Process for converting carbon source to serine
By implanting specific gene sequences into cyanobacteria and electroporation, the modified cyanobacteria was obtained, which solved the problem that the prior art could not convert the carbon source into serine, and achieved efficient and low-cost serine production.
Patent Information
- Application Number
- CN202410026926.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-12-27
- Filing Date
- 2024-01-08
- Publication Date
- 2025-06-27
AI Technical Summary
The prior art cannot convert carbon sources into serine using cyanobacteria, resulting in the inability to be used in production, and existing methods for manufacturing serine have high costs and contamination problems.
By synthesizing DNA sequences, implanting them into the plasmid, making the plasmid include a specific gene sequence, and implanting them into the cyanobacteria by electroporation, obtaining a modified cyanobacteria and providing a carbon source, so that the modified cyanobacteria converts the carbon source into serine.
The use of modified cyanobacteria to convert carbon sources into L-serine is achieved, reducing production costs, avoiding pollution, and improving the purity of the product.
Smart Images
Figure CN120210254A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for converting a carbon source into serine, and particularly to a method for converting a carbon source into serine by using cyanobacteria. 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, cyanobacteria, which have the ability to fix carbon dioxide into metabolites, 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 a carbon source into serine, cyanobacteria cannot be applied to the production of serine in the prior art.
[0003] Serine can promote the metabolism of fats and fatty acids and helps maintain the immune system, thus having a wide range of uses in medicine. The methods for manufacturing serine in the prior art mainly include fermentation methods, protein hydrolysis methods, chemical synthesis methods, etc. However, these methods still have many drawbacks, resulting in limited applications. For example, the fermentation method uses glycine as a raw material for fermentation, and the protein hydrolysis method uses natural proteins as raw materials, and the obtained products are mixtures of various amino acids, and further purification and separation steps are still required, resulting in high process costs. The chemical synthesis method has high production costs, serious pollution, and it is not easy to separate the simultaneously produced D-serine and L-serine.
[0004] Therefore, how to improve the process to use cyanobacteria to convert a carbon source into serine, so as to produce high-economic-value chemicals while treating carbon-containing waste gas, 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 converting a carbon source into serine in view of the deficiencies of the prior art.
[0006] To solve the above technical problem, one of the technical solutions adopted by the present invention is to provide a method for converting a carbon source into serine, the method comprising: synthesizing a DNA sequence; implanting the DNA sequence into a plasmid such that the plasmid comprises the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; implanting the plasmid into cyanobacteria via electroporation treatment to obtain modified cyanobacteria; and providing the carbon source to the modified cyanobacteria such that the modified cyanobacteria convert the carbon source into serine.
[0007] Furthermore, the plasmid is an Escherichia coli plasmid.
[0008] Further, the method further includes the step of implanting the plasmid into Escherichia coli for mass production.
[0009] Further, the cyanobacterium is Synechococcus elongatus.
[0010] Further, the modified cyanobacterium has the ability to produce 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase, and phosphoserine transaminase.
[0011] Further, the electroporation treatment is performed at a voltage of 0.5 to 1.5 kV for 2 to 10 mSec.
[0012] Further, the electroporation treatment further includes adding polyethylene glycol at a concentration of 0.5 to 2%.
[0013] Further, the serine is L-serine.
[0014] Further, the carbon source is carbon dioxide, glucose, sucrose, fructose, or galactose.
[0015] To solve the above technical problems, another technical solution adopted by the present invention is to provide a method for converting a carbon source into serine, which uses a modified cyanobacterium to convert the carbon source into serine; wherein, the modified cyanobacterium includes the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
[0016] Further, the serine is L-serine.
[0017] Further, the carbon source is carbon dioxide, glucose, sucrose, fructose, or galactose.
[0018] Further, the modified cyanobacterium converts the carbon source into glyceraldehyde 3-phosphate (G3P) and has the ability to produce 3-phosphoglycerate dehydrogenase (Ser A) to convert the glyceraldehyde 3-phosphate (G3P) into 3-phosphohydroxypyruvate (3P-HP).
[0019] Further, the modified cyanobacterium has the ability to produce phosphoserine transaminase (Ser C) to convert the 3-phosphohydroxypyruvate (3P-HP) into phosphoserine (3P-Serine).
[0020] Further, the modified cyanobacterium has the ability to produce phosphoserine phosphatase (Ser B) to convert the phosphoserine (3P-Serine) into the serine.
[0021] One of the beneficial effects of the present invention is that the method for converting a carbon source into serine provided by the present invention can utilize the technical solutions of "modifying the gene sequences of cyanobacteria including SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3" and "providing the carbon source to the modified cyanobacteria" to convert the carbon source into L-serine by using the modified cyanobacteria, thereby achieving the benefits of carbon reduction while obtaining high-economic-value chemicals.
[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 intended to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 It is a flowchart of the method for converting a carbon source into serine 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] Reference numerals: S1 to S4: steps. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0027] The following are specific examples to illustrate the embodiments of the "method for converting a carbon source into serine" disclosed by the present invention. 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 dimensions, which is stated in advance. The following embodiments will further detail the related technical content of the present invention, but the disclosed content is not intended to limit the protection scope of the present invention.
[0028] 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 related 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.
[0029] As used herein, the term "exogenous gene" may also be referred to as a heterologous gene, which refers to a gene or nucleotide fragment that is not from the endogenous genome of 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 through genetic engineering techniques.
[0030] Refer to Figures 1 to 3 As shown, 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 through 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.
[0031] In Figure 2 and Figure 3 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 the ribosome-binding site.
[0032] In step S1 of synthesizing the DNA sequence, it refers to artificially synthesizing a DNA sequence with a recognition degree suitable for cyanobacteria for cyanobacteria to recognize and produce corresponding substances. In particular, a gene encoding suitable for recognition by Synechococcus elongates PCC7942 is synthesized. Further, the artificially synthesized DNA sequence can be mass-produced by polymerase chain reaction (PCR). In the 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 conditions of polymerization at 72°C for 10 minutes.
[0033] In addition, plasmid DNA was mass-produced and isolated from the native Escherichia coli DH5α strain, and in step S2 of implanting the DNA sequence into the plasmid, the designed DNA sequence was introduced into the plasmid of Escherichia coli, and the modified Escherichia coli was replicated and mass-produced to obtain a recombinant plasmid, which was 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 further taken out and transferred to the native cyanobacteria.
[0034] 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, and is integrated into the genome of cyanobacteria for expression through homologous recombination. Subsequently, antibiotics were used to screen for the successfully homologous recombination to obtain the modified cyanobacteria with SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In other words, the successfully modified cyanobacteria strains can grow on a solid medium containing antibiotics.
[0035] In step S3 of implanting the plasmid into cyanobacteria, the cyanobacteria can be cultured in BG11 medium, and the growth concentration of the strain can be measured by OD 730 Furthermore, the plasmid was implanted into cyanobacteria via electroporation to obtain modified cyanobacteria. Electroporation is to apply an electric current to the cyanobacteria cells within a very short time (microseconds to milliseconds), making them in an environment of high voltage and low capacitance. The cell membrane will generate a potential difference, causing a change in the cell membrane structure, resulting in the cell membrane being compressed and thinned, and then numerous tiny pores are generated, allowing the plasmid to pass through the cell membrane and enter the cells of cyanobacteria.
[0036] 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 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 cyanobacteria are 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 studied the amount of the successfully obtained modified cyanobacteria strains of the native cyanobacteria with a quantitative concentration of 1x10 6 under the treatment conditions of different voltages and times, as shown in Table 1 below.
[0037] Table 1 (1% PEG was added to each group)
[0038] 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
[0039] Based on the results in Table 1 above, in the case of adding 1% PEG, the electroporation treatment in the present invention is preferably carried out on cyanobacteria 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.
[0040] 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. In order to utilize cyanobacteria to process carbon sources and convert carbon sources into serine, the present invention prepares modified cyanobacteria.
[0041] In the present invention, the carbon source can 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 can 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).
[0042] 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 (SerA), phosphoserine phosphatase (SerB) and phosphoserine aminotransferase (SerC), and can independently carry out the following reaction of formula 1 to convert G3P into L-serine.
[0043]
[0044] The modified cyanobacteria of the present invention have multiple exogenous genes, which include the nucleotide sequence encoding the 3-phosphoglycerate dehydrogenase (SerA) gene, the nucleotide sequence encoding the phosphoserine phosphatase (SerB) gene, and the nucleotide sequence encoding the phosphoserine transaminase (SerC) gene, and these genes can be expressed or overexpressed in the modified cyanobacteria. In other words, the modified cyanobacteria of the present invention have an expression plasmid including SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
[0045] Specifically, the modified cyanobacteria of the present invention still retain the characteristic of the native cyanobacteria to convert a carbon source into glyceraldehyde 3-phosphate (G3P). Moreover, the modified cyanobacteria of the present invention can also produce 3-phosphoglycerate dehydrogenase (SerA), and thus can convert glyceraldehyde 3-phosphate (G3P) into 3-phosphohydroxypyruvate (3P-HP). Furthermore, the modified cyanobacteria of the present invention can also produce phosphoserine transaminase (SerC), and thus can convert 3-phosphohydroxypyruvate into phosphoserine (3P-Serine). In addition, the modified cyanobacteria of the present invention can also produce phosphoserine phosphatase (SerB), and thus can convert phosphoserine (3P-Serine) into serine, especially L-serine (L-Serine).
[0046] Therefore, the modified cyanobacteria of the present invention have the ability to convert a carbon source into L-serine and release it extracellularly, and L-serine can be obtained without cell disruption treatment. For example, the carbon source can be carbon dioxide, glucose, sucrose, fructose, or galactose. However, the present invention is not limited to the above examples. 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 as to obtain an economically beneficial chemical while treating industrial waste with the modified cyanobacteria.
[0047] 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.
[0048] Accordingly, the modified cyanobacteria can convert a carbon source into glyceraldehyde 3-phosphate (G3P). The cyanobacteria can also utilize the expression of the sequence of SEQ ID NO:1 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 sequence of SEQ ID NO:3 to produce phosphoserine transaminase (SerC) to convert 3-phosphohydroxypyruvate (3P-HP) into phosphoserine (3P-Serine). The cyanobacteria can also utilize the expression of the sequence of SEQ ID NO:2 to produce phosphoserine phosphatase (SerB) to convert phosphoserine (3P-Serine) into the serine.
[0049] Beneficial effects of the examples
[0050] One of the beneficial effects of the present invention is that the method for converting a carbon source into serine provided by the present invention can utilize the technical solutions of "the modified cyanobacteria include the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3" and "providing the carbon source to the modified cyanobacteria" to be able to convert the carbon source into L-serine by using the modified cyanobacteria, thus realizing the benefits of reducing carbon while obtaining high-economic chemicals.
[0051] Furthermore, the present invention utilizes a modified cyanobacteria strain, which includes the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and can exhibit the characteristic of converting a carbon source into serine. Specifically, culturing the modified cyanobacteria of the present invention at 38 °C, 3% CO2, and 25 mM NaHCO3 for 60 hours can produce up to 2.78 g / L of L-serine. In addition, since the modified cyanobacteria of the present invention can only produce L-serine and do not require the separation of D-serine and L-serine, compared with the chemical synthesis method, the method for converting a carbon source into serine by using the modified cyanobacteria of the present invention can also reduce the process steps of separating D-serine and L-serine.
[0052] The content disclosed above is only the preferred feasible embodiments of the present invention, and does not limit the protection scope 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 protection scope of the claims of the present invention.
Claims
1. A method for converting a carbon source into serine, characterized in that: The method for converting a carbon source into serine comprises: Synthesize DNA sequences; Implanting the DNA sequence into a plasmid, so that the plasmid includes the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; implanting the plasmid into cyanobacteria via electroporation to obtain modified cyanobacteria; and A carbon source is provided to the modified cyanobacteria, so that the modified cyanobacteria converts the carbon source into serine.
2. The method for converting a carbon source into serine according to claim 1, characterized in that The plasmid is an Escherichia coli plasmid.
3. The method for converting a carbon source into serine according to claim 1, characterized in that: The method further comprises the step of implanting the plasmid into Escherichia coli for mass production.
4. The method for converting a carbon source into serine according to claim 1, characterized in that The cyanobacteria are Synechococcus elongates.
5. The method for converting a carbon source into serine according to claim 1, characterized in that: The modified cyanobacteria has the ability to produce 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase and phosphoserine aminotransferase.
6. The method for converting a carbon source into serine according to claim 1, characterized in that: The electroporation treatment is performed at a voltage of 0.5 to 1.5 kV for 2 to 10 mSec.
7. The method for converting a carbon source into serine according to claim 1, characterized in that: The electroporation treatment further comprises adding polyethylene glycol at a concentration of 0.5 to 2%.
8. The method for converting a carbon source into serine according to claim 1, characterized in that: The serine is L-serine.
9. The method for converting a carbon source into serine according to claim 1, characterized in that: The carbon source is carbon dioxide, glucose, sucrose, fructose or galactose.
10. A method for converting a carbon source into serine, characterized in that: The method utilizes modified cyanobacteria to convert a carbon source into serine; wherein the modified cyanobacteria comprises gene sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:
3.
11. The method for converting a carbon source into serine according to claim 10, characterized in that: The serine is L-serine.
12. The method for converting a carbon source into serine according to claim 10, characterized in that: The carbon source is carbon dioxide, glucose, sucrose, fructose or galactose.
13. The method for converting a carbon source into serine according to claim 10, characterized in that: The modified cyanobacterium converts the carbon source into glyceraldehyde 3-phosphate and has the ability to produce 3-phosphoglycerate dehydrogenase to convert the glyceraldehyde 3-phosphate into 3-phosphohydroxypyruvate.
14. The method for converting a carbon source into serine according to claim 10, characterized in that: The modified cyanobacterium has the ability to produce phosphoserine aminotransferase to convert 3-phosphohydroxypyruvate into phosphoserine.
15. The method for converting a carbon source into serine according to claim 10, characterized in that: The modified cyanobacterium has the ability to produce phosphoserine phosphorylase to convert phosphoserine into the serine.