A phase separation system and a method for CO2 fixation and amino acid production via phase separation.

Abstract

This invention provides a phase separation system comprising H-Laf1, P-Laf1, and T-Laf1 fusion proteins. The H-Laf1 fusion protein is obtained by fusing the H protein of the GCS system with the IDR region of the Laf1 protein; the P-Laf1 fusion protein is obtained by fusing the P protein of the GCS system with the IDR region of the Laf1 protein; and the T-Laf1 fusion protein is obtained by fusing the T protein of the GCS system with the IDR region of the Laf1 protein. This invention also provides a method for CO2 fixation and amino acid production via phase separation. By constructing a phase separation system, this invention forms a dynamic membrane-free microreactor in vitro, achieving substrate concentration and mass transfer optimization, significantly increasing glycine yield. Simultaneously, based on the temperature response characteristics of the IDR tag, it enables rapid enzyme separation, truly realizing enzyme recycling.

Description

Technical Field

[0001] This invention relates to the field of carbon fixation, and more particularly to a phase separation system and a method for CO2 fixation and amino acid production via phase separation. Background Technology

[0002] In contemporary scientific research, carbon dioxide fixation pathways have attracted much attention. Among them, the reductive glycine pathway (RGP) stands out as the most efficient C1 assimilation pathway.

[0003] The glycine reduction pathway is a biological pathway that converts a simple carbon source into an amino acid through a specific enzymatic catalytic reaction. It is mainly divided into the "natural glycine reduction pathway" and the "artificial glycine reduction pathway." The natural pathway uses formic acid as a C1 substrate (formic acid to glycine), while the artificial pathway uses formaldehyde and CO2 as C1 substrates (formaldehyde, carbon dioxide to glycine). Synthesized glycine has wide applications in various fields, including food, medicine, cosmetics, industrial production, and biotechnology.

[0004] The core of the glycine reduction pathway is the reversible glycine cleavage system (GCS), composed of four enzymes: T protein (aminomethyltransferase), P protein (glycine decarboxylase), L protein (dihydroacetamide dehydrogenase), and H protein (aminomethyl carrier). However, in practical applications, traditional GCS enzymes face challenges in utilizing carbon dioxide for technical biosynthesis due to high energy (ATP, NADPH) requirements, low thermodynamic driving force, and limited biosynthetic rates. To address this, Liu et al. constructed a biocompatible electron transport chain mediated by dithiothreitol (DTT) (replacing L protein and NADH), overcoming thermodynamic limitations and significantly increasing the driving force, ultimately achieving a more than 50-fold increase in glycine production; however, at present, the glycine yield of this method remains relatively low.

[0005] Biomolecular condensates, as membrane-free microcompartments within eukaryotic cells, selectively concentrate and compartmentalize biomolecules through liquid-liquid phase separation (LLPS) mechanisms. These dynamic structures are driven by proteins rich in intrinsically disordered regions (IDRs), such as the P-granules in the germ cells of *C. elegans*, whose core component, DDX3 RNA helicase, forms droplet-like structures through in vitro phase separation to participate in RNA metabolic regulation. Similar to the principle of molecule enrichment by the RuBisCO (ribose-1,5-bisphosphate carboxylase) protein, these liquid condensates utilize physicochemical processes to selectively concentrate specific molecules while maintaining their ability to exchange substances with the cytoplasmic environment. In the field of in vitro cell-free catalysis, Liu's team innovatively constructed artificial microcompartments using a synthetic phase separation system based on neuronal postsynaptic density proteins (GKAP / Shank / Homer). They successfully anchored the menadione synthase system (MenF / D / H) and terpene synthase (Idi / IspA) within the condensate through peptide-peptide interactions, thereby improving catalytic efficiency and demonstrating the potential of liquid-liquid phase separation (LLPS) for the in vitro immobilization of multiple enzymes utilizing carbon dioxide. Based on this, it is possible to explore the construction of phase separation systems with autonomous phase separation capabilities, forming dynamic membrane-free microreactors in vitro to achieve substrate concentration and mass transfer optimization, thereby increasing glycine production.

[0006] Furthermore, focusing on the engineering design of biocatalytic systems, enzyme recycling is undoubtedly the core point for improving economic efficiency. However, neither traditional GCS enzymes nor the GCS enzyme system improved by Liu et al. can truly achieve enzyme recycling. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a phase separation system and a method for CO2 fixation and amino acid production through phase separation. The constructed phase separation system forms a dynamic membrane-free microreactor in vitro, realizing substrate concentration and mass transfer optimization, which greatly improves glycine production. At the same time, based on the temperature response characteristics of IDR tags, rapid enzyme separation is achieved, truly realizing enzyme recycling.

[0008] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0009] A phase-separated system includes H-Laf1, P-Laf1, and T-Laf1 fusion proteins; wherein the H-Laf1 fusion protein is obtained by fusing the H protein (aminomethyl carrier) of the GCS system with the IDR region of the Laf1 protein; the P-Laf1 fusion protein is obtained by fusing the P protein (glycine decarboxylase) of the GCS system with the IDR region of the Laf1 protein; and the T-Laf1 fusion protein is obtained by fusing the T protein (aminomethyltransferase) of the GCS system with the IDR region of the Laf1 protein; and the IDR region sequence of the Laf1 protein is shown in SEQ ID NO.1.

[0010] As one of the preferred embodiments of the present invention, PEG 8000 is also included.

[0011] As one of the preferred embodiments of the present invention, free Laf1 protein is also included.

[0012] A method for CO2 fixation and amino acid production via phase separation, based on the aforementioned phase separation system, includes the following steps:

[0013] (1) Construct a reaction system in a reaction vessel consisting of “H-Laf1, P-Laf1, T-Laf1 fusion protein, PEG 8000” or “H-Laf1, P-Laf1, T-Laf1 fusion protein, PEG 8000, free Laf1 protein”; wherein, PEG 8000 is used as a molecular crowding agent to induce phase separation and form a dynamic membrane-free microreactor;

[0014] (2) Place the reaction system constructed in step (1) in a water bath for reaction;

[0015] (3) Temperature-responsive separation technology is used to separate the protein phase and aqueous phase in the reaction product, and the H-Laf1, P-Laf1 and T-Laf1 fusion proteins in the protein phase are recovered and recycled.

[0016] As one of the preferred embodiments of the present invention, in step (1), the molar concentration ratio of H-Laf1, P-Laf1 and T-Laf1 fusion proteins is (5-200):(1-20):(1-20), the total concentration of the fusion protein is 5-1000 μM, and the concentration of PEG 8000 is 1-30%.

[0017] As one of the preferred embodiments of the present invention, in step (1), the concentration of free Laf1 protein is 20–30 μM.

[0018] As one of the preferred embodiments of the present invention, in step (1), the reaction systems comprising “H-Laf1, P-Laf1, T-Laf1 fusion protein, PEG 8000” and “H-Laf1, P-Laf1, T-Laf1 fusion protein, PEG 8000, free Laf1 protein” also include the substrate and buffer required for the glycine reduction pathway, respectively.

[0019] As one of the preferred embodiments of the present invention, the substrate includes tetrahydrofolate, dithiothreitol, C1 substrate, pyridoxal phosphate, and ammonium salt.

[0020] As one of the preferred embodiments of the present invention, in step (2), the reaction temperature is 10-80°C and the reaction time is 0.5-3h.

[0021] As one of the preferred embodiments of the present invention, step (3) is specifically operated as follows:

[0022] ① The protein phase and aqueous phase in the reaction product are separated by low-temperature centrifugation at 2-20℃; wherein, the aqueous phase is the target product - glycine; the protein phase is the H-Laf1, P-Laf1, T-Laf1 fusion protein to be recycled, or the H-Laf1, P-Laf1, T-Laf1 fusion protein and free Laf1 protein;

[0023] ② The separated protein phase is added back into a new reaction system to produce glycine again; this process is repeated several times.

[0024] Design principles:

[0025] This invention constructs a reaction system directly in a reaction vessel, utilizing the phase separation function of the IDR region of the Laf1 protein to form multiple membrane-free microreactors within the reaction system, enriching the substrate and protein. This accelerates the biochemical reaction in a more compact environment, exhibiting high dynamism and fluidity, expediting the exchange of substrate and product within the aggregates, and promoting increased glycine production. After the reaction, the phase-separated proteins are gelled at low temperature, and centrifugation separates H-Laf1, P-Laf1, and T-Laf1 proteins, which can then be used for further glycine production through multiple reactions.

[0026] The advantages of this invention compared to the prior art are:

[0027] This invention innovatively fuses the phase-separation functional module (IDR sequence) of the Laf1 protein with key enzymes (P, T, and H proteins) of the GCS system to construct H-Laf1, P-Laf1, and T-Laf1 fusion proteins with autonomous phase-separation capabilities. Through the synergistic effect of the PEG8000 molecular congestor and the fusion protein, a dynamic membrane-free microreactor is formed in vitro to enrich substrates and proteins, achieving substrate and protein concentration and optimized mass transfer. In the first reaction, glycine yield is increased by more than 60% compared to existing methods (Liu et al.'s method based on reversed GCS and dithiothreitol DTT). Simultaneously, utilizing the temperature-responsive characteristics of the IDR tag, this invention develops a temperature-responsive protein recovery technology, enabling rapid separation of the protein phases (H-Laf1, P-Laf1, and T-Laf1 fusion proteins) via low-temperature centrifugation, achieving recycling of the GCS enzyme system (maintaining 52.5% yield after 5 cycles), significantly reducing production costs. Specific innovations and advantages are summarized below:

[0028] I. Breakthrough in Technical Performance

[0029] A stable and robust phase separation system was constructed. By using the fusion phase transition tag method, more structurally stable fusion proteins P-Laf1, T-Laf1, and H-Laf1 can be obtained without destroying the function of the original domains. They can still undergo LLPS to concentrate the protein, and multiple droplet-like substances can be observed under a confocal microscope.

[0030] Significantly improved reaction efficiency: By synergistically inducing phase separation with fusion protein (H / P / T-Laf1) and PEG 8000 to form a dynamic microreactor, the yield reached 5.3 mM within 1 hour, an increase of 61%.

[0031] Low-temperature reversible recovery: Utilizing the temperature response characteristics of IDR tags, enzyme-product rapid separation is achieved through low-temperature centrifugation at 2–20℃, resulting in high protein recovery rates and avoiding activity loss caused by traditional immobilized enzyme carriers.

[0032] Economic benefits of recycling: The recovered fusion protein can be reused 5 times, with a secondary reaction yield of ≥80%, and the total yield of 5 cycles reaches 2.8 times the initial yield, reducing enzyme usage costs by 60%.

[0033] II. Technological Innovation

[0034] It pioneered the engineering application of LLPS.

[0035] Modular design: The IDR region of the Laf1 protein is used as a universal phase separation tag and fused with the GCS enzyme system, which breaks through the limitation of traditional LLPS relying on free scaffold proteins and avoids phase separation loss due to competitive binding.

[0036] Precise control: The phase separation degree is adjusted by regulating the concentration of PEG 8000 to achieve a balance and optimization of reaction kinetics and mass transfer efficiency.

[0037] III. Advantages of Membrane-Free Reactors

[0038] Dynamic mass transfer: The droplet-like aggregates combine high substrate enrichment capacity (concentration coefficient > 5×) with fluidity (droplet fusion time < 30s), and the product diffusion rate is 2.1 times higher than that of solid carriers. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the H-laf1 plasmid structure constructed in this invention;

[0040] Figure 2 This is a schematic diagram of the P-Laf1 plasmid structure constructed in this invention;

[0041] Figure 3 This is a schematic diagram of the T-Laf1 plasmid structure constructed in this invention;

[0042] Figure 4 This is a schematic diagram of the Laf1-IDR plasmid structure constructed in this invention;

[0043] Figure 5 The present invention takes the reaction system of Example 4 as an example and observes the phase separation and droplet flow under a microscope within 30 minutes (in the figure, a to f are the observation results during the time process from 0 to 30 minutes).

[0044] Figure 6 The present invention takes the reaction system of Example 7 as an example and observes the phase separation and droplet flow under a microscope within 30 minutes (in the figure, a to f are the observation results during the time process from 0 to 30 minutes);

[0045] Figure 7 The figures show the microscopic phenotypes of the reaction systems of series 1 and series 2 after the first reaction 1 hour and the corresponding glycine production results (Figure a shows the glycine production results of series 1 after the first reaction 1 hour; Figure b shows the glycine production results of series 2 after the first reaction 1 hour; Figure c shows the microscopic phenotype results, and Figures A to D show the microscopic phenotypes of series 1 after the reaction of different concentrations of free Laf1 protein system, and Figures E to H show the microscopic phenotypes of series 2 after the reaction of different concentrations of free Laf1 protein system).

[0046] Figure 8 The graph shows the glycine yield results of multiple cycles of the reaction system of the second reaction group of the present invention (Figure a shows the glycine yield results of multiple cycles of the reaction under the condition of 20 μM free Laf1; Figure b shows the glycine yield results of multiple cycles of the reaction under the condition of 30 μM free Laf1).

[0047] Figure 9 This invention takes series group 1 and series group 2 as examples to measure their OD. 600 The results of the values ​​changing with temperature (in the figure, a is the result of series group 1; b is the result of series group 2);

[0048] Figure 10 The present invention uses 4°C as the separation temperature and centrifuges the supernatant and precipitate gel electrophoresis results of the reaction products of series 1 and series 2 (Figure a shows the electrophoresis results of the supernatant; Figure b shows the electrophoresis results of the precipitate).

[0049] Figure 11 This is a micrograph of the reaction system based on the P-Laf1, T-Laf1, and H-Laf1 fusion protein of this invention, and which does not contain PEG 8000. Detailed Implementation

[0050] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents, methods, and equipment used in the present invention are commercially available reagents, methods, and equipment conventionally available in this technical field.

[0051] Example 1

[0052] This embodiment describes an H-Laf1, P-Laf1, and T-Laf1 fusion protein, obtained by fusing the H, P, and T proteins of the GCS system with the IDR region of the Laf1 protein (sequence shown in SEQ ID NO.1). The specific method is as follows:

[0053] (1) The coding genes for H, P, and T proteins were amplified from the genomic DNA of Escherichia coli BL21, respectively; the coding gene sequences for H, P, and T proteins are shown in SEQ ID NO.2 to 4, respectively.

[0054] (2) HIS tag sequences were inserted into the XhoI sites of the H, P, and T protein encoding genes, respectively. Then, the H, P, and T protein genes with added HIS tags were ligated into the pET-28a vector containing the RGG domain of the Laf1 protein (i.e., the IDR region of the Laf1 protein) using ligase, respectively, to construct the corresponding H-laf1-IDR, P-laf1-IDR, and T-laf1-IDR plasmids (e.g., Figures 1-3 (As shown). This step can be outsourced to a biotechnology company for synthesis.

[0055] (3) The H-laf1-IDR, P-laf1-IDR and T-laf1-IDR plasmids were introduced into Escherichia coli BL21 for expression and purified by nickel column to obtain the target H-Laf1, P-Laf1 and T-Laf1 fusion protein.

[0056] Example 2

[0057] This embodiment of the phase separation system includes the H-Laf1, P-Laf1, T-Laf1 fusion protein and PEG 8000 from the above embodiments.

[0058] Example 3

[0059] This embodiment of the phase separation system includes the H-Laf1, P-Laf1, and T-Laf1 fusion proteins, as well as PEG 8000 and free Laf1 protein, as described in the above embodiments.

[0060] The free Laf1 protein is the Laf1 protein after an HIS tag has been inserted into its IDR region. The method for obtaining this protein is as follows:

[0061] (1) Synthesize a pET-28a plasmid containing the sequence “Laf1 IDR region + HIS tag” (shown in SEQ ID NO.5), and name it laf1-IDR plasmid (e.g., Figure 4 (As shown). This step can be outsourced to a biotechnology company for synthesis.

[0062] (2) The Laf1-IDR plasmid was introduced into Escherichia coli BL21 for expression and purified by nickel column to obtain the target free Laf1 protein.

[0063] Example 4

[0064] This embodiment of the RGP reaction system includes the phase separation system of Example 2, as well as the basic substrate and buffer required for the reaction, as shown in Table 1.

[0065] Table 1. Composition of the reaction system

[0066]

[0067]

[0068] Example 5

[0069] The RGP reaction system of this embodiment is basically the same as that of Example 4, except that: (1) the total concentration of fusion protein is 5 μM, wherein the molar concentration ratio of H-Laf1, P-Laf1 and T-Laf1 fusion proteins is 5:1:1; (2) the concentration of PEG 8000 is 1%.

[0070] Example 6

[0071] The RGP reaction system of this embodiment is basically the same as that of Example 4, except that: (1) the total concentration of the fusion protein is 1000 μM, and the molar concentration ratio of H-Laf1, P-Laf1 and T-Laf1 fusion proteins is 10:1:1; (2) the concentration of PEG 8000 is 30%.

[0072] Example 7

[0073] This embodiment of the RGP reaction system includes the phase separation system of Example 3, as well as the basic substrate and buffer required for the reaction, as shown in Table 2.

[0074] Table 2 Composition of the reaction system

[0075]

[0076]

[0077] Example 8

[0078] The RGP reaction system in this embodiment is basically the same as that in Example 7, except that the concentration of free Laf1 protein is 25 μM.

[0079] Example 9

[0080] The RGP reaction system in this embodiment is basically the same as that in Example 7, except that the concentration of free Laf1 protein is 30 μM.

[0081] Example 10

[0082] This embodiment presents a method for CO2 fixation and amino acid production via phase separation:

[0083] (1) The RGP reaction system shown in the above embodiment is constructed in a reaction vessel; wherein, PEG 8000 is used as a molecular crowding agent to induce phase separation and form a dynamic membrane-free microreactor.

[0084] (2) Place the reaction system constructed in step (1) in a water bath at 37°C and react for 1 hour.

[0085] (3) Temperature-responsive separation technology is used to separate the protein phase and aqueous phase in the reaction product, and the H-Laf1, P-Laf1 and T-Laf1 fusion proteins in the protein phase are recovered and recycled.

[0086] ① The protein phase (precipitate) and aqueous phase (supernatant) in the reaction product were separated by centrifugation at 4℃; wherein, the aqueous phase (supernatant) is the target product - glycine; the protein phase (precipitate) is the H-Laf1, P-Laf1, T-Laf1 fusion protein to be recycled, or the H-Laf1, P-Laf1, T-Laf1 fusion protein and free Laf1 protein;

[0087] ② The separated protein phase is added back into a new reaction system (other components in the reaction system need to be added separately) to produce glycine again; the reaction is repeated 5 times.

[0088] Example 11

[0089] The method for CO2 fixation and amino acid production by phase separation in this embodiment is basically the same as that in Example 10, except that: (1) the reaction system is placed in a water bath at 10°C and reacted for 3 hours; (2) the protein phase and aqueous phase in the reaction product are separated by centrifugation at 2°C.

[0090] Example 12

[0091] The method for CO2 fixation and amino acid production by phase separation in this embodiment is basically the same as that in Example 10, except that: (1) the reaction system is placed in a water bath at 80°C for 0.5 h; (2) the protein phase and aqueous phase in the reaction product are separated by centrifugation at 20°C.

[0092] Experimental Example 1

[0093] This experimental example is used to verify the phase separation function of the H-Laf1, P-Laf1, and T-Laf1 fusion protein of the present invention.

[0094] I. Experimental Methods

[0095] Taking Examples 4 and 7 as examples, after constructing the corresponding reaction systems, the phase separation and droplet flow were observed under a microscope within 30 minutes.

[0096] II. Experimental Results

[0097] The results are as follows Figure 5 , Figure 6 As shown. By Figure 5 , Figure 6 It can be seen that whether it is "fusion protein P-Laf1, T-Laf1, H-Laf1" or "fusion protein P-Laf1, T-Laf1 and H-Laf1 with free Laf1 protein added at the same time", they all have phase separation function, and the solution has strong fluidity. Moreover, multiple small droplets rapidly converge into a large droplet within 30 minutes, which further proves that liquid-liquid phase separation has occurred in the solution.

[0098] Experimental Example 2

[0099] This experimental example is used to verify the effects of fusion addition, free addition, and the amount of free addition of Laf1 protein on the glycine production of the present invention.

[0100] I. Experimental Methods

[0101] Configure the following two series groups:

[0102] Group 1: Based on the original GCS proteins P, T, and H, different concentrations (0, 30, 60, and 90 μM) of free Laf1 protein were added to construct the reaction system (other components are the same as in Example 4).

[0103] Group 2: Based on the "P-Laf1, T-Laf1, H-Laf1 fusion protein of the present invention", different concentrations (0, 10, 20, 30 μM) of free Laf1 protein were added to construct the reaction system (other components are the same as in Example 4).

[0104] After constructing the corresponding RGP reaction systems according to the composition of each group, they were placed in a water bath at 37℃ for 1 hour for the first reaction. After the first reaction, the reaction products were centrifuged at 12000g for 3 minutes at 4℃. The supernatant after centrifugation was aspirated, and the glycine yield was measured. At the same time, the precipitate (containing H-Laf1, P-Laf1, and T-Laf1 fusion proteins) was resuspended with Tris to obtain the precipitate resuspension. 4 μL of the supernatant and the precipitate resuspension were taken for SDS-PAGE verification. Subsequently, the precipitate resuspension was added to a new reaction system to produce glycine again; the reaction was carried out 5 times in total, and the glycine yield obtained in each reaction was measured.

[0105] In this embodiment, the concentration of glycine in the reaction mixture was determined using the "pre-column dansyl chloride derivatization method": 40 μL of the reaction product was mixed with 160 μL of 0.2 M NaHCO3 and 200 μL of 5.4 mg / mL... -1 Dansyl chloride was mixed in acetonitrile. The derivatization reaction was carried out at 30 °C for 30 min. After the reaction was completed, 600 μL of 0.12 M HCl was added to adjust the pH of the sample to make it weakly acidic. After centrifugation at 12000 × g for 3 min, the supernatant was filtered through a 0.22 μm membrane.

[0106] Analysis was performed using an Agilent 1290 Infinity HPLC system (Agilent Technologies, USA), equipped with a Poroshell 120 high-performance liquid chromatography column (4.6 × 150 mm l. D. 2 μm). The mobile phase was 20 mM, pH 6.0 phosphate (A) and acetonitrile (B). Before sample analysis, defoaming was performed with 75% solution A and 25% solution B at a flow rate of 1.0 mL·min. -1 With the AB solution ratio unchanged, perform gradient flushing at (0.2-0.4-0.6-0.8) mL / min. -1 Then the sample is tested.

[0107] II. Experimental Results

[0108] The microscopic phenotypes of reaction systems in series 1 and series 2 after the first reaction 1 hour and the corresponding glycine yield detection results are as follows: Figure 7 As shown in the figure. In series group 1, the glycine yield in the second cycle of reactions with Laf1 at 0, 30, 60, and 90 μM was zero in all cases. The glycine yield results for multiple cycles in series group 2 are shown in the figure. Figure 8 As shown.

[0109] The results above show that (1) when the amount of free Laf1 protein added is 0, the glycine yield of the P-Laf1, T-Laf1, H-Laf1 fusion protein of the present invention is significantly increased compared with the original GCS protein P, T, H (traditional method); (2) when based on the original GCS protein P, T, H, the yield of free Laf1 protein and PEG is significantly increased. The phase separation produced by 8000 resulted in a glycine yield of up to 3.3 mM in the first reaction at 1 h; while in the phase separation system constructed from P-Laf1, T-Laf1, H-Laf1 proteins and free Laf1 protein, the glycine yield in the first reaction reached up to about 5.3 mM at 1 h, achieving a 61% increase in yield; (3) When based on the original GCS proteins P, T, and H, even with the addition of different concentrations of free Laf1 protein, the glycine yield in the second reaction was all 0; while based on the fusion protein of P-Laf1, T-Laf1, and H-Laf1 of this invention, under the condition of 20 μM free Laf1, the glycine yield in the second reaction was 85% of that in the first reaction, the third reaction still retained 57% of the yield, the fourth was 42%, and the fifth still had 32% of the yield; 30 μM The glycine yield of the Laf1 group samples can be up to 99% of the first time in the second reaction, 73% in the third reaction, 56% in the fourth reaction, and 52.5% in the fifth reaction; (4) The recovered fusion protein can be reused 5 times, the yield of the second reaction is ≥80%, the total yield of the 5 cycles is 2.8 times that of the initial, and the enzyme usage cost is reduced by 60%; (5) When based on the "original GCS protein P, T, H", the clusters or aggregations become more and more obvious as the concentration of free Laf1 increases; when based on the "P-Laf1, T-Laf1, H-Laf1 fusion protein of this invention", no obvious clumps are formed, but many obvious droplets are formed, which have better fluidity and mass transfer effect.

[0110] Experimental Example 3

[0111] This experimental example is used to verify the temperature response characteristics and protein enrichment effects of the P-Laf1, T-Laf1, and H-Laf1 fusion proteins of the present invention.

[0112] I. Temperature Response Characteristics

[0113] The P-Laf1, T-Laf1, and H-Laf1 fusion protein solution of this invention exhibits the physical property of "gelation at low temperature and subsequent transparentization at high temperature." This experimental example uses two series reaction systems (series 1 and series 2) from Experiment 2 as examples, and measures their OD... 600 The value was verified by varying with temperature (2–40℃).

[0114] OD 600 The results are as follows Figure 9 As shown, by Figure 9 It is known that the P-Laf1, T-Laf1, and H-Laf1 fusion protein of the present invention has temperature-responsive characteristics. The protein phase and aqueous phase in the reaction product can be separated by low-temperature centrifugation, and the H-Laf1, P-Laf1, and T-Laf1 fusion protein in the protein phase can be recovered. The separation effect is best at a temperature of 2 to 20°C, especially at 4°C.

[0115] II. Protein enrichment effect

[0116] Based on the above results, the reaction products of the two series reaction systems (Series 1 and Series 2) were centrifuged at a separation temperature of 4℃, and the resulting supernatant and precipitate were subjected to gel electrophoresis. The results are as follows: Figure 10 As shown. By Figure 10 It can be seen that the P, T, and H protein bands enriched in the precipitate of series 1 are very light, while the P-Laf1, T-Laf1, and H-Laf1 protein bands enriched in series 2 are very dark. This shows that the system of series 2 has a better protein enrichment effect.

[0117] Experiment Example 4

[0118] This experimental example is used to verify the effect of adding PEG 8000 on phase separation and glycine production.

[0119] I. Experimental Methods

[0120] Based on the "P-Laf1, T-Laf1, H-Laf1 fusion protein of this invention", a reaction system without PEG 8000 was constructed (other components are the same as in Example 4). Its microscopic phenotype was observed, and its OD activity was also investigated. 600nm The absorbance value was measured.

[0121] II. Experimental Results

[0122] Microphenotypic results as follows Figure 11 As shown, OD 600nm The absorbance values ​​are close to those of the clear solution. This indicates that simply adding H-Laf1, P-Laf1, or T-Laf1 proteins has a very weak phase separation effect; the solution at OD... 600nm The absorbance value is close to that of a transparent solution, and the addition of the molecular crowding agent PEG 8000 can cause the solution to become turbid immediately and undergo obvious phase separation (as can be seen from the results of the aforementioned experimental examples), which in turn affects the subsequent production of glycine.

[0123] In summary, this invention innovatively fuses the phase separation functional module (IDR sequence) of the Laf1 protein with key enzymes (P, T, and H proteins) of the GCS system to construct a H-Laf1, P-Laf1, and T-Laf1 fusion protein with autonomous phase separation capability. Through the synergistic effect of the fusion protein and the PEG 8000 molecular congestor, a dynamic membrane-free microreactor is formed in vitro, achieving substrate concentration and mass transfer optimization. In the first reaction, glycine yield is increased by more than 60% compared to existing methods. Simultaneously, utilizing the temperature-responsive characteristics of the IDR tag, this invention develops a temperature-responsive protein recovery technology, enabling rapid separation of the protein phases (H-Laf1, P-Laf1, and T-Laf1 fusion protein) via low-temperature centrifugation, achieving recycling of the GCS enzyme system (maintaining 52.5% yield after 5 cycles), significantly reducing production costs.

[0124] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A phase separation system, characterized by, The H-Laf1, P-Laf1 and T-Laf1 fusion proteins; wherein the H-Laf1 fusion protein is obtained by fusing the amino methyl carrier H protein of the GCS system with the IDR region of the Laf1 protein; the P-Laf1 fusion protein is obtained by fusing the glycine decarboxylase P protein of the GCS system with the IDR region of the Laf1 protein; the T-Laf1 fusion protein is obtained by fusing the amino methyltransferase T protein of the GCS system with the IDR region of the Laf1 protein; and the sequence of the IDR region of the Laf1 protein is shown as SEQ ID NO. 1, and the coding gene sequences of the H, P and T proteins are shown as SEQ ID NO. 2-4, respectively.

2. The phase separation system according to claim 1, characterized in that PEG 8000 is also included.

3. The phase separation system of claim 1, wherein, Free Laf1 protein is also included.

4. A method for CO2 fixation and amino acid production by phase separation, characterized by, The phase separation system according to any one of claims 1-3, comprising the following steps: (1) constructing a reaction system comprising "H-Laf1, P-Laf1, T-Laf1 fusion protein, PEG 8000" or "H-Laf1, P-Laf1, T-Laf1 fusion protein, PEG 8000, free Laf1 protein" in a reaction container; wherein PEG 8000 is used as a molecular crowding agent to induce phase separation and form a dynamic membrane-free microreactor; (2) placing the reaction system constructed in step (1) in a water bath for reaction; (3) separating the protein phase and the aqueous phase in the reaction product by using a temperature-responsive separation technology, and recycling the H-Laf1, P-Laf1 and T-Laf1 fusion proteins in the protein phase.

5. The method of CO2 fixation and amino acid production by phase separation according to claim 4, characterized by, In step (1), the molar concentrations of the H-Laf1, P-Laf1 and T-Laf1 fusion proteins are (5-200):(1-20):(1-20), respectively, and the total concentration of the fusion proteins is 5-1000 μM; the concentration of PEG 8000 is 1-30%.

6. The method of CO2 fixation and amino acid production by phase separation according to claim 4, characterized by, In step (1), the concentration of free Laf1 protein is 20-30 μM.

7. The method of CO2 fixation and amino acid production by phase separation according to claim 4, characterized by, In step (1), the reaction systems "H-Laf1, P-Laf1, T-Laf1 fusion protein, PEG 8000" and "H-Laf1, P-Laf1, T-Laf1 fusion protein, PEG 8000, free Laf1 protein" further comprise substrates and buffers required for the reduced glycine pathway, respectively.

8. The method of CO2 fixation and amino acid production by phase separation according to claim 7, characterized by, The substrates include tetrahydrofolate, dithiothreitol, carbon one substrate, pyridoxal phosphate and ammonium salt.

9. The method of CO2 fixation and amino acid production by phase separation according to claim 4, characterized by, In step (2), the reaction temperature is 10-80 ℃, and the reaction time is 0.5-3 h.

10. The method of CO2 fixation and amino acid production by phase separation according to claim 4, characterized by, In step (3), the specific operation is as follows: a. separating the protein phase and the aqueous phase in the reaction product by low-temperature centrifugation at 2-20 ℃; wherein the aqueous phase is the target product-glycine, and the protein phase is the H-Laf1, P-Laf1 and T-Laf1 fusion proteins to be recycled, or the H-Laf1, P-Laf1 and T-Laf1 fusion proteins and free Laf1 protein to be recycled; b. The separated protein phase is again put into a new reaction system to produce glycine again; and the above process is repeated for several times.

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