An optimized lipoic acid protein ligase for glycine production in reverse glycine cleavage system and application thereof

Abstract

The application discloses a lipoic acid protein ligase for optimizing production of glycine in a reverse glycine cleavage system and application thereof, and the lipoic acid protein ligase is derived from Citrobacter koseri. Citrobacter koseri The amino acid sequence is shown as SEQ ID NO. 1. Compared with the prior art, the lipoic acid ligase (ckLplA) derived from the Citrobacter koseri is first discovered and utilized to solve the rate-limiting step of H protein esterification in the rGCS system. After the enzyme of the application is added to the rGCS system, the in-vitro production rate of glycine can be increased to 3 times of the original level at the highest, and the increase is far more than the effect achieved by optimizing traditional conditions or modifying other components, and an unexpected technical effect is achieved.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to the optimization technology of the reverse glycine cleavage system (rGCS), and particularly to a novel lipoic acid protein ligase catalyst that overcomes the rate-limiting bottleneck of this synthetic pathway by enhancing the esterification efficiency of H protein, and its application. Background Technology

[0002] Glycine, as the simplest amino acid in structure, has extremely wide applications in medicine, food, feed, cosmetics, and chemical industries. Currently, large-scale industrial production of glycine mainly relies on chemical synthesis methods, with the Strecker amino acid synthesis and chloroacetic acid ammonolysis being the mainstream methods. However, chemical synthesis methods have many significant drawbacks, including environmental unfriendliness, complex processes, safety challenges, and difficulties in product purification.

[0003] To overcome the drawbacks of chemical methods and achieve a green, mild, and specific synthesis of glycine, Professor Zeng Anping's team at Westlake University constructed a cell-free multi-enzyme catalytic system in vitro. This system combines chemical and biocatalytic methods, utilizing a reversible glycine cleavage system related to one-carbon metabolism, and employing formaldehyde and bicarbonate as substrates to synthesize glycine. In this system, three core proteins participate in the reaction: P protein (glycine decarboxylase), H protein (acylcarrier protein), and T protein (aminomethyltransferase).

[0004] Despite the promising prospects of this multi-enzyme catalytic system, its industrial application has been limited by its low overall catalytic efficiency. Recent studies have shown that the catalytic cycle of rGCS has a significant rate-limiting step. Researchers in this field have also confirmed through in vitro reconstruction experiments that the esterification efficiency of the H protein is a crucial rate-limiting step in the entire rGCS cycle. The low efficiency of this step severely restricts the final yield of glycine.

[0005] Currently, research strategies to improve the efficiency of the rGCS system mainly focus on the engineering modification of the P protein or the optimization of reaction conditions (pH, temperature, etc.), but the effects are not significant, and the yield increase is limited. To date, there are no reports of systematically improving the overall efficiency of rGCS by directly enhancing the rate-limiting step of H protein esterification. Therefore, developing a novel enzyme catalyst capable of specifically and efficiently catalyzing the H protein esterification reaction is of urgent industrial demand and significant technological value for unleashing the full potential of the reverse glycine cleavage system and realizing the efficient and green biomanufacturing of glycine. Summary of the Invention

[0006] The purpose of this invention is to overcome the rate-limiting bottleneck of low H-protein esterification efficiency in existing rGCS systems, and to provide a novel enzyme catalyst capable of specifically and efficiently catalyzing this step. This invention provides the encoding gene of the enzyme, a recombinant expression system, and a preparation method thereof, thereby enhancing the rGCS system and significantly improving the in vitro synthesis rate and yield of glycine. This invention aims to provide a novel and significantly competitive technical solution for the green, efficient, and large-scale biomanufacturing of glycine.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides an octanoic acid protein ligase for optimizing the reverse glycine cleavage system to produce glycine, which is derived from Citrobacter causativeis (Citrobacter causativeis). Citrobacter koseri This enzyme, named ckLplA, can efficiently catalyze the esterification reaction of H protein in the reverse glycine cleavage system.

[0008] Preferably, the enzyme has the amino acid sequence shown in SEQ ID NO.1; Alternatively, the lipoic acid protein ligase may comprise a derivative protein having at least 80%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO.1 and retaining the activity of the lipoic acid protein ligase. Alternatively, the lipoic acid protein ligase may be a fusion protein with a tag sequence fused to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.1; the tag sequence may include, but is not limited to, a histidine tag (His-tag), a glutathione thiotransferase tag (GST-tag), or a maltose-binding protein tag (MBP-tag).

[0009] The present invention also provides biomaterials related to the above-mentioned lipoic acid protein ligase, wherein the biomaterials are any one of the following: (1) The nucleic acid molecule encoding the lipoic acid protein ligase mentioned above; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1), or a recombinant vector containing the expression cassette described in (2); (4) Recombinant microorganisms containing the nucleic acid molecule described in (1), or recombinant microorganisms containing the expression cassette described in (2), or recombinant microorganisms containing the recombinant vector described in (3); (5) A cell line containing the nucleic acid molecule described in (1), or a cell line containing the expression cassette described in (2), or a cell line containing the recombinant vector described in (3).

[0010] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2.

[0011] Specifically, a recombinant expression vector is constructed from the gene sequence shown in SEQ ID NO.2 using genetic engineering techniques; the vector includes, but is not limited to, pET28a, pET22b, pET21a, or pET30a plasmids.

[0012] Furthermore, the recombinant expression vector is transferred into a host cell; the host cell includes, but is not limited to, Escherichia coli, yeast, mammalian cells, or insect expression systems.

[0013] Furthermore, the host cells described above are cultured to induce the expression of the target protein, and then the enzyme is isolated and purified from the culture; the purification method includes, but is not limited to, nickel column affinity chromatography.

[0014] The present invention also provides the application of the lipoic acid protein ligase in enhancing the efficiency of acylcarrier protein esterification reaction.

[0015] The present invention also provides the application of the lipoic acid protein ligase in the preparation of enzyme catalysts that enhance the efficiency of esterification reactions of acyl carrier proteins.

[0016] The present invention also provides an enzyme catalyst for enhancing the efficiency of esterification reactions of acylcarrier proteins, including the aforementioned lipoic acid protein ligase.

[0017] Experimental results in the embodiments of the present invention show that, compared with LplA (ecLplA) derived from Escherichia coli, this enzyme exhibits a faster catalytic rate for the esterification of H protein.

[0018] The present invention provides a system for in vitro production of glycine, the system comprising: core enzyme components necessary for the reverse glycine cleavage system (rGCS), including P protein, H protein and T protein; and ckLplA or the enzyme catalyst described above.

[0019] In a preferred embodiment, the core enzyme component and the lipoic acid protein ligase together constitute an enhanced multi-enzyme catalytic system.

[0020] In a more preferred embodiment, the concentration ratio of P protein, T protein, H protein to ckLplA in the multi-enzyme catalytic system is 1-10 μM: 1-10 μM: 10-100 μM: 1-3 μM.

[0021] The present invention provides a method for producing glycine in vitro, comprising: thoroughly mixing a precursor substrate for glycine synthesis with the above-described system under reaction conditions suitable for the production of glycine, thereby obtaining glycine.

[0022] In one embodiment, the precursor substrate includes tetrahydrofolate, dithiothreitol, a C1 substrate, pyridoxal phosphate, an ammonium salt, lipoic acid, ATP, and magnesium ions, with the following concentration ratios: tetrahydrofolate 0.1-1 mM, dithiothreitol 1-20 mM, C1 substrate 1-20 mM, pyridoxal phosphate 1-25 μM, ammonium salt 10-50 mM, lipoic acid 0.1-2 mM, ATP 0.1-5 mM, and magnesium ions 0.1-5 mM.

[0023] In a preferred embodiment, the carbon-1 substrate is formaldehyde, or a substrate capable of generating formaldehyde in the reaction system, including methanol or formic acid.

[0024] In another preferred embodiment, the ammonium salt is one or more of ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate, ammonium fluoride, or ammonium carbonate.

[0025] In another preferred embodiment, the magnesium ions are derived from one or more of magnesium chloride and magnesium sulfate.

[0026] In another preferred embodiment, the reaction conditions include: a pH range of 6-8, a reaction temperature of 0-47°C, and preferably, a reaction temperature of 37-47°C; the pH range is adjusted using a buffer solution with a concentration of 20-100 mM, wherein the buffer solution is one of Tris-HCl, phosphate, citrate, or acetate buffer.

[0027] Compared to existing technologies, this invention is the first to discover and utilize a lipoic acid ligase (ckLplA) derived from *Citrobacter coccineus* to solve the rate-limiting step of H-protein esterification in the rGCS system. Adding this enzyme to the rGCS system can increase the in vitro production rate of glycine by up to three times, a significant improvement far exceeding what can be achieved by optimizing traditional conditions or modifying other components, resulting in unexpected technological advancements. This invention provides an enzymatic biocatalytic process with mild reaction conditions, eliminating the need for highly toxic chemicals and meeting the requirements of green chemistry and sustainable development. This enzyme can serve as a highly efficient catalytic component, easily integrated into existing in vitro synthetic biology systems without requiring radical changes to the entire process, facilitating industrial scale-up and application. It provides a new technological direction for the efficient biomanufacturing of glycine and has broad application prospects. Attached Figure Description

[0028] Figure 1The image shows the SDS-PAG analysis of purified thioctic acid protein ligase (ckLplA) derived from citrate rods, as described in this invention. In the image, M represents the protein molecular weight standard (Marker); 1 represents 5 μg of ckLplA protein obtained after purification by nickel column affinity chromatography; and 2 represents 2 μg of ckLplA protein obtained after purification by nickel column affinity chromatography.

[0029] Figure 2 This invention relates to the enzyme kinetic analysis of ckLplA enzyme; wherein, a: Michaelis constant and maximum reaction rate of ckLplA enzyme with lipoic acid; b: Michaelis constant and maximum reaction rate of ckLplA enzyme with ATP; c: Michaelis constant and maximum reaction rate of ckLplA enzyme with H protein derived from Escherichia coli; d: Michaelis constant and maximum reaction rate of ckLplA enzyme with H protein derived from Citrobacter causativeis.

[0030] Figure 3 The graph shows a comparison of the reaction rates of glycine production catalyzed by the ckLplA enzyme and the lipoic acid protein ligase (ecLplA) derived from Escherichia coli under the same conditions. In this paper, **** indicates p<0.0001.

[0031] Figure 4 : A comparison chart of the yield of glycine produced by the ckLplA enzyme of this invention at different temperatures. Detailed Implementation

[0032] Example 1: Construction, fermentation, and protein purification of genetically engineered bacteria In this embodiment, the target gene (encoding gene of lipoic acid protein ligase, amino acid sequence as shown in SEQ ID No. 1) of the sequence shown in SEQ ID No. 2 was cloned, an expression vector of the target protein was constructed, and the expression vector was introduced into Escherichia coli to achieve expression and production.

[0033] The specific operations in this embodiment include: obtaining the target gene sequence from NCBI and inserting an insertion at the N-terminus of the target sequence. Nco I restriction enzyme site, C-terminus inserted Xho I restriction enzyme sites are used for in vitro gene synthesis. Nco I and Xho Two restriction endonuclease sites were ligated to the pET28a expression vector via enzyme digestion and ligation, and a His tag was inserted at the C-terminus. The successfully constructed pET28a-ckLplA expression vector was then transformed into E. coli BL21(DE3) cells to construct recombinant engineered E. coli.

[0034] Escherichia coli BL21(DE3) containing the pET28a-ckLplA plasmid was inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37°C until OD200. 600 ≈0.6. Add 0.2mM IPTG (isopropyl-β-D-thiogalactoside) and induce expression at 30℃ for 8 hours. Collect bacterial cells by centrifugation, resuspend in lysis buffer (300mM NaCl, 50mM Tris-HCl, 20mM imidazole, pH 7.5), and sonicate. Filter the supernatant through Ni 2+ Affinity chromatography was used for purification. The target protein was eluted with buffer containing 300 mM imidazole, followed by ultrafiltration with a 30 kDa cutoff to replace the protein in phosphate buffer (20 mM, pH 7.2). Lipoic acid protein ligase with a purity ≥85% was obtained. SDS-PAGE results are shown below. Figure 1 As shown.

[0035] Example 2: Enzyme activity assay and kinetic analysis In a 200 μL reaction system, 10 mM PBS buffer (pH 7.5), 1 mM MgCl2, and 1 μM ckLplA enzyme were added, and different concentrations of substrate were added according to the following protocol: To determine the kinetic parameters of R-(+)-lipoic acid, the ATP concentration was fixed at 1 mM and the concentration of H protein (Hapo) derived from Citrobacter coccine was fixed at 100 μM, allowing the lipoic acid concentration to vary within the range of 0-1 mM.

[0036] To determine the kinetic parameters of ATP, the concentration of lipoic acid was fixed at 0.5 mM and the concentration of H protein derived from Citrobacter coccine was fixed at 100 μM, allowing the ATP concentration to vary within the range of 0-1 mM.

[0037] To determine the kinetic parameters of H protein, the concentration of lipoic acid was fixed at 0.5 mM and the concentration of ATP was fixed at 1 mM, and the concentration of H protein (ecHapo from Escherichia coli and ckHapo from Citrobacter clausus) was varied in the range of 0-500 μM.

[0038] The reaction was initiated by adding ATP, incubated at a constant temperature of 30°C for 5 minutes, and then terminated by heating in a boiling water bath for 1 minute.

[0039] The reaction product (lipidized H protein, Hlip) was quantified using high-performance liquid chromatography (HPLC). Nonlinear fitting was performed using the Michaelis-Menten equation by measuring the initial reaction rate at different substrate concentrations. Figure 2 The Michaelis constant (K) of the enzyme for each substrate was calculated. m ) and maximum reaction rate (Vmax The measurement results are summarized in Table 1.

[0040] Table 1 As shown in Table 1, the ckLplA enzyme described in this invention exhibits significant catalytic activity for all the substrates. Importantly, compared to LplA (ecLplA) derived from Escherichia coli, this enzyme demonstrates a faster catalytic rate for the esterification of H proteins.

[0041] Example 3: In vitro synthesis of glycine using the enzyme of the present invention A 1 mL reaction mixture was prepared in 10 mM PBS buffer (pH 7.5) with the following components and final concentrations: tetrahydrofolate (THF) 0.5 mM, dithiothreitol (DTT) 20 mM, formaldehyde 10 mM, pyridoxal phosphate (PLP) 50 μM, ammonium chloride (NH4Cl) 50 mM, adenosine triphosphate (ATP) 1 mM, lipoic acid 1 mM, magnesium chloride (MgCl2) 1 mM, H protein (Hapo, GenBank ID: NP_417380.1) 100 μM, P protein (GenBank ID: NP_417379.1) 5 μM, T protein (GenBank ID: NP_417381.1) 3 μM, and lipoic acid protein ligase (LplA) 1 μM. The reaction mixture was incubated in a 37°C water bath for 60 minutes. After the reaction was completed, the following were added in sequence: 16 μL of terminator (0.2 M sodium bicarbonate solution), 20 μL of derivatizing reagent (dansyl chloride acetonitrile solution, concentration 5.4 mg / mL), and 60 μL of dilute hydrochloric acid (obtained by diluting concentrated hydrochloric acid 100 times with ultrapure water, used to neutralize the pH of the reaction system).

[0042] After the above treatment, an appropriate amount of sample was taken, and the glycine concentration was detected by high performance liquid chromatography (HPLC). To evaluate the effect of the enzyme (ckLplA) of this invention, a control experiment was set up under exactly the same conditions. The control group used an equimolar concentration (1 μM) of lipoic acid protein ligase (ecLplA, GenBank number NP_418803.1) derived from Escherichia coli instead of ckLplA.

[0043] Experimental results show that ( Figure 3 In the experimental group using ckLplA of the present invention, the glycine production rate was 3.2 times that of the control group (ecLplA). This result clearly confirms that in the reverse glycine cleavage system, ckLplA provided by the present invention can significantly accelerate the glycine synthesis rate and effectively improve production efficiency.

[0044] In addition, the above reaction system was incubated in constant temperature water baths at 37°C, 42°C, and 47°C, respectively, while keeping other conditions constant, to investigate the yield of glycine produced by the ckLplA enzyme described in this invention at different temperatures. The results are as follows: Figure 4 As shown, the yield of glycine produced by this enzyme reaches its maximum at 42°C.

Claims

1. Application of lipoic acid protein ligase in enhancing the efficiency of acylcarrier protein esterification reaction, wherein the amino acid sequence of the lipoic acid protein ligase is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the lipoic acid protein ligase is shown in SEQ ID NO.

2.

3. Application of lipoic acid protein ligase in the preparation of enzyme catalysts that enhance the efficiency of esterification reaction of acyl carrier proteins, wherein the amino acid sequence of the lipoic acid protein ligase is shown in SEQ ID NO.

1.

4. The application as described in claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the lipoic acid protein ligase is shown in SEQ ID NO.

2.

5. A system for producing glycine in vitro, characterized in that, The system includes: a core enzyme component necessary for the reverse glycine cleavage system and a lipoic acid protein ligase, the amino acid sequence of which is shown in SEQ ID NO.1; The core enzyme components necessary for the reverse glycine cleavage system include glycine decarboxylase, acylcarrier protein, and aminomethyltransferase.

6. The system for producing glycine in vitro as described in claim 5, characterized in that, in, The concentration ratio of glycine decarboxylase, acylcarrier protein, aminomethyltransferase and lipoic acid protein ligase was 1-10 μM: 1-10 μM: 10-100 μM: 1-3 μM.

7. A method for producing glycine in vitro, characterized in that, The precursor substrate for glycine synthesis is mixed with the in vitro glycine production system described in claim 5 or 6 and reacted to obtain glycine.

8. The method for producing glycine in vitro as described in claim 7, characterized in that, The precursor substrates include tetrahydrofolate, dithiothreitol, C1 substrate, pyridoxal phosphate, ammonium salt, lipoic acid, ATP, and magnesium ions, with the following concentration ratios: tetrahydrofolate 0.1-1 mM, dithiothreitol 1-20 mM, C1 substrate 1-20 mM, pyridoxal phosphate 1-25 μM, ammonium salt 10-50 mM, lipoic acid 0.1-2 mM, ATP 0.1-5 mM, and magnesium ions 0.1-5 mM. The reaction conditions are: pH range of 6-8 and reaction temperature of 0-47℃.

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