A method for the continuous synthesis of glutamate and / or alanine in artificial vesicles

Abstract

The application belongs to the technical field of biotechnology, and specifically provides a method for continuously synthesizing glutamic acid and / or alanine in an artificial vesicle, which utilizes ethanol, alpha-ketoglutaric acid and pyruvic acid with membrane permeability to realize stable generation of the two amino acids through a multi-stage enzyme reaction, meets the demand of continuous supply of amino acids for a synthetic cell, and improves the functional integrity and application potential of the synthetic cell.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically provides a method for the continuous synthesis of glutamic acid and / or alanine in artificial vesicles. This method uses small molecules that can diffuse freely across the membrane as precursors to synthesize amino acids, without relying on membrane proteins; it has high stability and is simple to operate. Background Technology

[0002] A fundamental goal of synthetic biology is to construct synthetic cells from the smallest functional modules that realize core life processes, thereby exploring the origin and evolution of life. In recent years, reconstructing enzyme cascades and metabolic pathways within cell-sized compartments (such as artificial phospholipid vesicles) has enabled the mimicking of key life functions such as intracellular synthesis, energy metabolism, and morphological regulation, demonstrating significant application value in in vitro biosynthesis, drug delivery, and intelligent microreactors. However, a key obstacle to advancing from isolated functions to higher stages lies in sustainability—the ability of synthetic cells to continuously obtain energy and build modules from their environment. Amino acids are the basic building blocks of protein translation and are crucial for the growth and development of synthetic cells. Achieving sustainable amino acid synthesis within artificial vesicles remains a key bottleneck in synthetic cell research.

[0003] Recent literature reports the reconstruction of an archaic nonphosphorylated glycolysis pathway within artificial vesicles. This involves encapsulating three key enzymes—glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxyglucuronide aldolase (KDGA)—to convert glucose into pyruvate. Furthermore, the introduction of alanine dehydrogenase (AlaDH) and ammonium ions enables the synthesis of alanine from pyruvate within the vesicles. This approach represents the first successful implementation of complete metabolic synthesis from glucose to amino acids within artificial cells, demonstrating the feasibility of constructing an amino acid synthesis pathway within vesicles.

[0004] However, the aforementioned existing technologies still have significant shortcomings: (1) It can only utilize one molecule of glucose to metabolize one molecule of alanine, resulting in low energy utilization efficiency; (2) It relies on glucose pre-embedded in vesicles, cannot continuously obtain substrates from the outside, and has byproducts generated in the metabolic pathway, thus failing to achieve continuous and stable synthesis of alanine. (3) It only supports the synthesis of alanine and cannot generate other important amino acids such as glutamic acid at the same time, thus limiting its application scenarios; Therefore, there is an urgent need for a technical solution that can achieve continuous, stable, and parallel synthesis of multiple amino acids within artificial vesicles to address the shortcomings of existing systems, such as single product, low efficiency, and inability to produce continuously. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide a method for the continuous synthesis of glutamic acid and / or alanine in artificial vesicles. The method utilizes ethanol, α-ketoglutarate and pyruvate, which have membrane permeability, to achieve the stable generation of the two amino acids through a multi-cascade enzyme reaction, thereby meeting the continuous supply of amino acids required by the synthetic cells and improving the functional integrity and application potential of the synthetic cells.

[0006] This invention is achieved through the following technical solution: A method for the continuous synthesis of glutamate and / or alanine within artificial vesicles, comprising the following steps: 1) Preparation of phospholipid-containing mineral oil; 2) Prepare the inner phase solution and outer phase solution of artificial phospholipid vesicles; 3) Constructing the oil-water interface: Slowly drop the phospholipid-containing mineral oil prepared in step 1) onto the surface of the external phase solution prepared in step 2), and let it stand to form a stable oil-water interface; 4) Preparation of oil-water emulsion: Add the internal phase solution prepared in step 2) to the bottom of the phospholipid-containing mineral oil prepared in step 1), and form an oil-water emulsion by friction; 5) Preparation of artificial phospholipid vesicles: Immediately add the oil-water emulsion obtained in step 4) slowly to the oil-water interface obtained in step 3), centrifuge, remove the upper mineral oil, and shake and resuspend the lower aqueous solution to obtain an artificial phospholipid vesicle suspension. 6) Initiating the synthesis of glutamate and / or alanine within vesicles: Adding substrates to the artificial phospholipid vesicle suspension prepared in step 5) to achieve the continuous synthesis of glutamate and / or alanine under the catalysis of a multi-enzyme cascade; wherein, when synthesizing glutamate, the substrates are ethanol and α-ketoglutarate; when synthesizing alanine, the substrates are ethanol and pyruvate; when synthesizing glutamate and alanine simultaneously, the substrates are ethanol, α-ketoglutarate, and pyruvate.

[0007] In this system, the substrate freely crosses the membrane and enters the vesicle, along with ethanol and NAD. + NADH and acetaldehyde are synthesized under the action of alcohol dehydrogenase. α-ketoglutarate, NADH and ammonium ions are synthesized into glutamate under the action of glutamate dehydrogenase. Pyruvate, NADH and ammonium ions are synthesized into alanine under the action of alanine dehydrogenase. NADH is recycled and regenerated. Other substrates and products other than amino acids can cross the membrane to enter or exit vesicles, thereby realizing the continuous synthesis of glutamate and alanine under the catalysis of multiple enzyme cascades. The phospholipids in step 1) include phospholipids 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol in a mass ratio of (20-30):1, preferably in a mass ratio of 24.4:1.

[0008] Furthermore, in step 2), the internal phase solution contains Tris-HCl, magnesium chloride, ammonium acetate, alcohol dehydrogenase, amino acid dehydrogenase, and NAD. + An aqueous solution of sucrose, wherein sucrose is used to provide a density difference to facilitate centrifugal sedimentation during vesicle preparation; when synthesizing glutamic acid, the amino acid dehydrogenase is glutamate dehydrogenase; when synthesizing alanine, the amino acid dehydrogenase is alanine dehydrogenase; when synthesizing glutamic acid and alanine simultaneously, the amino acid dehydrogenases are glutamate dehydrogenase and alanine dehydrogenase. The external phase is an aqueous solution containing Tris-HCl, magnesium chloride, ammonium acetate, and glucose; the glucose concentration is adjusted to ensure that the osmotic pressure difference between the internal and external phases is below 50 osm / L.

[0009] Furthermore, in step 2), the internal phase solution contains 50 mM Tris-HCl, 5 mM magnesium chloride, 10 mM ammonium acetate, 0.02 g / L alcohol dehydrogenase, 0.2 g / L glutamate dehydrogenase and / or 20 U / L alanine dehydrogenase, and 4 mM NAD. + An aqueous solution of 200 mM sucrose, with the sucrose used to provide a density difference to facilitate centrifugal sedimentation during vesicle preparation; The external phase was an aqueous solution containing 50 mM Tris-HCl, 5 mM magnesium chloride, 10 mM ammonium acetate and 300 mM glucose. The osmotic pressure of the internal and external phases was measured, and the glucose concentration was adjusted to ensure that the osmotic pressure difference between the internal and external phases was below 50 osm / L.

[0010] Furthermore, the final substrate concentration in the system of step 6) is: (0.25-4) mM α-ketoglutarate and / or (0.25-4) mM alanine, 40 mM ethanol, preferably 4 mM α-ketoglutarate and / or 4 mM alanine, 40 mM ethanol.

[0011] Further, the process of preparing phospholipid-containing mineral oil in step 1) is as follows: phospholipids 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine POPC and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000 DOPE-PEG2000 are dissolved in chloroform and mixed to obtain a phospholipid chloroform solution. After vacuuming the above phospholipid chloroform solution, mineral oil is added, and the mixture is sonicated until the phospholipids are fully dissolved to obtain phospholipid-containing mineral oil with a total phospholipid concentration of 0.2-0.3 g / L.

[0012] Furthermore, in step 3), the volume ratio of the phospholipid-containing mineral oil to the external phase solution is 5:8; Furthermore, in step 4), the volume ratio of the phospholipid-containing mineral oil to the internal phase solution is 25:1; This invention also provides the application of the above method in in vitro protein synthesis.

[0013] Furthermore, the application is as follows: the application obtains the target protein by introducing DNA encoding the target protein as a template into an artificial vesicle and translating it into the artificial vesicle.

[0014] Furthermore, the specific operation of the application is as follows: A) Preparation of phospholipid-containing mineral oil; B) Preparation of the inner phase solution and outer phase solution of artificial phospholipid vesicles; C) Constructing the oil-water interface: Slowly add the phospholipid-containing mineral oil prepared in step A) to the surface of the external phase solution prepared in step B), and let it stand to form a stable oil-water interface; D) Preparation of oil-water emulsion: Add the internal phase solution prepared in step B) to the bottom of the phospholipid-containing mineral oil prepared in step A), and form an oil-water emulsion by friction; E) Preparation of artificial phospholipid vesicles: Immediately add the oil-water emulsion obtained in step D) slowly to the oil-water interface obtained in step C), centrifuge, remove the upper mineral oil, and shake and resuspend the lower aqueous solution to obtain an artificial phospholipid vesicle suspension. F) Initiate the synthesis of glutamate and alanine in vesicles: Add substrate ethanol, α-ketoglutarate and pyruvate to the artificial phospholipid vesicle suspension prepared in step E) to achieve the continuous synthesis of glutamate and alanine; The components of the inner phase solution described in step B) include: transfer RNA, 18 amino acids (excluding glutamate and alanine) that constitute human proteins, dextran 60, ammonium acetate, alcohol dehydrogenase, glutamate dehydrogenase, alanine dehydrogenase, and NAD. + and the DNA template encoding the target protein; The external phase solution described in step B) is an aqueous solution containing HEPES, magnesium acetate, ammonium acetate, sorbitol, 18 amino acids that constitute human proteins except glutamic acid and alanine, TCEP, folinic acid, spermidine, and glycerol.

[0015] Other reaction conditions are controlled under the same conditions as those specified in the above-described method for the continuous synthesis of glutamic acid and / or alanine within artificial vesicles.

[0016] Furthermore, the sequence of the eGFP DNA is shown in SEQ ID NO.1.

[0017] The advantages and beneficial effects of this invention compared to the prior art include: 1. Using uncharged phospholipid POPC as the main component of the phospholipid membrane allows uncharged small molecules to cross the membrane. Employing transmembrane-transferable small molecules such as ethanol, α-ketoglutarate, and pyruvate as substrates, ethanol, as an uncharged amphiphilic molecule, can freely cross the membrane. The undissociated molecular forms of the small-molecule acids α-ketoglutarate and pyruvate are uncharged and can pass through the electrically neutral POPC phospholipid membrane. Therefore, continuous substrate replenishment can be achieved without a complex transport system, ensuring stable and uninterrupted amino acid synthesis. This results in synthesis efficiency far exceeding existing technologies: compared to the system for synthesizing glutamic acid from pre-encapsulated D-ribose, the glutamic acid synthesis rate of this invention is increased by 57.6 times; compared to the system for synthesizing alanine from pre-encapsulated glucose via glycolysis, the alanine synthesis rate of this invention is increased by over 100 times. The accompanying NADH fluorescence quantitative standard curve allows for accurate calculation of amino acid yield, and the quantitative data directly demonstrates the order-of-magnitude improvement in synthesis rate achieved by this method.

[0018] That is, this invention does not rely on pre-encapsulated substrates, but utilizes a combination of specific vesicle membrane components and substrates to achieve exogenous substrate addition, with the substrate continuously supplying amino acid synthesis via transmembrane entry into the vesicles. Specifically, it employs electrically neutral phospholipid molecules (POPC) as vesicle membrane components. Small-molecule substrates capable of permeation, such as ethanol and weakly acidic α-ketoglutarate and / or pyruvate, are added to the outside of the vesicles. Ethanol and α-ketoglutarate and / or pyruvate then cross the membrane into the vesicles. Ethanol is converted to NADH by ADH enzymes, and NADH, along with α-ketoglutarate and / or pyruvate, are converted to glutamate and / or alanine by GDH enzymes and / or AlaDH enzymes. When the ethanol and α-ketoglutarate and / or pyruvate within the vesicles are consumed, the external substrate can be continuously supplied via transmembrane entry, thereby achieving continuous amino acid production.

[0019] 2. Simultaneously construct glutamic acid and alanine synthesis pathways to achieve simultaneous production of two amino acids, significantly improving the diversity of raw material supply for synthetic cells; 3. The preparation process is simple and reproducible, making it suitable for in vitro biosynthesis, biomimetic microreactors, synthetic cell construction, and metabolic function repair, with broad application prospects. Attached Figure Description

[0020] Figure 1 The flowchart is a method for synthesizing amino acids in artificial vesicles provided by the present invention; Figure 2 The image shows a fluorescence image of NADH synthesis monitored within vesicles in Example 1. Figure 3 This is a statistical graph showing the average fluorescence intensity of NADH in vesicles at different concentrations of α-ketoglutarate in Example 1. Figure 4 This is a statistical graph showing the average fluorescence intensity of NADH in vesicles at different concentrations of pyruvate in Example 1. Figure 5 This is a fluorescence image of the detection of eGFP synthesis within vesicles in Example 2; Figure 6 This is a statistical graph of the eGFP fluorescence intensity within the vesicles in Example 2; Figure 7 This is a schematic diagram illustrating how the synthesis of glutamate and alanine within vesicles drives eGFP synthesis in Example 2. Figure 8 This is a standard curve showing the fluorescence intensity and concentration of NADH in the vesicles in Example 1. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but the present invention does not limit the implementation thereto.

[0022] The Tris-HCl used in the following examples is 1 mol / L Tris-HCl at pH 7.5.

[0023] The system contains 18 amino acids, excluding glutamic acid and alanine, including glycine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, aspartic acid, lysine, arginine, and histidine.

[0024] The eGFP DNA (SEQ ID NO.1) used in this invention was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0025] Example 1: Synthesis and rate regulation of glutamate or alanine in vesicles 1) Preparation of phospholipid-containing mineral oil: 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000 (DOPE-PEG2000), and ATTO647-labeled 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (ATTO647-DOPE) were purchased from Avanti Lipids. Each phospholipid was pre-dissolved in chloroform and transferred to 2 mL amber glass vials, sealed with sealing film, and stored at -20°C for long-term storage. When using, 16 μL of 25 mg / mL POPC, 16.4 μL of 1 mg / mL DOPE-PEG2000, and 2 μL of 1 mg / mL ATTO647-DOPE were placed in 2 mL amber glass vials, and the vials were evacuated for 15 min until the chloroform evaporated. Add 1600 μL of mineral oil (purchased from Sigma, catalog number M8410), seal the sample vial with sealing film, and sonicate in a 45°C water bath for 120 min to fully dissolve the phospholipids, resulting in mineral oil containing 0.2615 g / L of phospholipids. The addition of ATTO647-labeled 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (ATTO647-DOPE) was to observe the vesicle membrane and determine the location of the vesicles.

[0026] 2) Preparation of the inner and outer phase solutions for artificial phospholipid vesicles: The inner phase solution is 100 μL containing a final concentration of 50 mM Tris-HCl, 5 mM magnesium chloride, 10 mM ammonium acetate, 0.02 g / L alcohol dehydrogenase (ADH), 0.2 g / L glutamate dehydrogenase (GDH, used for glutamate synthesis) or 20 U / L alanine dehydrogenase (AlaDH, used for alanine synthesis), and 4 mM NAD. + The external phase solution is a 200 μL aqueous solution containing 50 mM Tris-HCl, 5 mM magnesium chloride, 10 mM ammonium acetate, and 300 mM glucose. The reagents listed above, except for NAD+, are... + All external components were purchased from Sigma, NAD + Purchased from Roche, part number 10127981001. The osmotic pressure of the internal and external phases was measured using an OSMOMAT 3000 osmometer, and the osmotic pressure difference was found to be less than 50 osm / L, which meets the conditions for vesicle preparation.

[0027] 3) Constructing the oil-water interface: Add 200 μL of external phase solution to a 1.5 mL EP tube, and slowly add 125 μL of mineral oil containing 0.2615 g / L phospholipids to the surface of the external phase solution. Let it stand for 30 min to form a stable oil-water interface.

[0028] 4) Preparation of oil-water emulsion: Take a 1.5 mL EP tube, first add 250 μL of mineral oil containing 0.2615 g / L phospholipids, then slowly add 10 μL of internal phase solution to the bottom of the tube, attach the bottom of the tube to the left well wall of the 96-well PCR plate, and rub vigorously from left to right 15 times until the solution becomes a uniform milky white, thus completing the preparation of the oil-water emulsion.

[0029] 5) Preparation of artificial phospholipid vesicles: Immediately, slowly stack the oil-water emulsion obtained in step 4) onto the oil-water interface formed in step 3), centrifuge at 2000 rcf for 8 min at room temperature, remove most of the upper mineral oil with a 200 μL pipette tip, and then completely remove the remaining mineral oil with a 20 μL pipette tip. Then gently vortex and resuspend the lower aqueous layer to obtain an artificial phospholipid vesicle suspension containing an amino acid synthase chain system.

[0030] 6) Initiate the synthesis of glutamate or alanine within vesicles: For glutamate synthesis: 45 μL, 44.875 μL, 44.5 μL, and 43 μL of the above vesicle suspensions were transferred to the wells of a 384-well glass-bottom plate pretreated with 30 μL of 1 mg / mL PLL20-g-PEG(7)2K (purchased from Ruixi Biotechnology Co., Ltd., catalog number R-PL1152-2K-200mg) for 30 min. The plate was placed under a Nikon confocal microscope, and the integrity of the vesicle membrane was observed by excitation at 647 nm. Subsequently, 5 μL of 1M ethanol was added to each of the four wells, followed by 100 mM α-ketoglutarate (0 μL, 0.125 μL, 0.5 μL, and 2 μL were added to the four wells respectively). The final ethanol concentration was 100 mM, and the final concentrations of α-ketoglutarate were 0 mM, 0.25 mM, 1 mM, and 4 mM respectively. For alanine synthesis, α-ketoglutarate is replaced with pyruvate, while other conditions remain unchanged. During the process, the substrates ethanol, pyruvate, and α-ketoglutarate freely cross the membrane into the vesicle, where they continuously synthesize glutamate or alanine under the catalysis of a multi-enzyme cascade.

[0031] 7) Vesicle structure observation and amino acid synthesis verification: Artificial phospholipid vesicles were observed using a Nikon AX laser confocal microscope with dual-channel time-series imaging. ATTO647-DOPE was embedded in the vesicle membrane and excited by a 647 nm laser to locate the vesicles and observe the membrane structure integrity. Utilizing the intrinsic fluorescence properties of NADH, it was excited by a 405 nm laser, and the dynamic changes in fluorescence intensity in this channel were used to monitor the changes in NADH content during the synthesis of glutamate or alanine within the vesicles in real time.

[0032] Imaging parameters were set as follows: resolution 1024×1024, 405nm laser intensity 15, gain 50, 647nm laser intensity 2, gain 5. A frame was acquired every 3 minutes, with continuous time-series imaging for 45 minutes to avoid photobleaching and stably capture reaction dynamics. Single-vesicle fluorescence intensity was quantitatively analyzed using Fiji software. Data statistics and plotting were performed using GraphPad Prism10. Three independent replicate experiments were conducted, with error bars representing ± standard deviation. Each experiment included more than 200 vesicles.

[0033] In this embodiment, the accumulation of NADH within the vesicles is as follows: Figure 2 As shown, when only ethanol is added, ethanol enters the vesicles and, under the action of alcohol dehydrogenase, continuously synthesizes NADH, which then accumulates in the vesicles. Figure 2 (Top left, bottom left) When ethanol and pyruvate are added simultaneously, pyruvate also crosses the membrane into the vesicle and is synthesized into alanine by alanine dehydrogenase along with NADH and ammonium ions. This slows down the accumulation of NADH in the vesicle. Figure 2 The upper and lower middle sections indicate that alanine continuously synthesizes and consumes NADH within the vesicles. When ethanol and α-ketoglutarate are added simultaneously, α-ketoglutarate also crosses the membrane into the vesicles and, together with NADH and ammonium ions, is catalyzed by glutamate dehydrogenase to synthesize glutamate. NADH levels within the vesicles remain low. Figure 2 (Top right, bottom right) indicates that glutamate is continuously synthesized and consumes NADH within the vesicles.

[0034] In this embodiment, the changes in fluorescence intensity of NADH within vesicles after the addition of different concentrations of α-ketoglutarate are statistically analyzed as follows: Figure 3 As shown, the accumulation rate of NADH is highest when α-ketoglutarate is not added; as the concentration of α-ketoglutarate increases, the accumulation rate of NADH gradually decreases until it approaches 0. The magnitude of the decrease in the NADH accumulation rate reflects the synthesis rate of glutamate. The higher the concentration of α-ketoglutarate, the greater the decrease in the NADH accumulation rate, indicating that the concentration of α-ketoglutarate is positively correlated with the synthesis rate of glutamate.

[0035] In addition, the vesicles for encapsulating NADH standards were prepared according to the above steps, with the following differences: ① The internal phase solution was 100 μL of an aqueous solution containing 50 mM Tris-HCl, 5 mM magnesium chloride, 10 mM ammonium acetate, 0-4 mM NADH standards, and 200 mM sucrose at a final concentration; the external phase solution was 200 μL of an aqueous solution containing 50 mM Tris-HCl, 5 mM magnesium chloride, 10 mM ammonium acetate, and 300 mM glucose at a final concentration. The NADH standards were purchased from Roche, catalog number 10128023001; ② The operation in step 6) was not required.

[0036] Standard curves of NADH fluorescence intensity versus NADH concentration within vesicles were obtained by encapsulating NADH standards at different concentrations, as follows: Figure 8 As shown in the figure. Based on the standard curve, it was calculated that without the addition of α-ketoglutarate, 3.78 ± 0.32 mM of NADH was synthesized from ethanol within 42 min. With the addition of 4 mM α-ketoglutarate, there was virtually no NADH accumulation, indicating that α-ketoglutarate consumed ≥3.78 mM of NADH in the synthesis of glutamate. Since glutamate dehydrogenase consumes one molecule of NADH to synthesize one molecule of glutamate, it indicates that ≥3.78 mM of glutamate was synthesized within the vesicles. The calculated glutamate synthesis rate was approximately 5.40 mM / h. Compared to Liu Y et al., who synthesized 184.32 μM of glutamate in 120 min using pre-encapsulated D-ribose and α-ketoglutarate as substrates within vesicles, the synthesis rate increased by approximately 57.6 times, demonstrating the high efficiency of this invention in continuously synthesizing amino acids using permeabilizing substances.

[0037] In this embodiment, the changes in fluorescence intensity of NADH within vesicles after the addition of different concentrations of pyruvate are statistically analyzed as follows: Figure 4 As shown, a phenomenon similar to that observed in glutamate synthesis vesicles was observed, indicating a positive correlation between pyruvate concentration and alanine synthesis rate. Based on the standard curve, it was calculated that without pyruvate, 4.05 ± 0.49 mM NADH was synthesized from ethanol within 42 min. With the addition of 4 mM pyruvate, the NADH accumulation was 0.98 ± 0.36 mM, indicating that pyruvate consumption for alanine synthesis consumed ≥3.07 mM of NADH. Since alanine dehydrogenase consumes one molecule of NADH to synthesize one molecule of alanine, this indicates that ≥3.07 mM of alanine was synthesized within the vesicles. The calculated alanine synthesis rate is approximately 4.39 mM / h. Compared to Yang B et al., who synthesized 210 ± 39 μM alanine in 18 h using pre-encapsulated glucose as a substrate within vesicles, this represents a synthesis rate increase of over 100 times, demonstrating the high efficiency of this invention in continuously synthesizing amino acids using permeabilizing substances.

[0038] Example 2: Continuous synthesis of glutamate and alanine within vesicles for application in green fluorescent protein synthesis 1) Preparation of phospholipid-containing mineral oil: Same as in Example 1.

[0039] 2) Preparation of the inner and outer phase solutions of artificial phospholipid vesicles: The NEB PURExpress (ΔAA, tRNA) protein in vitro transcription and translation kit (catalog number E6840S) was used for the synthesis of enhanced green fluorescent protein (eGFP), which contains Solution A, Solution B, amino acids and transfer RNA.

[0040] The internal phase solution was 10 μL containing 1 × Solution A, 1 × Solution B, 1 × transfer RNA (each at 0.3 mM), 18 amino acids (excluding glutamate and alanine), 25 g / L dextran 60, 20 mM ammonium acetate, 0.1 g / L alcohol dehydrogenase (ADH), 0.085 g / L glutamate dehydrogenase (GDH), 20 U / L alanine dehydrogenase (AlaDH), and 4 mM NAD. + An aqueous solution of 4nMeGFP DNA.

[0041] The external phase was a 200 μL aqueous solution containing 200 mM HEPES (pH 7.5), 29.52 mM magnesium acetate, 20 mM ammonium acetate, 500 mM sorbitol, 18 amino acids (excluding glutamic acid and alanine) at 0.3 mM each, 25 mM TCEP, 50 μM folinic acid, 5 mM spermidine, and 2% glycerol by volume.

[0042] 3) Constructing the oil-water interface: Same as in Example 1.

[0043] 4) Preparation of oil-water emulsion: Same as in Example 1.

[0044] 5) Preparation of artificial phospholipid vesicles: Same as in Example 1.

[0045] 6) Initiation of glutamate and alanine synthesis within vesicles: 44 μL of vesicles were transferred to the wells of a 384-well glass-bottom plate pretreated with 30 μL of 1 mg / mL PLL20-g-PEG(7)2K for 30 min. 2 μL of 1 M ethanol, 2 μL of 100 mM α-ketoglutarate, and 2 μL of 100 mM pyruvate were added, resulting in a final concentration of 40 mM ethanol, 4 mM α-ketoglutarate, and 4 mM pyruvate. Simultaneously, a control experiment was performed with substrates missing: ethanol, α-ketoglutarate, and pyruvate were removed, as well as α-ketoglutarate and pyruvate were removed simultaneously. The missing substrates were replaced with an equal volume of sterile water. The vesicles were then incubated at 37°C for 3 hours to initiate protein synthesis.

[0046] 7) Vesicle structure observation and eGFP synthesis detection: After vesicle incubation for 3 hours, observation was performed using a Nikon AX laser confocal microscope. Vesicle location and morphology were examined using fluorescence excitation at 647nm wavelength. eGFP synthesis was detected by fluorescence excitation within the vesicles at 488nm wavelength. Microscope imaging parameters were as follows: resolution 1024×1024, 488nm laser intensity 10, gain value 20, 647nm laser intensity 2, gain value 5, and a shooting time of 3.5 seconds per image. Three different locations were selected for each group of images. Image processing was performed using Fiji software, and statistical graphs were plotted using Graphpadprism 10 software. Three independent replicate experiments were conducted, with more than 200 vesicles counted in each group.

[0047] In this embodiment, the fluorescence image of the synthesized eGFP vesicles is shown below. Figure 5 As shown, the fluorescence intensity statistics of vesicles expressing eGFP are as follows: Figure 6 As shown in the diagram, the vesicle system is as follows: Figure 7 As shown, ethanol and NAD + NADH and acetaldehyde are synthesized under the action of alcohol dehydrogenase. α-ketoglutarate, NADH, and ammonium ions are synthesized into glutamate under the action of glutamate dehydrogenase. Pyruvate, NADH, and ammonium ions are synthesized into alanine under the action of alanine dehydrogenase. The synthesized glutamate and alanine are used by the in vitro transcription-translation system (PURE) lacking glutamate and alanine for the synthesis of eGFP. When ethanol, α-ketoglutarate, and pyruvate are all added, the vesicle eGFP expression level is the highest; the expression level of vesicles without any one substrate decreases significantly; the expression level of eGFP is lower when both α-ketoglutarate and pyruvate are missing than when only one substrate is missing. This demonstrates that the method of the present invention can continuously synthesize glutamate and alanine in vesicles and supply them to the in vitro protein synthesis system lacking amino acids to restore protein synthesis capacity. The above results indicate that the method of the present invention can continuously synthesize glutamic acid and alanine in artificial vesicles using transmembrane substrates, and the synthesis rate of glutamic acid and alanine in artificial vesicles can be adjusted by regulating the substrate concentration. Furthermore, it is adaptable to in vitro protein synthesis systems, has a simple preparation process, good reproducibility, and broad application prospects.

[0048] Those skilled in the art will readily understand that the above description is merely 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 scope of protection of the present invention.

Claims

1. A method for the continuous synthesis of glutamic acid and / or alanine within artificial vesicles, characterized in that, Includes the following steps: 1) Preparation of phospholipid-containing mineral oil; 2) Prepare the inner phase solution and outer phase solution of the artificial phospholipid vesicles; wherein the inner phase solution contains Tris-HCl (tris-HCl), magnesium chloride, ammonium acetate, alcohol dehydrogenase, amino acid dehydrogenase, and nicotinamide adenine dinucleotide (NAD). + An aqueous solution of sucrose; the external phase solution is an aqueous solution containing Tris-HCl, magnesium chloride, ammonium acetate and glucose; 3) Constructing an oil-water interface: The phospholipid-containing mineral oil prepared in step 1) is slowly added dropwise to the surface of the external phase solution prepared in step 2), and allowed to stand to form a stable oil-water interface; 4) Preparation of oil-water emulsion: The internal phase solution prepared in step 2) is added to the bottom of the phospholipid-containing mineral oil prepared in step 1), and an oil-water emulsion is formed by friction; 5) Preparation of artificial phospholipid vesicles: The oil-water emulsion obtained in step 4) is immediately and slowly added to the oil-water interface obtained in step 3), centrifuged, the upper mineral oil is removed, and the lower aqueous solution is shaken and resuspended to obtain an artificial phospholipid vesicle suspension. 6) Initiate the synthesis of glutamate and / or alanine in vesicles: Add the substrate to the artificial phospholipid vesicle suspension prepared in step 5). The substrate permeates through the membrane into the vesicles, realizing the continuous synthesis of glutamate and / or alanine under the catalysis of a multi-enzyme cascade. In the synthesis of glutamic acid, the substrate is ethanol and α-ketoglutarate, and the amino acid dehydrogenase is glutamate dehydrogenase; in the synthesis of alanine, the substrate is ethanol and pyruvate, and the amino acid dehydrogenase is alanine dehydrogenase; in the simultaneous synthesis of glutamic acid and alanine, the substrate is ethanol, α-ketoglutarate, and pyruvate, and the amino acid dehydrogenase is glutamate dehydrogenase and alanine dehydrogenase; in step 6), after adding the substrate, the final concentration of the substrate is: (0.25-4) mM α-ketoglutarate and / or (0.25-4) mM pyruvate, 40 mM ethanol; The phospholipids in step 1) include phospholipids 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol in a mass ratio of (20-30):

1.

2. The method according to claim 1, characterized in that, The process for preparing phospholipid-containing mineral oil in step 1) is as follows: phospholipids 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000 are dissolved separately in chloroform and mixed to obtain a phospholipid chloroform solution. After vacuuming the above phospholipid chloroform solution, mineral oil is added, and the mixture is sonicated until the phospholipids are fully dissolved to obtain phospholipid-containing mineral oil with a total phospholipid concentration of 0.2-0.3 g / L.

3. The method according to claim 1, characterized in that, The internal phase solution mentioned in step 2) contains 50 mM Tris-HCl, 5 mM magnesium chloride, 10 mM ammonium acetate, 0.02 g / L alcohol dehydrogenase, 0.2 g / L glutamate dehydrogenase and / or 20 U / L alanine dehydrogenase, and 4 mM NAD. + A 200mM aqueous solution of sucrose; The external phase solution is an aqueous solution containing 50 mM Tris-HCl, 5 mM magnesium chloride, 10 mM ammonium acetate and 300 mM glucose.

4. The application of the method according to any one of claims 1-3 in in vitro protein synthesis, wherein the application involves introducing DNA encoding the target protein as a template into an artificial vesicle, and obtaining the target protein through translation in the artificial vesicle, characterized in that... The specific operation of the application is as follows: A) Preparation of phospholipid-containing mineral oil; B) Preparation of the inner phase solution and outer phase solution of artificial phospholipid vesicles; C) Constructing the oil-water interface: Slowly add the phospholipid-containing mineral oil prepared in step A) to the surface of the external phase solution prepared in step B), and let it stand to form a stable oil-water interface; D) Preparation of oil-water emulsion: Add the internal phase solution prepared in step B) to the bottom of the phospholipid-containing mineral oil prepared in step A), and form an oil-water emulsion by friction; E) Preparation of artificial phospholipid vesicles: Immediately add the oil-water emulsion obtained in step D) slowly to the oil-water interface obtained in step C), centrifuge, remove the upper mineral oil, and shake and resuspend the lower aqueous solution to obtain an artificial phospholipid vesicle suspension. F) Initiate the synthesis of glutamate and alanine in vesicles: Add substrate ethanol, α-ketoglutarate and pyruvate to the artificial phospholipid vesicle suspension prepared in step E) to achieve the continuous synthesis of glutamate and alanine; The components of the inner phase solution described in step B) include: Solution A and Solution B from the PURExpress (ΔAA, tRNA) Protein In Vitro Transcription and Translation Kit, transfer RNA, 18 amino acids that constitute human proteins except glutamate and alanine, dextran 60, ammonium acetate, alcohol dehydrogenase, glutamate dehydrogenase, alanine dehydrogenase, NAD+, and DNA template encoding the target protein; The external phase solution described in step B) is an aqueous solution containing 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), magnesium acetate, ammonium acetate, sorbitol, 18 amino acids that constitute human proteins except glutamic acid and alanine, tris(2-carboxyethyl)phosphine (TCEP), folinic acid, spermidine, and glycerol.

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