ID NO:1 sequence to produce 3-phosphoglycerate dehydrogenase (SerA) to further convert glyceraldehyde 3-phosphate (G3P) into 3-phosphohydroxypyruvate (3P-HP). The cyanobacteria can also utilize the expression of the SEQ ID NO:3 sequence to produce phosphoserine transaminase (SerC) to convert 3-phosphohydroxypyruvate (3P-HP) into phosphoserine (3P-Serine). The cyanobacteria can also utilize the expression of the SEQ ID NO:2 sequence to produce phosphoserine phosphatase (SerB) to convert phosphoserine (3P-Serine) into the serine.

[0051] On the other hand, the present invention can also include a method for producing cephalin using a carbon source. Refer to Figure 1 As shown, the method can include: S10: culturing modified cyanobacteria; S20: fermentation light treatment; S30: filtration treatment; S40: synthesizing cephalin and S50: oil-water separation treatment. In other words, in step S10, the modified cyanobacteria are cultured in a BG11 medium.

[0052] Specifically, the fermentation light treatment in step S20 is to culture the modified cyanobacteria in an environment of flue gas (industrial waste gas) containing CO2. In a preferred embodiment, sodium bicarbonate (NaHCO3) can be added to the culture medium of the modified cyanobacteria to help the flue gas dissolve into the culture medium of the modified cyanobacteria. Further, please refer to Figure 4As shown, the concentration of NaHCO3 can be 25 to 50 mM (any positive number between 25 and 50, for example). After culturing with the addition of NaHCO3 for 24 hours, the growth of the modified cyanobacteria has a higher OD value than that without the addition of NaHCO3, indicating that the addition of 25 to 50 mM of NaHCO3 also contributes to the growth of the modified cyanobacteria, thereby increasing the yield of L-serine. 730 The value shows that the addition of 25 to 50 mM of NaHCO3 also helps the growth of the modified cyanobacteria, thereby increasing the yield of L-serine.

[0053] In the filtration step S30, membrane filtration can be used to achieve this. For example, the culture solution or reaction solution can be introduced into a backwash filter, and filtered using a 0.45 μm filter membrane to remove the cells of the modified cyanobacteria and obtain a filtrate containing serine or cephalin. The filtered modified cyanobacteria can be recycled to the fermentation photoreaction tank for reuse in the fermentation light treatment, thereby saving process costs.

[0054] Specifically, in one embodiment, the culture solution can be filtered first to remove the cells of the cyanobacteria, and the filtered culture solution, lecithin, and phospholipase can be mixed to perform the cephalin synthesis step. In this embodiment, the reaction of the modified cyanobacteria converting the carbon source into serine and the cephalin synthesis reaction can be prevented from interfering with each other. In another embodiment, lecithin and phospholipase can be directly added to the culture solution of the modified cyanobacteria to perform cephalin synthesis, and the reaction can be carried out in one pot to save equipment costs.

[0055] In the cephalin synthesis step of step S40, the modified cyanobacteria and the L-serine produced by them are mixed with lecithin and phospholipase in the photoreaction tank, or the filtered culture solution containing L-serine (without the cells of the modified cyanobacteria) is mixed with lecithin and phospholipase in the photoreaction tank to obtain a cephalin mixture. In one embodiment of the present invention, the temperature of the fermentation light treatment can be 30 to 41 °C, for example, any temperature between 30 and 41 such as 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 °C, etc. It is worth mentioning that, as Figure 5 shown, the optimal temperature for the fermentation light treatment can be 38 °C to obtain the best cell growth.

[0056] Furthermore, the phospholipase of the present invention is added in the form of a magnetic catalyst. The preparation method of the magnetic catalyst includes: (A) preparing magnetic nanoparticles (MNP); (B) surface modification; and (C) enzyme immobilization, which are described in detail as follows.

[0057] (A) Preparation of magnetic nanoparticles

[0058] 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 into a 500 mL serum bottle, and stir in a water bath at 90 °C for 120 minutes. After collecting the generated MNP by magnetic separation, rinse it alternately with the same volume of deionized water and 95% alcohol three times. After each washing, separate and adsorb the MNP with a magnet for about 5 minutes, and then aspirate and remove the washing solution.

[0059] (B) Surface modification

[0060] After adjusting the concentration of the prepared MNP to 10 mg / mL, aspirate 1 mL and place it in a microcentrifuge tube. After collecting and adsorbing the MNP with a magnet for about 5 minutes, remove the supernatant, and then rinse the MNP three times with 1 mL of deionized water to completely remove the alcohol. After each washing, collect and adsorb the MNP with a magnet for about 5 minutes, and then remove the supernatant. After removing the alcohol, add 500 μL of deionized water to prepare an MNP suspension.

[0061] Weigh 2.5 mg of Dopamine hydrochloride (DA), place it in a small glass bottle, and add 500 μL of deionized water to stir and dissolve to make a dopamine hydrochloride solution. Add the MNP suspension to the dopamine hydrochloride solution, then add 1 μL of 10 N NaOH to adjust the pH to 8.5, and then stir for 3 hours. Since the pH will drop to 7 after stirring for about 2 hours, NaOH must be added again to adjust the pH back to 8.5. After collecting the modified DA-MNP with a magnet, rinse it 5 times with 1 mL of deionized water. After each washing, separate and collect the DA-MNP with a magnet for about 5 minutes, and then remove the supernatant. After washing, resuspend the volume of DA-MNP with deionized water to 1 mL, and ultrasonically treat it for 15 minutes, and then store it at 4 °C.

[0062] (C) Immobilization of Phospholipase D

[0063] Mix DA-MNP and phospholipase D in different ratios in 5 mM phosphate buffer (pH 6.8), and then let it stand for 5 minutes. Subsequently, use a magnet to adsorb and separate the DA-MNP for 5 minutes, and repeat the rinsing and magnetic separation with ultrapure water three times. Then, dissolve the DA-MNP coated with phospholipase D in the same volume of ultrapure water for later use.

[0064] In one embodiment of the present invention, 1 mol of lecithin and 0.05 - 5 wt% (relative to lecithin) of nanomagnetic beads are added to 1 L of cyanobacteria / L-serine (containing 5 mol of serine) to replace the trimethylethanaminium functional group in lecithin through the catalysis of phospholipase to produce cephalin. Specifically, it is shown in Formula 2 below.

[0065]

[0066] It is worth mentioning that in the present invention, through pH adjustment, the cephalin obtained after the reaction is immiscible with water, and can be purified by an oil-water separation system. Specifically, the pH of the filtrate obtained through filtration treatment is 1 to 3.

[0067] Therefore, in the step S50 of oil-water separation treatment, the filtrate can be placed in an oil-water separator to stand for stratification. The oil-water separator has a partition plate, so that the upper organic phase overflows to the right side of the separator through the partition plate, and the lower aqueous phase remains in the oil-water separator, achieving the purpose of oil-water separation and obtaining the cephalin in the organic phase. In addition, the separated aqueous phase can be recycled for preparing an aqueous NaHCO3 solution for repeated use, saving process costs. In one embodiment of the present invention, the time for standing stratification is 10 minutes or more.

[0068] 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 when cultured in an environment of 38 °C, 3% CO2, and 25 mM NaHCO3 for 60 hours. Further, when soybean lecithin and L-serine are reacted in a ratio of 1:5 in an environment of 10 mM Ca 2+ ions and 45 °C for 12 hours, 1.86 g / L of cephalin can be produced, and the conversion rate is about 83.2%. That is, when the weight ratio of soybean lecithin to L-serine is 1:5, the conversion rate of converting serine from modified cyanobacteria into cephalin can be further increased, thereby improving economic benefits.

[0069] Beneficial effects of the embodiment

[0070] One of the beneficial effects of the present invention is that the method for producing cephalin using a carbon source provided by the present invention can produce cephalin using L-serine produced from the carbon source as a reaction precursor through the technical solutions of "culturing a modified cyanobacteria in an environment containing a carbon source so that the modified cyanobacteria convert the carbon source into serine" and "the modified cyanobacteria include the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3", improving the economic benefits of waste gas treatment.

[0071] Furthermore, the present invention utilizes a modified cyanobacteria strain that includes the sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, enabling it to exhibit the characteristic of converting a carbon source into serine. Specifically, in an environment with sufficient carbon source, the modified cyanobacteria of the present invention can produce 35.2 mg / L to 46.3 mg / L of serine per hour. Moreover, when lecithin and L-serine are formulated in 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% can be achieved. In addition, the present invention modifies phospholipase on a magnetic catalyst, allowing the direct introduction of phospholipase into the fermentation photoreaction tank for a one-pot reaction. The operation process is simple and recyclable, thereby saving process costs.

[0072] The content disclosed above is only a preferred and feasible embodiment of the present invention, and does not limit the scope of protection of the claims of the present invention. Therefore, all equivalent technical changes made by using the content of the specification and drawings of the present invention are included in the scope of protection of the claims of the present invention.

Claims

1. A method for producing cephalin using a carbon source, characterized in that: The method for producing cephalin using a carbon source comprises: Cultivating the modified cyanobacteria in a culture solution; Fermentation light treatment, providing a carbon source to the modified cyanobacteria, so that the modified cyanobacteria converts the carbon source into serine; Filtration treatment, filtering the culture solution to separate the modified cyanobacteria and obtain a filtered culture solution; Synthesis of phospholipids, mixing the filtered culture fluid, the serine, phosphatidylcholine and phospholipase in a photoreaction tank to obtain a phospholipid mixture; and Oil-water separation treatment, placing the cephalin mixture in an oil-water separator and letting it stand for a predetermined time, taking the upper layer to obtain the cephalin; Wherein, the modified cyanobacteria include the gene sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:

3.

2. The method for producing cephalin using a carbon source according to claim 1, characterized in that: The modified blue-green bacteria is obtained by the following steps: Synthesize DNA sequences; inserting the DNA sequence into a plasmid; and The plasmid is implanted into cyanobacteria via electroporation to obtain the modified cyanobacteria.

3. The method for producing cephalin using a carbon source according to claim 2, characterized in that: The electroporation treatment is performed at a voltage of 0.5 to 1.5 kV for 2 to 10 mSec.

4. The method for producing cephalin using a carbon source according to claim 2, characterized in that: The electroporation treatment further comprises adding polyethylene glycol at a concentration of 0.5 to 2%.

5. The method for producing cephalin using a carbon source according to claim 1, characterized in that: The culture solution includes 25 to 50 mM sodium bicarbonate.

6. The method for producing cephalin using a carbon source according to claim 1, characterized in that: The cyanobacteria are Synechococcus elongates.

7. The method for producing cephalin using a carbon source according to claim 1, characterized in that: The phospholipase is a magnetic catalyst.

8. The method for producing cephalin using a carbon source according to claim 1, characterized in that: The temperature of the fermentation light treatment is 30 to 41°C.

9. The method for producing cephalin using a carbon source according to claim 1, characterized in that: The modified cyanobacteria has the ability to produce 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase and phosphoserine aminotransferase.

10. The method for producing cephalin using a carbon source according to claim 1, characterized in that: The carbon source is carbon dioxide, glucose, sucrose, fructose or galactose.

11. A method for producing cephalin using a carbon source, characterized in that: The method for producing cephalin using a carbon source comprises: Fermentation light treatment, using modified cyanobacteria to produce serine; Synthesis of phospholipids: mixing the serine, phosphatidylcholine and phospholipase in a photoreaction tank to obtain a phospholipid mixture; Filtration treatment, passing the cephalin mixture through a filter membrane to obtain a filtrate; and The filtrate is placed in an oil-water separator for a predetermined period of time, and the upper layer is taken to obtain the cephalin.

12. The method for producing cephalin using a carbon source according to claim 11, characterized in that: The modified cyanobacteria include gene sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:

3.

13. The method for producing cephalin using a carbon source according to claim 11, characterized in that: The weight ratio of the lecithin to the serine is 1:

5.

14. The method for producing cephalin using a carbon source according to claim 11, characterized in that: The pore size of the filter membrane is 0.45 μm.

15. The method for producing cephalin using a carbon source according to claim 11, characterized in that: The predetermined time is more than 10 minutes.

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