Phase separation system and method for CO2 immobilization and amino acid production through phase separation

Abstract

The invention provides a phase separation system. The phase separation system comprises H-Lab1 fusion protein, P-Lab1 fusion protein and T-Lab1 fusion protein, wherein the H-Lab1 fusion protein is obtained by fusing an H protein of a GCS system and an IDR region of a Lab1 protein; the P-Laf1 fusion protein is obtained by fusing a P protein of a GCS system and an IDR region of a Laf1 protein; the T-Lab1 fusion protein is obtained by fusing a T protein of a GCS system and an IDR region of a Lab1 protein. The invention also provides a method for CO2 immobilization and amino acid production by phase separation. According to the method, a dynamic membrane-free microreactor is formed in vitro through a constructed phase separation system, substrate concentration and mass transfer optimization are achieved, the yield of glycine is greatly increased, meanwhile, rapid separation of enzyme is achieved based on the temperature response characteristic of an IDR tag, and cyclic utilization of the enzyme is truly achieved.

Description

Technical Field

[0001] The present invention relates to the field of carbon sequestration, and in particular to a phase separation system and a method for CO fixation and amino acid production by phase separation. 2 Background Art

[0002] In current scientific research, carbon dioxide fixation pathways have received much attention. Among them, the Reductive Glycine pathway (RGP) stands out and is regarded as the most effective C1 assimilation pathway.

[0003] The Reductive Glycine pathway is a biological pathway that converts simple carbon sources into amino acids through specific enzyme-catalyzed reactions. It is mainly divided into the "natural Reductive Glycine pathway" and the "artificial Reductive Glycine pathway". Among them, the natural pathway proceeds through formic acid as the C1 substrate (from formic acid to glycine), and the artificial pathway uses formaldehyde and CO 2 as the C1 substrate (from formaldehyde and carbon dioxide to glycine). The synthesized glycine has a wide range of applications in many fields such as food, medicine, cosmetics, industrial production, and biotechnology.

[0004] The core part of the Reductive Glycine pathway is the reversible glycine cleavage system (GCS), which consists of four enzymes: T protein (aminomethyltransferase), P protein (glycine decarboxylase), L protein (dihydrolipoamide dehydrogenase), and H protein (aminomethyl carrier). However, in actual applications, due to some obstacles such as high energy (ATP, NADPH) requirements, low thermodynamic driving force, and limited biosynthesis rate, it is difficult to use carbon dioxide for technical biosynthesis. In this regard, Liu et al. constructed a biocompatible electron transfer chain mediated by dithiothreitol DTT (replacing L protein and NADH), which overcame the thermodynamic limitation and significantly increased the driving force, ultimately achieving a more than 50-fold leap in the productivity of glycine; however, at present, the glycine yield of this method is still at a relatively low level.

[0005] Biomolecular condensates, as membraneless microcompartments within eukaryotic cells, achieve the selective concentration and compartmentalization of biomacromolecules through the liquid-liquid phase separation (LLPS) mechanism. These dynamic structures are driven by proteins rich in intrinsically disordered regions (IDRs). For example, P granules in the germ cells of Caenorhabditis elegans, whose core component is the DDX3 RNA helicase, form droplet-like structures through in vitro phase separation and participate in the regulation of RNA metabolism. Similar to the principle of protein-rich molecules such as RuBisCO (ribulose-1,5-bisphosphate carboxylase), these liquid condensates utilize physicochemical interactions to selectively concentrate specific molecules while maintaining the ability to exchange substances with the cytoplasmic environment. In the field of in vitro cell-free catalysis, the Liu team innovatively used the synthetic phase separation system of postsynaptic density proteins (GKAP / Shank / Homer) in neurons to construct artificial microcompartments, successfully anchoring the menaquinone synthase system (MenF / D / H) and terpene synthase (Idi / IspA) in the condensates through peptide-peptide interactions, achieving an improvement in catalytic efficiency and demonstrating the potential of liquid-liquid phase separation (LLPS) in the in vitro fixation of multiple enzymes for carbon dioxide utilization. Accordingly, an attempt can be made to construct a phase separation system with autonomous phase separation ability to form a dynamic membraneless microreactor in vitro, realizing substrate concentration and mass transfer optimization, and improving glycine production.

[0006] In addition, focusing on the engineering design of biocatalytic systems, the recycling of enzymes is undoubtedly the core point to improve economy. However, neither the traditional GCS enzyme nor the improved GCS enzyme system by Liu et al. can truly achieve enzyme recycling. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a phase separation system and a method for CO 2 fixation and amino acid production through phase separation, which forms a dynamic membraneless microreactor in vitro through the constructed phase separation system, realizes substrate concentration and mass transfer optimization, greatly improves glycine production, and at the same time realizes the rapid separation of enzymes based on the temperature-responsive characteristics of IDR tags, truly achieving enzyme recycling.

[0008] The present invention adopts the following technical solutions to solve the above technical problems:

[0009] A phase separation 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; 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 sequence of the IDR region of the Laf1 protein is as shown in SEQ ID NO.1.

[0010] As one of the preferred embodiments of the present invention, it further includes PEG 8000.

[0011] As one of the preferred embodiments of the present invention, it further includes free Laf1 protein.

[0012] A method for CO 2 fixation and amino acid production by phase separation, based on the above phase separation system, includes the following steps:

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

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

[0015] (3) Use a temperature-responsive separation technique to separate the protein phase and the aqueous phase in the reaction product, and recover the H-Laf1, P-Laf1, and T-Laf1 fusion proteins in the protein phase for recycling.

[0016] As one of the preferred embodiments of the present invention, in step (1), the molar concentration ratios of the H-Laf1, P-Laf1, and T-Laf1 fusion proteins are respectively (5-200):(1-20):(1-20), and the total concentration of the fusion proteins is 5-1000 μM; 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 the free Laf1 protein is 20-30 μM

[0018] As one of the preferred embodiments of the present invention, in the step (1), in the reaction systems including "H-Laf1, P-Laf1, T-Laf1 fusion protein, PEG 8000" and "H-Laf1, P-Laf1, T-Laf1 fusion protein, PEG 8000, free Laf1 protein", substrates required for the reductive glycine pathway and buffers are further included respectively.

[0019] As one of the preferred embodiments of the present invention, the substrates include tetrahydrofolic acid, dithiothreitol, one-carbon substrate, pyridoxal phosphate, and ammonium salt.

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

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

[0022] ① Separate the protein phase and the aqueous phase in the reaction product by low-temperature centrifugation at 2 - 20 °C; 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, free Laf1 protein;

[0023] ② Put the separated protein phase into a new reaction system again to react to produce glycine again; cycle and use it several times in sequence.

[0024] Design principle:

[0025] In the present invention, by directly constructing a reaction system in a reaction vessel and utilizing the phase separation function of the IDR region of Laf1 protein, multiple membrane-less microreactors are formed in the reaction system to enrich substrates and proteins. The biochemical reaction is accelerated in a closer environment. This process has high dynamics and fluidity, accelerating the exchange of substrates and products in the condensate and promoting the increase in glycine production. After the reaction, the phase-separated protein becomes gelatinous by low temperature, and the H-Laf1 protein, P-Laf1 protein, and T-Laf1 protein can be centrifuged and separated and can be used for multiple reactions to continue producing glycine.

[0026] The advantages of the present invention compared with the prior art are as follows:

[0027] The present invention innovatively fuses the phase separation functional module (IDR sequence) of Laf1 protein with the key enzymes (P, T, H proteins) of the GCS system to construct H-Laf1, P-Laf1, and T-Laf1 fusion proteins with autonomous phase separation ability. Through the synergistic action of the PEG8000 molecular crowding agent and the fusion proteins, a dynamic membraneless microreactor is formed in vitro to enrich substrates and proteins, achieving the concentration of substrates and proteins and optimizing mass transfer. In the first reaction, the glycine production is increased by more than 60% compared with the existing method (the method based on reversed GCS and dithiothreitol DTT by Liu et al.). At the same time, utilizing the temperature-responsive characteristics of the IDR tag, the present invention develops a temperature-responsive protein recovery technology. The protein phase (H-Laf1, P-Laf1, T-Laf1 fusion proteins) can be rapidly separated by low-temperature centrifugation, realizing the recycling of the GCS enzyme system (after 5 cycles, the yield still remains at 52.5%), and significantly reducing the production cost. The specific innovation points and advantages are summarized as follows:

[0028] I. Breakthrough in technical performance

[0029] A stable and firm phase separation system is constructed. By using the method of fusing phase transition tags, more structurally stable fusion proteins P-Laf1, T-Laf1, and H-Laf1 can be obtained. Without destroying the functions of the original domains, they can still undergo LLPS to concentrate proteins, and multiple droplet-like substances can be observed under a confocal microscope.

[0030] The reaction efficiency is significantly improved: Through the synergistic induction of phase separation by the fusion proteins (H / P / T-Laf1) and PEG 8000 to form a dynamic microreactor, the yield reaches 5.3 mM within 1 h, an increase of 61%.

[0031] Low-temperature reversible recovery: Utilizing the temperature-responsive characteristics of the IDR tag, low-temperature centrifugation at 2 - 20 °C enables rapid separation of the enzyme-product, with a high protein recovery rate, avoiding the activity loss caused by traditional immobilized enzyme carriers.

[0032] Economy of recycling: The recovered fusion proteins can be reused 5 times, the yield of the secondary reaction is ≥80%, the total yield after 5 cycles reaches 2.8 times that of the initial, and the enzyme usage cost is reduced by 60%.

[0033] II. Technical innovation

[0034] The first engineering application of LLPS is initiated.

[0035] Modular design: Taking the IDR region of Laf1 protein as a general phase separation tag and fusing it with the GCS enzyme system, breaking through the limitation of traditional LLPS relying on free scaffold proteins and avoiding the out-of-control phase separation caused by competitive binding.

[0036] Precise regulation: By adjusting the degree of phase separation through the concentration of PEG 8000, the balance optimization of reaction kinetics and mass transfer efficiency is achieved.

[0037] III. Advantages of the membrane-free reactor

[0038] Dynamic mass transfer: Droplet-like condensates have both high substrate enrichment ability (concentration factor > 5×) and fluidity (droplet fusion time < 30 s), and the product diffusion rate is 2.1 times higher than that of solid carriers. Brief description of the drawings

[0039] Figure 1 It is a schematic diagram of the structure of the H-laf1 plasmid constructed by the present invention;

[0040] Figure 2 It is a schematic diagram of the structure of the P-Laf1 plasmid constructed by the present invention;

[0041] Figure 3 It is a schematic diagram of the structure of the T-Laf1 plasmid constructed by the present invention;

[0042] Figure 4 It is a schematic diagram of the structure of the Laf1-IDR plasmid constructed by the present invention;

[0043] Figure 5 It is a diagram showing the phase separation and droplet fluidity observed under a microscope within 30 min for the reaction system of Example 4 of the present invention (in the figure, a-f are the observation results during the time process from 0 to 30 min);

[0044] Figure 6 It is a diagram showing the phase separation and droplet fluidity observed under a microscope within 30 min for the reaction system of Example 7 of the present invention (in the figure, a-f are the observation results during the time process from 0 to 30 min);

[0045] Figure 7 It is the microscopic phenotype and the corresponding glycine yield detection results of the reaction systems of Series Group 1 and Series Group 2 of the present invention after the first reaction for 1 h (in the figure, a is the glycine yield detection result of Series Group 1 after the first reaction for 1 h; b is the glycine yield detection result of Series Group 2 after the first reaction for 1 h; c is the microscopic phenotype result, and A-D are the microscopic phenotypes after the reaction of different concentrations of free Laf1 protein systems in Series Group 1, and E-H are the microscopic phenotypes after the reaction of different concentrations of free Laf1 protein systems in Series Group 2);

[0046] Figure 8 It is a graph of the glycine yield results of multiple cycle reactions of the reaction system of Series Group 2 of the present invention (in the figure, a is the glycine yield result of multiple cycle reactions under the condition of 20 μM free Laf1; b is the glycine yield result of multiple cycle reactions under the condition of 30 μM free Laf1);

[0047] Figure 9 In the present invention, taking Series Group 1 and Series Group 2 as examples, the OD 600 values are measured as the results of temperature change (in the figure, Figure a shows the results of Series Group 1; Figure b shows the results of Series Group 2);

[0048] Figure 10 In the present invention, taking 4°C as the separation temperature, the supernatant and precipitate after centrifugation of the reaction products of Series Group 1 and Series Group 2 are subjected to SDS-PAGE (in the figure, Figure a shows the SDS-PAGE results of the supernatant; Figure b shows the SDS-PAGE results of the precipitate);

[0049] Figure 11 It is a micrograph of the reaction system based on the P-Laf1, T-Laf1, and H-Laf1 fusion proteins without PEG 8000 in the present invention. Detailed implementation mode

[0050] The embodiments of the present invention will be described in detail below. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation modes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments. At the same time, unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional commercially available reagents, methods, and equipment in this technical field.

[0051] Example 1

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

[0053] (1) The coding genes of the H, P, and T proteins are respectively amplified from the genomic DNA of Escherichia coli BL21; the coding gene sequences of the H, P, and T proteins are shown in SEQ ID NOs. 2-4 respectively;

[0054] (2) HIS tag sequences are respectively inserted at the XhoI sites of the H, P, and T protein coding genes, and then the "H, P, and T protein genes with HIS tags added" are respectively ligated to the "pET-28a vector containing the RGG domain of the Laf1 protein (i.e., the IDR region of the Laf1 protein)" through a ligase to construct H-laf1-IDR, P-laf1-IDR, and T-laf1-IDR plasmids (as Figures 1 to 3 shown). This step can be synthesized by a biological company.

[0055] (3) Respectively introduce the H-laf1-IDR, P-laf1-IDR, and T-laf1-IDR plasmids into Escherichia coli BL21 for expression, and purify them through nickel columns to obtain the H-Laf1, P-Laf1, and T-Laf1 fusion proteins required for the target.

[0056] Example 2

[0057] A phase separation system in this example includes the H-Laf1, P-Laf1, and T-Laf1 fusion proteins in the above example and PEG 8000.

[0058] Example 3

[0059] A phase separation system in this example includes the H-Laf1, P-Laf1, and T-Laf1 fusion proteins in the above example, as well as PEG 8000 and free Laf1 protein.

[0060] Among them, the free Laf1 protein is the protein after inserting the HIS tag into the IDR region of the Laf1 protein, and the obtaining method is as follows:

[0061] (1) Synthesize the pET-28a plasmid containing the sequence of "IDR region of Laf1 + HIS tag" (shown in SEQ ID NO.5), and name it the laf1-IDR plasmid (as Figure 4 shown). This step can be entrusted to a biological company for synthesis.

[0062] (2) Introduce the Laf1-IDR plasmid into Escherichia coli BL21 for expression, and purify it through nickel columns to obtain the free Laf1 protein required for the target.

[0063] Example 4

[0064] A RGP reaction system in this example includes the phase separation system in Example 2 and the basic substrates and buffers required for the reaction, as shown in Table 1 specifically.

[0065] Table 1 Composition of the reaction system

[0066]

[0067]

[0068] Example 5

[0069] A RGP reaction system in this example is basically the same as that in Example 4. The main differences are as follows: (1) The total concentration of the fusion protein is 5 μM, and among them, the molar concentrations of the H-Laf1, P-Laf1, and T-Laf1 fusion proteins are in a ratio of 5:1:1 respectively; (2) The concentration of PEG 8000 is 1%.

[0070] Example 6

[0071] An RGP reaction system of this example is basically the same as that of Example 4, and the main differences are as follows: (1) The total concentration of the fusion protein is 1000 μM, and among them, the molar concentrations of the H-Laf1, P-Laf1, and T-Laf1 fusion proteins are in a ratio of 10:1:1 respectively; (2) The concentration of PEG 8000 is 30%.

[0072] Example 7

[0073] An RGP reaction system of this example includes the phase separation system of Example 3 and the basic substrates and buffers required for the reaction, as shown in Table 2 specifically.

[0074] Table 2 Composition of the reaction system

[0075]

[0076]

[0077] Example 8

[0078] An RGP reaction system of this example is basically the same as that of Example 7, and the main difference is that the concentration of free Laf1 protein is 25 μM.

[0079] Example 9

[0080] An RGP reaction system of this example is basically the same as that of Example 7, and the main difference is that the concentration of free Laf1 protein is 30 μM.

[0081] Example 10

[0082] A method for CO 2 fixation and amino acid production by phase separation:

[0083] (1) Construct the RGP reaction system shown in the above examples in a reaction vessel; among them, PEG 8000 is used as a molecular crowding agent to induce phase separation to 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 h.

[0085] (3) Adopt temperature-responsive separation technology to separate the protein phase and the aqueous phase in the reaction product, and recover the H-Laf1, P-Laf1, and T-Laf1 fusion proteins in the protein phase for recycling.

[0086] ①Separate the protein phase (precipitate) and the aqueous phase (supernatant) in the reaction product by low-temperature centrifugation at 4°C; among them, 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] ②Put the separated protein phase back into a new reaction system (other components in the reaction system need to be supplemented additionally), and react again to produce glycine; perform the cyclic reaction 5 times.

[0088] Example 11

[0089] A method for CO 2 fixation and amino acid production by phase separation in this example is basically the same as that in Example 10, and the main differences are: (1) Place the reaction system in a water bath at 10°C and react for 3 h; (2) Separate the protein phase and the aqueous phase in the reaction product by low-temperature centrifugation at 2°C.

[0090] Example 12

[0091] A method for CO 2 fixation and amino acid production by phase separation in this example is basically the same as that in Example 10, and the main differences are: Place the reaction system in a water bath at 80°C and react for 0.5 h; (2) Separate the protein phase and the aqueous phase in the reaction product by low-temperature 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, T-Laf1 fusion protein of the present invention.

[0094] I. Experimental method

[0095] Taking Example 4 and Example 7 as examples, after constructing the corresponding reaction systems respectively, observe the phase separation and the fluidity of the droplets under a microscope within 30 min.

[0096] II. Experimental results

[0097] The results are as Figure 5 、 Figure 6 shown. From Figure 5 、 Figure 6It can be seen that whether it is the "fusion proteins P-Laf1, T-Laf1, H-Laf1" or the "fusion proteins P-Laf1, T-Laf1, and H-Laf1 added with free Laf1 protein simultaneously", they all have the function of phase separation, and the fluidity of the solution is relatively strong. Moreover, multiple small droplets quickly converge into a large droplet within 30 minutes, further proving 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 the fusion addition, free addition, and free addition amount of Laf1 protein on the production of glycine in the present invention.

[0100] I. Experimental Method

[0101] Set the following two series of groups:

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

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

[0104] After constructing the corresponding RGP reaction systems according to the compositions of each group, place them in a water bath at 37°C for 1 h for the first reaction. After the first reaction ends, centrifuge the reaction product at 4°C and 12,000 g for 3 min. Aspirate the supernatant after centrifugation, detect the glycine yield, and at the same time resuspend the precipitate (containing H-Laf1, P-Laf1, T-Laf1 fusion proteins) with Tris to obtain a precipitate resuspension. Take 4 μL of each of the aforementioned supernatant and precipitate resuspension for SDS-PAGE verification. Subsequently, put the precipitate resuspension into a new reaction system to produce glycine again; the reaction is carried out 5 times in total, and the glycine yield obtained from each reaction is detected.

[0105] In this example, the "pre-column dansyl chloride derivatization method" is specifically used to determine the glycine concentration in the reaction mixture: Mix 40 μL of the reaction product with 160 μL of 0.2 M NaHCO 3 and 200 μL of 5.4 mg mL -1Dansyl chloride was mixed in acetonitrile. The derivatization reaction was carried out at 30 °C for 30 min. After the reaction, 600 μL of 0.12 M HCl was added to adjust the pH of the sample to be 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, 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 detection, degassing was performed with 75% solution A and 25% solution B, and the flow rate was 1.0 mL·min -1 . The ratio of solution A and solution B remained unchanged, and the column was flushed with a gradient, (0.2 - 0.4 - 0.6 - 0.8) mL·min -1 , and then the sample was detected.

[0107] II. Experimental Results

[0108] The microscopic phenotypes of the reaction systems of Series Group 1 and Series Group 2 after the first reaction for 1 h and the corresponding detection results of glycine production are as Figure 7 shown. The second glycine production of Laf1 with 0, 30, 60, 90 μM in Series Group 1 after cyclic reactions was all 0. The glycine production results of the multiple cyclic reactions of the reaction system of Series Group 2 are as Figure 8 shown.

[0109] From the above results, it can be seen that: (1) When the addition amounts of free Laf1 protein are all 0, compared with the original GCS proteins P, T, and H (traditional method), the glycine yields using the P-Laf1, T-Laf1, and H-Laf1 fusion proteins of the present invention are significantly increased; (2) When based on the "original GCS proteins P, T, and H", using the phase separation generated by free Laf1 protein and PEG 8000, the glycine yield in the first reaction reaches a maximum of 3.3 mM at 1 h; while in the phase separation system constructed by P-Laf1, T-Laf1, H-Laf1 proteins and free Laf1 protein, the first glycine yield reaches a maximum of about 5.3 mM at 1 h, with a 61% increase in yield; (3) When based on the "original GCS proteins P, T, and H", even when adding different concentrations of free Laf1 protein additionally, the second glycine yields are all 0; while when based on the "P-Laf1, T-Laf1, H-Laf1 fusion proteins of the present invention", under the condition of 20 μM free Laf1, the second reaction glycine yield is 85% of the first time, the third reaction still retains 57% of the yield, the fourth is 42%, and the fifth remains 32% of the yield; for the samples in the 30 μM Laf1 group, the second glycine yield can be retained as high as 99% of the first time, the third still retains 73%, the fourth is 56%, and the fifth is 52.5%; (4) The recovered fusion protein can be reused 5 times, the yield of the second reaction is ≥80%, the total yield of 5 cycles reaches 2.8 times of the initial, and the enzyme usage cost is reduced by 60%; (5) When based on the "original GCS proteins P, T, and H", as the concentration of free Laf1 increases, its clusters or aggregations become more and more obvious; when based on the "P-Laf1, T-Laf1, H-Laf1 fusion proteins of the present invention", no obvious lumps are formed, but many obvious droplets are formed, with better fluidity and mass transfer effect.

[0110] Experimental Example 3

[0111] This experimental example is used to verify the temperature-responsive characteristics of the P-Laf1, T-Laf1, and H-Laf1 fusion proteins of the present invention and their enrichment effect on proteins.

[0112] I. Temperature-responsive characteristics

[0113] The P-Laf1, T-Laf1, and H-Laf1 fusion protein solutions of the present invention have the physical property of "forming a gel at low temperature and becoming transparent again at high temperature". In this experimental example, taking the reaction systems of two series groups (Series Group 1 and Series Group 2) in Experimental Example 2 as examples, by measuring the change of its OD 600 value with temperature (2 - 40 °C) for verification.

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

[0115] II. Enrichment effect on proteins

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

[0117] Experimental Example 4

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

[0119] I. Experimental method

[0120] Based on "the P-Laf1, T-Laf1, and H-Laf1 fusion proteins of the present invention", a reaction system without PEG 8000 was constructed (other components were the same as in Example 4), and its microscopic phenotype was observed. At the same time, the absorbance value at OD 600nm was detected.

[0121] II. Experimental results

[0122] The microscopic phenotype results are as Figure 11 shown, and the absorbance value results at OD 600nm are close to the value of a transparent solution. Thus, it can be known that the phase separation effect of simply adding H-Laf1 protein, P-Laf1 protein, and T-Laf1 protein is very weak, and the absorbance value of the solution at OD 600nm is close to the value of a transparent solution. The addition of the molecular crowding agent PEG 8000 can cause the solution to become turbid immediately and obvious phase separation occurs (combined with the results of the previous experimental examples), which further affects the subsequent glycine production.

[0123] In summary, the present invention creatively fuses the phase separation functional module (IDR sequence) of Laf1 protein with the key enzymes (P, T, H proteins) of the GCS system to construct H-Laf1, P-Laf1, and T-Laf1 fusion proteins with autonomous phase separation ability. Through the synergistic effect of PEG 8000 molecular crowding agent and the fusion proteins, a dynamic membraneless microreactor is formed in vitro to achieve substrate concentration and mass transfer optimization. In the first reaction, the glycine production is increased by more than 60% compared with the existing method. At the same time, by utilizing the temperature-responsive characteristics of the IDR tag, the present invention develops a temperature-responsive protein recovery technology, and the protein phase (H-Laf1, P-Laf1, T-Laf1 fusion proteins) can be quickly separated by low-temperature centrifugation, realizing the recycling of the GCS enzyme system (after 5 cycles, the yield still remains 52.5%), significantly reducing the production cost.

[0124] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A phase separation system, characterized in that: It includes H-Laf1, P-Laf1 and T-Laf1 fusion proteins; wherein the H-Laf1 fusion protein is obtained by fusing the aminomethyl 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 aminomethyltransferase T protein 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.

2. The phase separation system according to claim 1, characterized in that Also includes PEG 8000.

3. The phase separation system according to claim 1, characterized in that Free Laf1 protein was also included.

4. A method for CO2 fixation and amino acid production by phase separation, characterized in that: The phase separation system according to any one of claims 1 to 3 comprises the following steps: (1) constructing a reaction system including "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 to form a dynamic membraneless microreactor; (2) placing the reaction system constructed in step (1) in a water bath for reaction; (3) Using temperature-responsive separation technology, the protein phase and the aqueous phase in the reaction product are separated, and the H-Laf1, P-Laf1, and T-Laf1 fusion proteins in the protein phase are recovered and recycled.

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

7. The method for CO2 fixation and amino acid production by phase separation according to claim 4, characterized in that: In the 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 respectively comprise substrates and buffers required for the reduction of glycine pathway.

8. The method for CO2 fixation and amino acid production by phase separation according to claim 7, characterized in that: The substrates include tetrahydrofolic acid, dithiothreitol, a carbon-one substrate, pyridoxal phosphate, and ammonium salt.

9. The method for CO2 fixation and amino acid production by phase separation according to claim 4, characterized in that: In the step (2), the reaction temperature is 10 to 80° C. and the reaction time is 0.5 to 3 h.

10. The method for CO2 fixation and amino acid production by phase separation according to claim 4, characterized in that: In the step (3), the specific operations are as follows: ① Separating the protein phase and the aqueous phase in the reaction product by low-temperature centrifugation at 2-20°C; 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 H-Laf1, P-Laf1, T-Laf1 fusion protein, free Laf1 protein; ② The separated protein phase is put back into a new reaction system to react again to produce glycine; this process is cyclically used several times.

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