Method for converting carbon sorce into serine

Abstract

This invention discloses a method for converting a carbon source into serine. The method includes synthesizing a DNA sequence; inserting the DNA sequence into a plasmid, such that the plasmid includes gene sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; inserting the plasmid into a cyanobacterium via electroporation to obtain a modified cyanobacterium; and providing the modified cyanobacterium with the carbon source, enabling the modified cyanobacterium to convert the carbon source into serine.

Description

Technical Field

[0001] The present invention relates to a method for converting a carbon source into serine, and in particular to a method for converting a carbon source into serine using cyanobacteria. Prior Art

[0002] Cyanobacteria are self-sustaining organisms capable of synthesizing their own nutrients through photosynthesis. To mitigate greenhouse gas emissions and environmental damage, existing technologies utilize cyanobacteria's ability to fix carbon dioxide into metabolic products and apply them to the production of alcohols and organic acids such as ethanol, butanol, 2,3-butanediol, succinic acid, lactic acid, and isopropylene. However, due to their lack of ability to convert carbon sources into serine, existing technologies have limited their application in serine production.

[0003] Serine promotes the metabolism of fats and fatty acids, helps maintain the immune system, and has a wide range of medical applications. Existing methods for producing serine primarily include fermentation, protein hydrolysis, and chemical synthesis. However, these methods still have numerous drawbacks, limiting their application. For example, fermentation uses glycine as a raw material, while protein hydrolysis uses natural protein as a raw material. The resulting product is a mixture of various amino acids, requiring further purification and separation steps, resulting in high production costs. Chemical synthesis methods are costly and polluting, and the simultaneous production of D-serine and L-serine is difficult to separate.

[0004] Therefore, how to improve the process and utilize cyanobacteria to convert carbon sources into serine, thereby treating carbon-containing waste gas while producing high-value chemicals, has become one of the key issues that this industry aims to address. 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 response to the deficiencies of the existing technology.

[0006] To solve the above-mentioned technical problems, one of the technical solutions adopted by the present invention is to provide a method for converting a carbon source into serine, comprising: synthesizing a DNA sequence; implanting the DNA sequence into a plastid, so that the plastid includes the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; implanting the plastid into a cyanobacterium via electroporation to obtain a modified cyanobacterium; and providing the carbon source to the modified cyanobacterium, so that the modified cyanobacterium converts the carbon source into serine.

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

[0008] In one embodiment of the present invention, the method further comprises the step of implanting the plasmid into Escherichia 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 has the ability to produce 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase and phosphoserine transaminase.

[0011] In one 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.

[0012] In one embodiment of the present invention, the electroporation treatment 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] In order to solve the above-mentioned technical problems, another technical solution adopted by the present invention is to provide a method for converting a carbon source into serine, which utilizes a modified cyanobacterium to convert a 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] 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) and has the ability to produce 3-phosphoglycerate dehydrogenase (Ser A) to convert the glyceraldehyde 3-phosphate (G3P) into 3-phosphohydroxypyruvate (3P-HP).

[0019] In one embodiment of the present invention, the modified cyanobacteria has the ability to produce phosphoserine aminotransferase (Ser C) to convert the 3-phosphohydroxypyruvate (3P-HP) into phosphoserine (3P-Serine).

[0020] In one embodiment of the present invention, the modified cyanobacteria has the ability to produce phosphoserine phosphorylase (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 cyanobacteria to 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 convert the carbon source into L-serine, thereby achieving the benefits of reducing carbon emissions while simultaneously obtaining highly economical 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 drawings provided are for reference and illustration only and are not intended to limit the present invention. Simple diagram description

[0023] FIG1 is a flow chart of the method for converting a carbon source into serine according to the present invention.

[0024] FIG2 is a schematic diagram of the metabolic pathway of the modified cyanobacteria of the present invention.

[0025] FIG3 is a schematic diagram of the plasmid construction of the present invention. Implementation Method

[0026] The following describes specific embodiments of the present invention's "method for converting a carbon source into serine." Those skilled in the art will appreciate the advantages and benefits of the present invention based on the disclosure herein. The present invention may be implemented or applied through various other specific embodiments, and the details herein may be modified and altered based on different perspectives and applications without departing from the scope of the present invention. Furthermore, the drawings herein are for illustrative purposes only and are not intended to be drawn to actual size. The following embodiments further illustrate the relevant technical aspects of the present invention, but the disclosure is not intended to limit the scope of protection of the present invention.

[0027] It should be understood that the term "or" as used herein may include any one or more combinations of the associated listed items, as appropriate. Unless the context requires otherwise, the term "comprising" should be understood to imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps. In this specification, the terms "comprising," "containing," "including," or "having" may be used interchangeably.

[0028] The term "exogenous gene" as used herein, also known as a heterologous gene, refers to a gene or nucleotide fragment that is not derived from the endogenous genome of the host cell or target cell itself, but is taken from another species or cell, or is artificially synthesized and introduced into the host cell or target cell through genetic engineering technology.

[0029] 1 to 3 , a first embodiment of the present invention provides a method for converting a carbon source into serine, comprising: step S1: synthesizing a DNA sequence; step S2: implanting the DNA sequence into a plastid, such that the plastid includes the sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; step S3: implanting the plastid into cyanobacteria via electroporation to obtain modified cyanobacteria; and step S4: providing a carbon source to the modified cyanobacteria, such that the modified cyanobacteria converts the carbon source into serine.

[0030] In Figures 2 and 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; Cytb 6f refers to cytochrome b 6f, which is the core of the light-dependent reactions of oxygenic photosynthesis; RuBP is ribulose-1,5-bisphosphate; CA refers to carbonic anhydrase; and rbs refers to ribosome-binding site.

[0031] In step S1 of synthesizing a DNA sequence, a DNA sequence with a gene encoding a recognition level suitable for cyanobacteria is artificially synthesized, allowing the bacteria to recognize and produce the corresponding substance. Specifically, the gene encoding a gene encoding a gene with a recognition level suitable for recognition by Synechococcus elongates PCC7942 is synthesized. Furthermore, the artificially synthesized DNA sequence can be mass-produced using a polymerase chain reaction (PCR). In one embodiment of the present invention, the PCR conditions may include: denaturation at 98°C for 30 seconds, followed by 30 cycles of denaturation at 98°C for 10 seconds, cold annealing at 56°C for 20 seconds, and polymerization at 72°C for 45 seconds, followed by polymerization at 72°C for 10 minutes.

[0032] Separately, plasmid DNA was mass-produced and isolated from a native E. coli DH5 strain. In step S2, the designed DNA sequence was introduced into the E. coli plasmids for replication and mass production in the modified E. coli, resulting in a recombinant plasmid named pSerSyn. However, E. coli cannot utilize CO₂, and even with the designed DNA sequence, it is unable to convert the carbon source into serine. Therefore, the mass-produced modified plasmid DNA must be removed and colonized in native cyanobacteria.

[0033] Specifically, the plasmid construct used in the present invention can use pSyn_1 as a backbone and carry the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, which are then integrated into the genome of a cyanobacterium via homologous exchange for expression. Subsequently, antibiotics are used to screen for successfully homologous exchange, resulting in modified cyanobacteria expressing SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In other words, successfully modified cyanobacterial strains can be grown on solid media containing antibiotics.

[0034] In step S3 of implanting the plasmid into the cyanobacteria, the cyanobacteria can be cultured in BG11 medium, and the growth concentration of the strain can be measured using OD 730. Furthermore, the plasmid is implanted into the cyanobacteria via electroporation to obtain modified cyanobacteria. Electroporation involves applying an electric current to the cyanobacterial cells for an extremely short period of time (microseconds to milliseconds), exposing them to a high-voltage, low-capacitance environment. This creates a potential difference across the cell membrane, altering its structure and causing it to compress and thin. This creates countless tiny pores, allowing the plasmid to penetrate the cell membrane and enter the cyanobacterial cell.

[0035] To achieve optimal plastid permeabilization, 0.5 to 2% polyethylene glycol (PEG) can be further added during this step, for example, at any concentration between 0.5 and 2%, such as 1.0% or 1.5%. Preferably, electroporation is performed at a voltage of 0.5 to 1.5 kV, for example, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, or 1.4 kV, for 2 to 10 milliseconds, for example, 3, 4, 5, 6, 7, 8, or 9 milliseconds. The present invention further investigated the successful electroporation of modified cyanobacteria using a quantitative concentration of 1x10⁶ native cyanobacteria under different voltage and time conditions, as shown in Table 1 below.

[0036] Table 1 (1% PEG was added to each group) Voltage (kV) Time (mSec) Number of colonies (colony) 0.5 2 6 0.5 5 12 0.5 10 twenty one 1.0 2 19 1.0 5 36 1.0 10 11 1.5 2 17 1.5 5 32 1.5 10 8

[0037] Based on the results in Table 1, when 1% PEG was added, the electroporation treatment in the present invention was preferably performed at a voltage of 0.5 kV 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.

[0038] Generally speaking, native cyanobacteria have the ability to reduce carbon dioxide to glyceraldehyde 3-phosphate (G3P). However, due to a lack of the relevant metabolic enzymes, native cyanobacteria are unable to further metabolize G3P into L-serine. The present invention prepares modified cyanobacteria to utilize cyanobacteria to process carbon sources and convert them into serine.

[0039] In the present invention, the carbon source can be industrial waste gas, i.e., a mixture of hydrogen, acetylene, methane, hydrogen sulfide, and acetaldehyde. Furthermore, the mixture can 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).

[0040] In step S4 of providing a carbon source to the modified cyanobacteria, the modified cyanobacteria of the present invention has the ability to produce 3-phosphoglycerate dehydrogenase (SerA), phosphoserine phosphatase (SerB), and phosphoserine aminotransferase (SerC), and can independently perform the reaction of the following formula 1 to convert G3P into L-serine. Formula 1

[0041] The modified cyanobacteria of the present invention contain multiple exogenous genes, including a nucleotide sequence encoding a 3-phosphoglycerate dehydrogenase (SerA) gene, a nucleotide sequence encoding a phosphoserine phosphatase (SerB) gene, and a nucleotide sequence encoding a phosphoserine aminotransferase (SerC) gene. 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 comprising SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0042] Specifically, the modified cyanobacteria of the present invention retain the native cyanobacteria's ability to convert carbon sources into glyceraldehyde 3-phosphate (G3P). Furthermore, the modified cyanobacteria of the present invention can produce 3-phosphoglycerate dehydrogenase (SerA), which can convert glyceraldehyde 3-phosphate (G3P) into 3-phosphohydroxypyruvate (3P-HP). Furthermore, the modified cyanobacteria of the present invention can produce phosphoserine aminotransferase (SerC), which can convert 3-phosphohydroxypyruvate into phosphoserine (3P-Serine). Furthermore, the modified cyanobacteria of the present invention can produce phosphoserine phosphorylase (SerB), which can convert phosphoserine (3P-Serine) into serine, particularly L-serine (L-Serine).

[0043] Therefore, the modified cyanobacteria of the present invention have the ability to convert carbon sources into L-serine and release it extracellularly, eliminating the need for bacterial disruption to obtain L-serine. For example, the carbon source can be carbon dioxide, glucose, sucrose, fructose, or galactose. However, the present invention is not limited to these examples. Preferably, the modified cyanobacteria of the present invention can utilize carbon sources in carbon-containing industrial waste gas to convert the carbon source in the industrial waste gas into L-serine, thereby simultaneously utilizing the modified cyanobacteria to treat industrial waste and produce economically profitable chemicals.

[0044] Another embodiment of the present invention also provides a method for converting a carbon source into serine, comprising at least using the modified cyanobacteria of the present invention to convert the carbon source into serine. The modified cyanobacteria comprises the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In other words, the modified cyanobacteria has an expression plasmid comprising SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0045] Accordingly, the modified cyanobacteria can convert a carbon source into glyceraldehyde 3-phosphate (G3P). The cyanobacteria can also produce 3-phosphoglycerate dehydrogenase (SerA) by expressing the sequence of SEQ ID NO: 1 to further convert glyceraldehyde 3-phosphate (G3P) into 3-phosphohydroxypyruvate (3P-HP). The cyanobacteria can also produce phosphoserine aminotransferase (SerC) by expressing the sequence of SEQ ID NO: 3 to convert 3-phosphohydroxypyruvate (3P-HP) into phosphoserine (3P-Serine). The cyanobacteria can also produce phosphoserine phosphorylase (SerB) by expressing the sequence of SEQ ID NO: 2 to convert phosphoserine (3P-Serine) into the serine.

[0046] [Beneficial Effects of Embodiments]

[0047] 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 cyanobacteria to 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 convert the carbon source into L-serine, thereby achieving the benefits of reducing carbon emissions while simultaneously obtaining highly economical chemicals.

[0048] Furthermore, the present invention utilizes a modified cyanobacterium strain comprising the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, capable of converting carbon sources into serine. Specifically, the modified cyanobacterium of the present invention can produce up to 2.78 g / L of L-serine when cultured for 60 hours at 38°C, 3% CO₂, and 25 mM NaHCO₃. Furthermore, because the modified cyanobacterium of the present invention produces only L-serine, eliminating the need to separate D-serine from L-serine, the present method of converting carbon sources into serine using modified cyanobacteria can reduce the number of steps required to separate D-serine from L-serine compared to chemical synthesis methods.

[0049] The contents disclosed above are only preferred feasible embodiments of the present invention and do not limit the scope of the patent application of the present invention. Therefore, all equivalent technical changes made using the contents of the description and drawings of the present invention are included in the scope of the patent application of the present invention.

[0050] S1~S4: Steps

[0051] TW202526027A_112150902_SEQL.xml

Claims

1. A method for converting a carbon source into serine, comprising: Synthesize a DNA sequence; The DNA sequence was inserted into a plastid, such that the plastid included the gene sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the plastid was then inserted into a cyanobacterium (Cyanobacteriota) by electroporation to obtain a modified cyanobacterium. and providing the carbon source to the modified cyanobacteria, enabling the modified cyanobacteria to convert the carbon source into serine; wherein the carbon source comprises acetylene, methane, or acetaldehyde; wherein the nucleotide sequence shown in SEQ ID NO: 1 is 2. The method as described in request item 1, wherein, The plastid is an Escherichia coli plastid.

3. The method as described in claim 1 further includes the step of implanting the plasmid into Escherichia coli for mass production.

4. The method as described in request item 1, wherein, The cyanobacteria mentioned are Synechococcus elongates.

5. The method as described in request item 1, wherein, The modified cyanobacteria have the ability to produce 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase, and phosphoserine transaminase.

6. The method as described in request item 1, wherein, The electroporation process is performed at a voltage of 0.5 to 1.5 kV for 2 to 10 mSec.

7. The method as described in request item 1, wherein, The electroporation process further includes adding polyethylene glycol at a concentration of 0.5% to 2%.

8. The method as described in request item 1, wherein, The serine in question is L-serine.

9. The method as described in claim 1, wherein, The carbon source further includes carbon dioxide, glucose, sucrose, fructose, or galactose.

10. The method as described in claim 1, wherein, The modified blue-green bacteria convert the carbon source into glyceraldehyde-3-phosphate (G3P) and have the ability to produce 3-phosphoglycerate dehydrogenase (Ser A) to convert the glyceraldehyde-3-phosphate (G3P) into 3-phosphohydroxypyruvate (3P-HP).

11. The method as described in claim 10, wherein, The modified blue-green bacteria have the ability to produce phosphoserine transaminase (Ser C) to convert 3-phosphohydroxypyruvate (3P-HP) into phosphoserine (3P-Serine).

12. The method as described in claim 11, wherein, The modified cyanobacteria have the ability to produce phosphoserine phosphorylase (Ser B) to convert phosphoserine (3P-Serine) into serine.

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