Method for converting carbon source into serine

Abstract

To provide a method for converting a carbon source into serine.SOLUTION: A method for converting a carbon source into serine includes a step of synthesizing a DNA sequence, a step of implanting the DNA sequence into a plasmid so that the plasmid includes specific gene sequences, a step of implanting the plasmid into cyanobacteria through electroporation treatment to obtain modified cyanobacteria, and a step of providing the carbon source to the modified cyanobacteria so that the modified cyanobacteria convert the carbon source into serine.SELECTED DRAWING: Figure 1

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 using cyanobacteria.

Background Art

[0002] Cyanobacteria are autotrophic organisms that perform photosynthesis and synthesize nutrients required for themselves. In order to mitigate the greenhouse effect and reduce the impact on the environment, in existing technologies, the ability of cyanobacteria to fix carbon dioxide into metabolites is utilized, and cyanobacteria are used to produce alcohols such as ethanol, butanol, 2,3-butanediol, succinic acid, lactic acid, isopropene, and organic acids. However, cyanobacteria lack the ability to convert a carbon source into serine, and the production of serine using cyanobacteria is impossible with existing technologies.

[0003] Serine is widely used in the pharmaceutical field because it promotes the metabolism of fats and fatty acids and helps maintain the immune system. Existing methods for producing serine mainly include fermentation methods, protein hydrolysis methods, chemical synthesis methods, etc. However, these methods have many drawbacks and their applications are limited. For example, the fermentation method ferments using glycine as a raw material, and the protein hydrolysis method uses natural proteins as raw materials. The obtained products are mixtures of multiple amino acids, and further purification and separation steps are required, resulting in high production costs. The chemical synthesis method has high production costs, serious pollution, and difficulty in separating D-serine and L-serine.

[0004] Therefore, through process improvement, a method for converting a carbon source into serine using cyanobacteria and producing high-economic-value chemicals while treating carbon emission gases is one of the important issues to be solved in this field.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The technical problem to be solved by the present invention is to remedy the deficiencies of the existing technology and provide a method for converting a carbon source into serine.

Means for Solving the Problem

[0006] In order 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 the steps of synthesizing a DNA sequence, introducing the DNA sequence into a plasmid such that the plasmid contains the gene sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, introducing the plasmid into cyanobacteria by electroporation to obtain modified cyanobacteria, providing the carbon source to the modified cyanobacteria such that the modified cyanobacteria convert the carbon source into serine.

[0007] In one embodiment of the present invention, the plasmid is an E. coli plasmid.

[0008] In one embodiment of the present invention, the method further comprises the step of introducing the plasmid into E. coli for mass production.

[0009] In one embodiment of the present invention, the cyanobacteria are Synechococcus elongatus.

[0010] In one embodiment of the present invention, the modified cyanobacteria have the ability to produce 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase and phosphoserine transferase.

[0011] In one embodiment of the present invention, the electroporation is performed at a voltage of 0.5 to 1.5 kV for 2 to 10 mSec.

[0012] In one embodiment of the present invention, the electroporation further comprises adding polyethylene glycol at a concentration of 0.5% to 2%.

[0013] In one embodiment of the present invention, the serine is L-serine.

[0014] In one embodiment of the present invention, 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 by using modified cyanobacteria, and the modified cyanobacteria includes the gene sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0016] In one embodiment of the present invention, the serine is L-serine.

[0017] In one embodiment of the present invention, the carbon source is carbon dioxide, glucose, sucrose, fructose or galactose.

[0018] In one embodiment of the present invention, the modified cyanobacteria converts the carbon source into glyceraldehyde 3-phosphate (G3P), has the production ability of glyceraldehyde 3-phosphate dehydrogenase (SerA), and converts the glyceraldehyde 3-phosphate (G3P) into 3-phosphohydroxypropionic acid (3P-HP).

[0019] In one embodiment of the present invention, the modified cyanobacteria has the ability to produce phosphoserine transferase (SerC), and converts the 3-phosphohydroxypropionic acid (3P-HP) into phosphoserine (3P-Serine).

[0020] In one embodiment of the present invention, the modified cyanobacteria has the ability to produce phosphoserine phosphatase (SerB), and converts the phosphoserine (3P-Serine) into the serine.

[0021] One beneficial effect of the present invention is to provide a method for converting a carbon source into serine. The method includes the technical solutions of "the modified cyanobacterium contains 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 cyanobacterium", and uses the modified cyanobacterium to convert the carbon source into L-serine, achieving the effect of obtaining chemicals with high economic value while performing carbon reduction.

[0022] To better understand the features and technical content of the present invention, the following provides a detailed description of the present invention with reference to the accompanying drawings. However, the provided accompanying drawings are only for reference and explanation purposes and do not limit the scope of the patent claims of the present invention.

Brief Description of the Drawings

[0023]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0024] The following describes the embodiments disclosed by the present invention. Those skilled in the art can understand the merits and effects of the present invention based on the disclosure in this specification. The present invention can be implemented or applied in other different embodiments. Each detail in this specification can also be equally modified and varied based on various viewpoints or applications without departing from the spirit of the present invention. Also, the drawings of the present invention are for simple and schematic illustration purposes and do not show actual dimensions. In the following embodiments, the technical matters related to the present invention are 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 multiple items according to the actual situation.

[0025] It should be understood that the term "or" as used herein may, in actual circumstances, include any one or a combination of multiple items listed in relation thereto. 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 not excluding 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" may be used interchangeably.

[0026] The term "exogenous gene" used herein, also called a heterologous gene, refers to a gene or nucleic acid fragment that is not part of 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 such 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 the synthesis of 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 the identification of the long and spherical cyanobacterium Synechococcus elongates PCC7942 is synthesized for gene code recognition. Furthermore, the artificially synthesized DNA sequence can be mass-produced using the polymerase chain reaction (PCR). In an 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 polymerization at 72° C. for 10 minutes is performed for PCR amplification.

[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 produced in large quantities 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, using antibiotic selection, cyanobacteria that have successfully undergone homologous recombination and have SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 are screened and obtained. In other words, cyanobacterial strains that have successfully undergone modification can grow on solid media 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 modified cyanobacteria. Electroporation applies an electric current to cyanobacterial cells for a very short time (from microseconds to milliseconds), creating a potential difference across the cell membrane in a high-voltage and low-volume environment, thereby changing the structure of the cell membrane. As a result, the cell membrane is compressed and thinned, creating numerous tiny pores through which the plasmid can pass into the cyanobacterial cells.

[0033] To achieve the optimal plasmid permeation effect, in this step, 0.5% to 2% polyethylene glycol (PEG) can be further added. For example, 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 to 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 to 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. In the present invention, furthermore, during the electroporation process, 1×10 6Research was conducted on treating native cyanobacteria under different voltage and time conditions to obtain the amount of modified cyanobacterial strains that were successful. The results are shown in Table 1 below.

[0034]

Table 1

[0035] Based on the results in Table 1 above, under the condition of adding 1% PEG, 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, and the maximum number of colonies can be obtained.

[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 the carbon source. This gas is a mixture of hydrogen, acetylene, methane, hydrogen sulfide, and acetaldehyde. Further, 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 carry out 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 sequence of the gene of 3-phosphoglycerate dehydrogenase (SerA), the nucleic acid sequence of the gene of phosphoserine phosphatase (SerB), and the nucleic acid sequence of the gene of 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 property of the native cyanobacteria to convert a carbon source into glyceraldehyde 3-phosphate (G3P). Further, the modified cyanobacteria of the present invention produce 3-phosphoglycerate dehydrogenase (SerA), thereby enabling the conversion of glyceraldehyde 3-phosphate (G3P) into 3-phosphohydroxypyruvate (3P-HP). Additionally, the modified cyanobacteria of the present invention produce phosphoserine transferase (SerC), which can convert 3-phosphohydroxypyruvate into phosphoserine (3P-Serine). Moreover, 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 and release it into the extracellular space without the need for a bacteriophage lysis step to obtain 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, thereby treating industrial waste using the modified cyanobacteria while obtaining a chemically valuable substance.

[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 corresponding modified cyanobacteria contain the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In other words, the corresponding modified cyanobacteria contain the expression plasmids of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

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

[0044] One beneficial effect of the present invention is that in the method for converting a carbon source into serine provided by the present invention, 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" and "providing a carbon source to the corresponding modified cyanobacteria", the modified cyanobacteria have the ability to convert the carbon source into L-serine. Thereby, the effect of obtaining economically valuable chemicals while reducing carbon dioxide is realized.

[0045] More specifically, the present invention utilizes a modified cyanobacterial strain that contains the gene sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 and expresses the property of converting a carbon source into serine. Specifically, when the modified cyanobacteria of the present invention are cultured for 60 hours under the conditions of 38°C, 3% carbon dioxide, and 25 mM sodium bicarbonate, up to 2.78 g / L of L-serine can be produced. Furthermore, the modified cyanobacteria of the present invention produce only L-serine and there is no need to separate D-serine and L-serine. Therefore, compared with the chemical synthesis method, the method for converting a carbon source into serine using the modified cyanobacteria of the present invention can reduce the steps for separating D-serine and L-serine.

[0046] The content disclosed above is only a preferred feasible 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 Reference Numerals

[0047] Steps S1 to S4

Claims

1. Synthesizing a DNA sequence, introducing the DNA sequence into a plasmid such that the plasmid contains the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, introducing the plasmid into cyanobacteria by electroporation to obtain modified cyanobacteria, providing a carbon source to the modified cyanobacteria such that the modified cyanobacteria convert the carbon source into serine, A method for converting a carbon source into serine, comprising the above steps.

2. The method according to claim 1, wherein the plasmid is an E. coli plasmid.

3. The method according to claim 1, further comprising the step of introducing the plasmid into E. coli for mass production.

4. The method according to claim 1, wherein the cyanobacteria are elongated spherical cyanobacteria.

5. The method according to claim 1, wherein the modified cyanobacteria have the ability to produce 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase, and phosphoserine transferase.

6. The method according to claim 1, wherein in the electroporation treatment, the treatment is performed at a voltage of 0.5 to 1.5 kV for 2 to 10 mSec.

7. The method according to claim 1, wherein the electroporation treatment further comprises adding 0.5% to 2% polyethylene glycol.

8. The method according to claim 1, wherein the serine is L-serine.

9. The method according to claim 1, wherein the carbon source is carbon dioxide, glucose, sucrose, fructose, or lactose.

10. A method for converting a carbon source into serine using modified cyanobacteria, wherein the modified cyanobacteria contain the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:

3.

11. The method according to claim 10, wherein the serine is L-serine.

12. The method according to claim 10, wherein the carbon source is carbon dioxide, glucose, sucrose, fructose, or lactose.

13. The method according to claim 10, wherein the modified cyanobacterium has the ability to convert the carbon source into glyceraldehyde 3-phosphate (G3P) and produce 3-phosphoglycerate dehydrogenase (SerA), and converts the glyceraldehyde 3-phosphate (G3P) into 3-phosphohydroxypyruvate (3P-HP).

14. The method according to claim 13, wherein the modified cyanobacterium has the ability to produce phosphoserine transferase (SerC), and converts the 3-phosphohydroxypyruvate (3P-HP) into 3-phosphoserine (3P-Serine).

15. The method according to claim 14, wherein the modified cyanobacterium has the ability to produce phosphoserine phosphatase (SerB), and converts the 3-phosphoserine (3P-Serine) into the serine.

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