Method for biosynthesizing glyoxylic acid

Abstract

The invention discloses a method for biosynthesizing glyoxylic acid. According to the method, mutants (W86R, N87T, L109G, L110E, A460M, H281V and Q282F) of benzoyl formate decarboxylase (BFD) are used for condensing formaldehyde and carbon dioxide, and glyoxylic acid is generated through a reaction. Therefore, an artificial carbon sequestration way is provided, carbon dioxide can be converted into a two-carbon compound glyoxylic acid, the glyoxylic acid can enter a center for metabolism through a glyceric acid way, and theoretical support and technical basis are provided for construction of an artificial one-carbon cell factory and a new one-carbon resource biotransformation way. Meanwhile, glyoxylic acid is an important precursor for synthesizing glycine, so that a way for synthesizing glycine from carbon dioxide can be constructed based on the reaction of condensing carbon dioxide with formaldehyde, and a foundation is laid for conversion of carbon dioxide into a large amount of amino acid.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology and bioengineering, and in particular relates to a method for biosynthesizing glyoxylic acid. Background Art

[0002] One-carbon (C1) compounds include CO2, CO, formic acid, formaldehyde and methanol, etc. They have the characteristics of wide sources and low value, and are recognized as the most ideal industrial biomanufacturing raw materials. Natural organisms usually use cyclic pathways for one-carbon utilization, such as the Calvin cycle, serine cycle, xylulose monophosphate cycle and ribulose monophosphate cycle, etc. However, natural cyclic carbon fixation pathways often have problems such as complex pathways, low efficiency and poor atom economy. Linear and orthogonal carbon fixation pathways are considered to be the most promising carbon fixation pathways. Taking the reductive glycine pathway discovered in recent years as an example, its core carbon fixation enzyme is the glycine cleavage system (GCS), which can reversely utilize CO2, 5,10-methylenetetrahydrofolate and ammonia (NH3) to synthesize glycine. The glycine cleavage system is a multi-enzyme complex composed of three enzymes (P protein, T protein, L protein) and a carrier H protein. It accepts and transfers CO2 through the lipoyl lysine arm on the carrier H protein as an active nucleophilic group. The reduced glycine pathway is considered to be one of the most advantageous pathways in carbon fixation. It has fewer reaction steps, lower ATP requirements, and the enzymes in the pathway have good tolerance to oxygen, which has little interference with central metabolism and has good potential for industrial application. Recently, the reduced glycine pathway has been successfully expressed in microorganisms such as E. coli, Saccharomyces cerevisiae, and Clostridium drakei, allowing host cells to use formic acid and CO2 to synthesize primary metabolites such as organic acids and amino acids.

[0003] The earth is rich in one-carbon resources. In 2013, the global formaldehyde production capacity was about 64 million tons, the global methanol production capacity was about 103.24 million tons, the recoverable reserves of natural gas were about 185.7 trillion cubic meters, and the recoverable reserves of raw coal were about 891.531 billion tons. There is no enzyme in nature that can catalyze the reaction of formaldehyde and carbon dioxide to produce glyoxylic acid. Therefore, exploring the production of glyoxylic acid using formaldehyde and carbon dioxide as raw materials and enzymes as catalysts will lay the foundation for the efficient utilization of one-carbon resources. Summary of the invention

[0004] To solve the problems of the above-mentioned prior art, the object of the present invention is to provide a method for biosynthesizing glyoxylic acid. The present invention uses mutants (W86R, N87T, L109G, L110E, A460M, H281V, Q282F) of benzoylformate decarboxylase (BFD, Pseudomonas putida) to condense formaldehyde and carbon dioxide to react and generate glyoxylic acid. Glyoxylic acid can enter central metabolism through the glycerate pathway, and can also be converted into glycine to construct an artificial glycine pathway. Therefore, the present invention will lay the foundation for the efficient utilization of one-carbon resources.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for biosynthesizing glyoxylic acid, comprising: synthesizing glyoxylic acid from formaldehyde and carbon dioxide under the action of hydroxyacetaldehyde synthase (GALS), and / or its mutant or an enzyme having the catalytic function thereof;

[0007] According to an embodiment of the present invention, the hydroxyacetaldehyde synthase or a mutant thereof may be a BFD mutant obtained by directed evolution of benzoylformate decarboxylase (BFD, Pseudomonas putida).

[0008] According to an embodiment of the present invention, the BFD mutant is selected from any one of the following:

[0009] (a) The amino acid sequence is derived from the sequence shown in SEQ ID NO: 1, with mutations at the following positions: W86R, N87T, L109G, L110E, A460M, H281V and Q282F;

[0010] or

[0011] (b) the BFD mutant has 95%, preferably 98%, more preferably 99% sequence identity with the amino acid sequence of (a), and has the function of the BFD mutant of (a);

[0012] or

[0013] (c) The BFD mutant is composed of 1-30, more preferably 1-10, even more preferably 1-6, and most preferably 1-3 amino acid residues added or deleted at the C-terminus and / or N-terminus of the amino acid sequence of (a), and has the function of the methanol dehydrogenase mutant of (a);

[0014] It is to be understood that in the context of the present invention, glycolaldehyde synthase (GALS) has the amino acid sequence defined above.

[0015] According to an embodiment of the present invention, the glycolaldehyde synthase used therein may be in the form of a purified enzyme, an enzyme lysis supernatant or whole cells.

[0016] According to an embodiment of the present invention, the method can be carried out in a "one-pot" manner and can be carried out in a buffer system. The buffer can be a sodium bicarbonate / potassium bicarbonate / ammonium bicarbonate buffer. The pH value of the buffer can be 8.5-9.5, for example, a pH of 9. The buffer is exemplarily a sodium bicarbonate buffer with a pH of 9.

[0017] According to an embodiment of the present invention, the concentration of the substrate formaldehyde in the reaction system may be 10-100 mM, for example, illustratively 50 mM.

[0018] According to an embodiment of the present invention, the buffer system further comprises a cofactor thiamine pyrophosphate (TPP), which acts as a reaction catalyst.

[0019] According to an embodiment of the present invention, the concentration of the glycolaldehyde synthase used may be 1-5 mg / mL, for example, exemplarily 2 mg / mL.

[0020] According to an embodiment of the present invention, the "one-pot" reaction may be carried out at 30-40°C, for example, illustratively at 37°C.

[0021] According to an embodiment of the present invention, the "one-pot" reaction can be carried out in 0.5-3 hours, for example, illustratively for 1 hour.

[0022] According to an embodiment of the present invention, in the "one-pot" reaction, after the reaction is completed, an equal volume of acetonitrile is added to the reaction system, the protein is denatured and precipitated, and the reaction is centrifuged at 12000 rpm for 1 hour, and the supernatant is taken to detect glyoxylic acid.

[0023] In a second aspect, the present invention provides use of the hydroxyacetaldehyde synthase or its mutant or related biological materials described in the first aspect in biosynthesis of glyoxylic acid.

[0024] According to an embodiment of the present invention, the above-mentioned hydroxyacetaldehyde synthase or its mutant or related biological materials are used to biosynthesize glyoxylic acid using formaldehyde and carbon dioxide as substrates.

[0025] According to an embodiment of the present invention, the biomaterial related to the hydroxyacetaldehyde synthase is a nucleic acid molecule encoding the hydroxyacetaldehyde synthase, or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

[0026] According to an embodiment of the present invention, the recombinant bacteria are obtained by introducing a nucleic acid molecule encoding the hydroxyacetaldehyde synthase or a mutant thereof into a host cell; for example, the nucleic acid molecule is introduced into the host cell in the form of a recombinant vector.

[0027] Wherein, the recombinant vector is a bacterial plasmid, a bacteriophage, a yeast plasmid or a retrovirus packaging plasmid carrying a nucleic acid molecule encoding the hydroxyacetaldehyde synthase or a mutant thereof.

[0028] The host cell may be a prokaryotic cell such as a bacterium, more specifically Escherichia coli, or a lower eukaryotic cell such as a yeast cell.

[0029] In the context of the present invention, amino acids are represented by single-letter or three-letter codes and have the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamate); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0030] In the context of the present invention, mutants are described according to their mutations at specific residues, the positions of which are determined by alignment with the wild-type enzyme sequence or with reference to the wild-type enzyme sequence. In the context of the present invention, any variants carrying these same mutations at functionally equivalent residues are also contemplated.

[0031] In this article, the mutation site and its replacement are expressed by the position number of the mutation site and the type of amino acid at the site. For example, W86R indicates that the tryptophan corresponding to the 86th position of SEQ ID NO: 1 is mutated to arginine in comparison with SEQ ID NO: 1; N87T indicates that the asparagine corresponding to the 87th position of SEQ ID NO: 1 is mutated to threonine in comparison with SEQ ID NO: 1. In the present invention, "," is used to indicate the combination of mutation sites. For example, "W86, N87" indicates that both the tryptophan at the 86th position and the asparagine at the 87th position are mutated, including two mutation sites, which is a double mutant. By analogy, "W86R, N87T, L109G, L110E, A460M, H281V, Q282F" indicates that the corresponding seven sites are mutated at the same time, which is a seven mutant.

[0032] Beneficial effects of the present invention:

[0033] The present invention found that the BFD mutant (W86R, N87T, L109G, L110E, A460M, H281V, Q282F) can condense formaldehyde and react with carbon dioxide to generate glyoxylic acid. Therefore, an artificial carbon fixation pathway is provided, which can convert carbon dioxide into a two-carbon compound glyoxylic acid, and glyoxylic acid can enter the central metabolism through the glyceric acid pathway. Therefore, the present invention provides theoretical support and technical basis for the construction of an artificial one-carbon cell factory and a new pathway for the biological conversion of one-carbon resources. At the same time, glyoxylic acid is an important precursor for the synthesis of glycine. Based on the formaldehyde condensation carbon dioxide reaction of the present invention, a pathway for the synthesis of glycine from carbon dioxide can be constructed, which lays the foundation for the conversion of carbon dioxide to bulk amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The technical roadmap for the biosynthesis of glyoxylic acid in the present invention is shown in FIG.

[0035] Figure 2 This is the plasmid map of pET-28a-GALS.

[0036] Figure 3 GALS catalyzes formaldehyde and CO2 to produce glyoxylic acid;

[0037] A is the glyoxylic acid standard, and B is the reaction of formaldehyde and carbon dioxide catalyzed by GALS. DETAILED DESCRIPTION

[0038] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0039] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0040] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0041] The inventors conducted directed evolution of benzoylformate decarboxylase (BFD, Pseudomonas putida) and obtained (W86R, N87T, L109G, L110E, A460M, H281V, Q282F), which successfully achieved the formaldehyde condensation reaction. We defined this BFD mutant as hydroxyacetaldehyde synthase (GALS), which can condense formaldehyde and react with carbon dioxide to produce glyoxylic acid. Glyoxylic acid can enter the central metabolism through the glycerate pathway, or it can be converted into glycine to construct an artificial glycine pathway.

[0042] Example 1: Construction of GALS expression vector

[0043] The GALS enzyme encoding gene is derived from Pseudomonas putida, the amino acid sequence of the GALS enzyme is shown in SEQ ID No. 2, and the nucleotide sequence of the GALS enzyme encoding gene is shown in SEQ ID No. 3. The vector for expressing the GALS enzyme is obtained by inserting the above GALS enzyme encoding gene (SEQ ID No. 3) between the NdeI and XhoI restriction sites of the pET-28a vector, and is named pET-28a-GALS (such as Figure 2 shown).

[0044] Example 2. Gene expression

[0045] In order to detect and verify the activity and function of GALS enzyme in vitro, the enzyme was exogenously expressed in Escherichia coli.

[0046] (1) The E. coli expression recombinant plasmid pET-28a-GALS prepared in Example 1 was transferred into E. coli BL21 (DE3) to obtain recombinant bacteria expressing GALS enzyme. Positive clones were screened using kanamycin resistance plates (Kanamycin resistant plates). + , 100 mg / mL), cultured overnight at 37°C;

[0047] (2) Pick a single clone and transfer it to 5 mL LB liquid medium (Kan + , 100 mg / mL), culture at 37°C, 220 r / min until OD600 is 0.6-0.8. Transfer 5 mL of LB culture medium to 800 mL of 2YT medium (Kan + , 100 mg / mL), cultured at 37°C, 220 rpm until OD600 was 0.6-0.8, cooled to 16°C, added IPTG to a final concentration of 0.5 mM, and induced expression for 16 h;

[0048] (3) Collect the above culture solution into a collection bottle and centrifuge at 5500 rpm for 15 min;

[0049] (4) Discard the supernatant, suspend the obtained bacterial precipitate with 35 mL of protein buffer (50 mM phosphate buffer, pH 7.4), pour it into a 50 mL centrifuge tube, and store it in a -80°C refrigerator to obtain bacteria expressing the GALS enzyme.

[0050] Example 3. Protein purification

[0051] (1) Bacterial disruption: The bacteria expressing the GALS enzyme obtained in Example 2 were disrupted twice using a high-pressure low-temperature disruptor at a pressure of 1200 bar and 4° C. The bacterial suspension after disruption was centrifuged at 4° C. and 10,000 rpm for 45 min;

[0052] (2) Purification: The supernatant was filtered through a 0.45 μm microporous filter membrane and purified by nickel affinity chromatography. The specific steps are as follows:

[0053] a: Column balancing: Before loading the supernatant, wash the Ni affinity column with ddH2O for 2 column volumes, and then balance the Ni affinity column with protein buffer for 1 column volume;

[0054] b: Sample loading: slowly pass the supernatant through the Ni affinity chromatography column at a flow rate of 0.5 mL / min, repeat once;

[0055] c: Elution of impurities: Wash one column volume with protein buffer, and then use 50 mL of protein buffer containing 50 mM and 100 mM imidazole to elute the strongly bound impurities;

[0056] d: Elution of target protein: Elute the target protein with 20 mL of protein buffer (50 mM potassium phosphate, pH=7.4, 5 mM MgSO4) containing 200 mM imidazole, take the first few drops of flow sample, prepare the sample, and detect it with 12% SDS-PAGE.

[0057] (3) Replacement of liquid: The collected target protein was concentrated by centrifugation (4°C, 3400 r / min) using a 50 mL Amicon ultrafiltration tube (30 kDa, Millipore) to 1 mL. 15 mL of protein buffer without imidazole was added and concentrated to 1 mL. Repeat once to obtain the GALS protein.

[0058] (4) The protein concentration after concentration was detected using a Nondrop 2000 micro-spectrophotometer and diluted to 10 mg / mL to obtain GALS protein.

[0059] Example 4. Functional detection of GALS protein catalyzing the production of glyoxylate

[0060] GALS protein catalyzes the production of glyoxylate from formaldehyde and CO2.

[0061] Sample: 2 mg / mL GALS, 200 mM sodium bicarbonate buffer (200 mM NaHCO3, pH = 9), cofactor: 1 mM thiamine pyrophosphate (TPP), pH 9, 50 mM formaldehyde.

[0062] Control: 200 mM sodium bicarbonate buffer, 1 mM TPP, pH 9, 50 mM formaldehyde.

[0063] Standard: 10 mM glyoxylic acid.

[0064] After the reaction system is mixed, it is placed at 37°C for 1 hour. After the reaction is completed, an equal volume of acetonitrile is added to the reaction system, and centrifuged at 12000 rpm for 1 hour. 100 μL of the supernatant is taken and tested by HPLC.

[0065] HPLC detection: The detection system is Agilent liquid chromatograph 7890A; the detection conditions are: Agilent chromatographic column is Aminex 87-H (300×7.8mm, 9μm); the detector is divided into an ultraviolet detector (Agilent, G1315D) with an ultraviolet detection wavelength of 210nm, and the flow rate of the mobile phase is 0.6ml / min; the injection volume is 20μl; and the column temperature is 35°C.

[0066] HPLC analysis showed that GALS can catalyze the condensation of formaldehyde with CO2 to produce glyoxylic acid. Figure 3 .

[0067] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A use of hydroxyacetaldehyde synthase or its mutant or related biological materials in the biosynthesis of glyoxylic acid using formaldehyde and carbon dioxide as substrates, characterized in that: The hydroxyacetaldehyde synthase or a mutant thereof may be a BFD mutant obtained by directed evolution of benzoylformate decarboxylase, wherein the BFD mutant is selected from any one of the following: (a) The amino acid sequence is derived from the sequence shown in SEQ ID NO: 1, with mutations at the following positions: W86R, N87T, L109G, L110E, A460M, H281V and Q282F; or (b) the BFD mutant has 95%, preferably 98%, more preferably 99% sequence identity with the amino acid sequence of (a), and has the function of the BFD mutant of (a); or (c) The BFD mutant is composed of 1-30, more preferably 1-10, even more preferably 1-6, and most preferably 1-3 amino acid residues added or deleted at the C-terminus and / or N-terminus of the amino acid sequence described in (a), and has the function of the methanol dehydrogenase mutant described in (a).

2. The use according to claim 1, characterized in that: The biological material related to the hydroxyacetaldehyde synthase is a nucleic acid molecule encoding the hydroxyacetaldehyde synthase, or an expression box, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

3. The use according to claim 2, characterized in that: The recombinant vector is a bacterial plasmid, a bacteriophage, a yeast plasmid or a retrovirus packaging plasmid carrying a nucleic acid molecule encoding the hydroxyacetaldehyde synthase or a mutant thereof.

4. The use according to claim 2, characterized in that: The recombinant bacteria are obtained by introducing a nucleic acid molecule encoding the hydroxyacetaldehyde synthase or a mutant thereof into a host cell; for example, the nucleic acid molecule is introduced into the host cell in the form of a recombinant vector; Preferably, the host cell may be a prokaryotic cell such as a bacterium, more specifically Escherichia coli, or a lower eukaryotic cell such as a yeast cell.

5. A method for biosynthesizing glyoxylic acid, comprising: Formaldehyde and carbon dioxide synthesize glyoxylic acid under the action of hydroxyacetaldehyde synthase (GALS), and / or its mutants or enzymes having this catalytic function; The hydroxyacetaldehyde synthase or its mutant may be a BFD mutant obtained by directed evolution of benzoylformate decarboxylase, and the BFD mutant is selected from any one of the following: (a) The amino acid sequence is derived from the sequence shown in SEQ ID NO: 1, with mutations at the following positions: W86R, N87T, L109G, L110E, A460M, H281V and Q282F; or (b) the BFD mutant has 95%, preferably 98%, more preferably 99% sequence identity with the amino acid sequence of (a), and has the function of the BFD mutant of (a); or (c) The BFD mutant is composed of 1-30, more preferably 1-10, even more preferably 1-6, and most preferably 1-3 amino acid residues added or deleted at the C-terminus and / or N-terminus of the amino acid sequence of (a), and has the function of the methanol dehydrogenase mutant of (a); Preferably, the glycolaldehyde synthase used may be in the form of a purified enzyme, an enzyme lysis supernatant or whole cells.

6. The method according to claim 5, characterized in that The method can be carried out in a "one-pot" manner and can be carried out in a buffer system. The buffer can be a sodium bicarbonate / potassium bicarbonate / ammonium bicarbonate buffer. The pH value of the buffer can be 8.5-9.5, for example, the buffer is exemplarily a sodium bicarbonate buffer with a pH value of 9.

7. The method according to claim 5, characterized in that The concentration of the substrate formaldehyde in the reaction system may be 10-100 mM, for example, illustratively 50 mM.

8. The method according to claim 5, characterized in that The buffer system also contains a cofactor thiamine pyrophosphate (TPP) as a reaction catalyst.

9. The method according to claim 5, characterized in that The concentration of glycolaldehyde synthase used may be 1-5 mg / mL, for example, exemplarily 2 mg / mL.

10. The method according to claim 5, characterized in that The "one-pot" reaction can be carried out at 30-40°C, for example, illustratively at 37°C.

11. The method according to claim 5, characterized in that The "one-pot" reaction can be carried out in 0.5-3 hours, for example, illustratively for 1 hour.

12. The method according to claim 5, characterized in that In the one-pot reaction, after the reaction is completed, an equal volume of acetonitrile is added to the reaction system to denature the protein and precipitate it. The reaction is centrifuged at 5000-15000 rpm for 0.5-3 h, and the supernatant is collected to detect glyoxylic acid.

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