Proteins having tetrahydrofolate-dependent demethylase activity and their use in l-5-methyltetrahydrofolate synthesis

Abstract

This invention belongs to the field of enzyme engineering technology, specifically relating to proteins with tetrahydrofolate-dependent demethylase activity and their application in the synthesis of L-5-methyltetrahydrofolate. This invention utilizes two proteins with tetrahydrofolate-dependent demethylase activity, namely *Sphingosine monocytogenes* (…). Sphingomonas paucimobilis Vanillic acid O-demethylase and Amorpha fruticosa intermediate rhizobia (from) Mesorhizobium amorphae The aminomethyltransferase derived from 6S-THF uses inexpensive vanillic acid or dicamba as methyl donors to directly methylate 6S-THF to synthesize L-5-MTHF. Combined with highly active dihydrofolate reductase and NADP cofactor regeneration system, this method effectively achieves efficient and green synthesis of L-5-methyltetrahydrofolate, and has good industrialization prospects.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a protein with tetrahydrofolate-dependent demethylase activity and its application in the synthesis of L-5-methyltetrahydrofolate. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] L-5-methyltetrahydrofolate (L-5-MTHF) is the main bioactive form of folic acid (vitamin B9) in the human body. Unlike synthetically produced oxidized folic acid (FA), L-5-MTHF can directly enter the cellular metabolic cycle without the stepwise reduction by dihydrofolate reductase (DHFR) and methyltransferases. It participates in the transfer of one-carbon units and various key biological processes, including purine and pyrimidine synthesis, amino acid metabolism, and DNA methylation in epigenetic regulation, thus exhibiting higher bioavailability and physiological availability. Currently, L-5-MTHF is widely used in dietary supplements, foods for special medical purposes, and drugs for treating megaloblastic anemia, cardiovascular diseases, and neural tube defects, and market demand continues to grow.

[0004] l-5 The synthesis of MTHF primarily uses folic acid as a precursor, encompassing three pathways: chemical synthesis, microbial fermentation, and enzymatic synthesis. Chemical synthesis is the main industrial method, using folic acid as a raw material and involving steps such as catalytic hydrogenation or reduction with strong reducing agents, and methylation. However, this process involves high-pressure hydrogen, unstable reducing agents, and transition metal catalysts, posing significant safety risks and environmental pollution problems. Furthermore, complex chiral resolution is necessary to obtain physiologically active L-5. L-5-MTHF synthesis, particularly with modified engineered bacteria, leads to low overall yield and significantly increased costs. Microbial fermentation, utilizing engineered microorganisms for biosynthesis, holds promise for green and sustainable production. However, its bottleneck lies in the difficulty of controlling the complex metabolic network within the strain, resulting in insufficient supply of key precursors and methyl groups, and the easy decomposition and consumption of the target product, leading to extremely low yields that fall far short of industrial production requirements. Enzymatic synthesis is an emerging green alternative, enabling the efficient and selective synthesis of L-5-MTHF using folic acid as a substrate through the construction of multi-enzyme cascades in vitro. However, these methods typically rely on extremely expensive dimethyl mercaptopropionic acid (DMSP) or S-adenosylmethionine (SAM) as direct methyl donors, or require the construction of complex cofactor regeneration systems, resulting in high overall process costs and limiting their industrial application. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides proteins with tetrahydrofolate-dependent demethylase activity and their application in the synthesis of L-5-methyltetrahydrofolate. Specifically, this invention screened and obtained two proteins with tetrahydrofolate-dependent demethylase activity, and used inexpensive vanillic acid or dicamba as methyl donors to directly methylate 6S-THF to synthesize L-5-MTHF, achieving efficient and green synthesis of L-5-MTHF. Based on the above research results, this invention is thus completed.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a protein having tetrahydrofolate-dependent demethylase activity, said protein having any of the amino acid sequences (a1)-(a3): (a1) The amino acid sequence shown in either SEQ ID NO. 1 or 3; (a2) A protein derived from the amino acid sequence shown in SEQ ID NO.1 or 3 by substitution and / or deletion and / or addition of one or more amino acid residues, and having the same function as the amino acid sequence shown in SEQ ID NO.1 or 3; (a3) Other genes encode proteins that have a similarity of more than 90% to the amino acid sequence composition shown in either SEQ ID NO.1 or 3 and have the enzyme activity shown in SEQ ID NO.1 or 3.

[0007] In this invention, the protein having the amino acid sequence shown in SEQ ID NO.1 is a vanillic acid O-demethylase protein, named LigM. The protein LigM originates from *Sphingomonas oligosporus* (…). Sphingomonas paucimobilis SYK-6 uses tetrahydrofolate as a methyl acceptor to convert vanillic acid into protocatechuic acid, or 3-O-methylgallic acid into gallic acid.

[0008] The protein having the amino acid sequence shown in SEQ ID NO.3 is an aminomethyltransferase protein, named Dmt. The protein Dmt is derived from *Amorpha fruticosa* intermediate rhizobium (…). Mesorhizobium amorphae It uses tetrahydrofolate as a methyl acceptor to convert dicamba into 3,6-dichlorosalicylic acid.

[0009] In a second aspect, the present invention provides a gene that encodes the protein having the above-mentioned tetrahydrofolate-dependent demethylase activity.

[0010] The gene has any of the nucleotide sequences described in (b1)-(b4): (b1) A nucleotide sequence as shown in either SEQ ID NO. 2 or 4; (b2) A sequence formed by substitution, deletion and / or addition of one or more nucleotides as shown in (b1); (b3) has 80% or more identity with the nucleotide sequence defined in (b1) or (b2) and is a nucleic acid molecule encoding the protein; (b4) A nucleotide sequence that, under stringent conditions, can hybridize with any of the nucleotide sequences described in (b1)-(b3) and encode a protein with the same function.

[0011] A third aspect of the present invention provides a recombinant expression vector comprising the gene.

[0012] A fourth aspect of the present invention provides a host cell containing the recombinant expression vector or a chromosome integrated with the gene or capable of expressing the protein having tetrahydrofolate-dependent demethylase activity.

[0013] The host cell is a bacterial cell or a fungal cell.

[0014] A fifth aspect of the invention provides the use of genes, recombinant expression vectors, or host cells in the preparation of proteins having tetrahydrofolate-dependent demethylase activity.

[0015] A sixth aspect of the present invention provides a method for preparing the protein having tetrahydrofolate-dependent demethylase activity, comprising: culturing the host cells to express the protein having tetrahydrofolate-dependent demethylase activity; and isolating and purifying the protein having tetrahydrofolate-dependent demethylase activity.

[0016] A seventh aspect of the invention provides the use of proteins, genes, recombinant expression vectors, or host cells having tetrahydrofolate-dependent demethylase activity in the synthesis of L-5-methyltetrahydrofolate or its derivatives.

[0017] An eighth aspect of the present invention provides a method for synthesizing L-5-methyltetrahydrofolate, the synthetic route of which is as follows:

[0018] The method includes constructing a host bacterium containing the protein-coding gene with tetrahydrofolate-dependent demethylase activity, and using wet bacterial cells obtained through fermentation culture of the host bacterium or enzyme-containing liquid obtained by cell disruption as a catalyst to catalyze the production of L-5. MTHF.

[0019] The beneficial technical effects of one or more of the above technical solutions are as follows: The above technical solution utilizes a protein with tetrahydrofolate-dependent demethylase activity, using inexpensive vanillic acid or dicamba as methyl donors, to directly methylate 6S-THF to synthesize L-5-MTHF. Combined with a highly active dihydrofolate reductase and NADP cofactor regeneration system, it effectively achieves efficient and green synthesis of L-5-methyltetrahydrofolate, thus having significant industrial application value. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 This is the HPLC chromatogram of the vanillic acid standard of the present invention; Figure 2 This is the HPLC chromatogram of the dicamba standard of the present invention; Figure 3 This is the HPLC chromatogram of the tetrahydrofolate standard of this invention; Figure 4 This is the HPLC chromatogram of the L-5-methyltetrahydrofolate standard of the present invention; Figure 5 This is a chromatogram of HPLC detection results catalyzed by vanillic acid O-demethylase in this invention; Figure 6 This is a chromatogram of HPLC detection results catalyzed by the aminomethyltransferase of the present invention. Detailed Implementation

[0022] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for the purpose of describing specific embodiments and not for limiting the scope of protection of the present invention. Experimental methods in the following specific embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, and such techniques and conditions are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer.

[0024] In a typical embodiment of the present invention, a protein having tetrahydrofolate-dependent demethylase activity is provided, said protein having any of the amino acid sequences (a1)-(a3): (a1) The amino acid sequence shown in either SEQ ID NO. 1 or 3; (a2) A protein derived from the amino acid sequence shown in SEQ ID NO.1 or 3 by substitution and / or deletion and / or addition of one or more amino acid residues, and having the same function as the amino acid sequence shown in SEQ ID NO.1 or 3; (a3) Other genes encode proteins that have a similarity of more than 90% to the amino acid sequence composition shown in either SEQ ID NO.1 or 3 and have the enzyme activity shown in SEQ ID NO.1 or 3.

[0025] The plurality of amino acid residues may be no more than 20 amino acid residues, and more particularly no more than 10 amino acid residues.

[0026] The proteins shown in (a1)-(a3) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0027] In one specific embodiment of the invention, the protein further comprises a signal peptide and / or a tag protein.

[0028] In another specific embodiment of the present invention, a gene is provided that encodes the protein having the above-mentioned tetrahydrofolate-dependent demethylase activity.

[0029] The gene has any of the nucleotide sequences described in (b1)-(b4): (b1) A nucleotide sequence as shown in either SEQ ID NO. 2 or 4; (b2) A sequence formed by substitution, deletion and / or addition of one or more nucleotides as shown in (b1); (b3) has 80% or more identity with the nucleotide sequence defined in (b1) or (b2) and is a nucleic acid molecule encoding the protein; (b4) A nucleotide sequence that, under stringent conditions, can hybridize with any of the nucleotide sequences described in (b1)-(b3) and encode a protein with the same function.

[0030] It should be noted that the term "identity" refers to sequence similarity to an amino acid / nucleotide sequence. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0031] The aforementioned 80% or more identity can be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0032] In another specific embodiment of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector contains the gene.

[0033] According to the present invention, the recombinant expression vector is obtained by effectively linking the above-mentioned gene to an expression vector, wherein the expression vector is any one or more of a viral vector, plasmid, bacteriophage, granule, or artificial chromosome; the viral vector may include an adenovirus vector, a retrovirus vector, or an adeno-associated virus vector, and the artificial chromosome includes a bacterial artificial chromosome (BAC), a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC); further, the expression vector is a plasmid, such as pET-28a.

[0034] In another specific embodiment of the present invention, a host cell is provided, wherein the host cell contains the recombinant expression vector or a chromosome integrated with the gene or is capable of expressing the protein having tetrahydrofolate-dependent demethylase activity.

[0035] The host cell can be a prokaryotic cell or a eukaryotic cell.

[0036] In another specific embodiment of the present invention, the host cell is a bacterial cell or a fungal cell; The bacterial cells mentioned therein are any one of the following genera: Bacteroides, Escherichia, Agrobacterium, Bacillus, Streptomyces, Pseudomonas, or Staphylococcus. In another specific embodiment of the present invention, the bacterial cells are Escherichia coli (such as Escherichia coli BL21(DE3)), Agrobacterium tumefaciens (such as GV3101), Agrobacterium rhizogenes, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, or Pseudomonas fluorescens.

[0037] The fungal cells include yeasts and filamentous fungi.

[0038] Furthermore, the present invention also protects the fermentation products or metabolites of the host cells mentioned above.

[0039] In this invention, the term "fermentation product" is used to refer to fermentation products. The corresponding fermentation product can be a liquid obtained from the process of fermenting host cells, and therefore can also be called a fermentation broth; the liquid may contain host cells (cell bodies), but is not necessarily required to contain host cells (cell bodies). The liquid preferably contains metabolites produced by the host cells of this invention, said metabolites particularly including the proteins described above that have tetrahydrofolate-dependent demethylase activity.

[0040] In another specific embodiment of the present invention, the above-mentioned gene, recombinant expression vector or host cell is provided for use in the preparation of proteins with tetrahydrofolate-dependent demethylase activity.

[0041] In another specific embodiment of the present invention, a method for preparing the protein having tetrahydrofolate-dependent demethylase activity is provided, comprising: culturing the host cells to express the protein having tetrahydrofolate-dependent demethylase activity; and isolating and purifying the protein having tetrahydrofolate-dependent demethylase activity.

[0042] In another specific embodiment of the present invention, the use of proteins, genes, recombinant expression vectors or host cells having tetrahydrofolate-dependent demethylase activity in the synthesis of L-5-methyltetrahydrofolate or its derivatives is provided.

[0043] In another specific embodiment of the present invention, a method for synthesizing L-5-methyltetrahydrofolate is provided, the synthetic route of which is as follows:

[0044] In another specific embodiment of the present invention, the method includes constructing a host bacterium containing the protein-coding gene with tetrahydrofolate-dependent demethylase activity, and using wet bacterial cells obtained by fermentation culture of the host bacterium or enzyme-containing liquid obtained by cell disruption as a catalyst to catalyze the production of L... 5 MTHF.

[0045] In another specific embodiment of the present invention, the above method further includes other small molecule compounds as required, including but not limited to substrate folic acid, hydrogen donors, antioxidants, methyl donors, etc.

[0046] In another specific embodiment of the present invention, the hydrogen donor may be NADPH. Further, the NADPH may be derived from NADP. + It is obtained by converting the hydrogen removed from glucose.

[0047] In another specific embodiment of the present invention, the antioxidant may be at least one of sodium ascorbate, dithiothreitol, and β-mercaptoethanol, thereby effectively preventing the oxidation of the product tetrahydrofolic acid.

[0048] In another specific embodiment of the present invention, the reaction pH is 5.0-8.0.

[0049] In another specific embodiment of the present invention, the reaction temperature is 30℃-40℃ and the reaction time is 2-24h.

[0050] In another specific embodiment of the present invention, the method further includes the addition of methyl donor vanillic acid or dicamba.

[0051] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the materials, reagents, carriers, strains, etc., used in the following examples are commercially available.

[0052] Example 1: Construction of recombinant Escherichia coli engineered strain with vanillic acid O-demethylase Retrieved from the GenBank database were samples from *Sphingosine monocytogenes* (…). Sphingomonas paucimobilis The vanillic acid O-demethylase LigM of SYK-6, namely GenBank accession BAD61059, has the amino acid sequence shown in SEQ ID NO.1. After codon optimization, the coding sequence of the LigM gene was obtained, as shown in SEQ ID NO.2. This gene was submitted to BGI Genomics for gene synthesis and cloned into the pET28a plasmid to obtain the recombinant expression plasmid pET28a-LigM. This plasmid was transformed into the expression host *Escherichia coli* (E. coli). Escherichia coli Recombinant strains were obtained from BL21(DE3) strain. E. coli BL21(DE3) / pET28a-LigM.

[0053] Example 2: Induced expression and activity analysis of vanillic acid O-demethylase The recombinant Escherichia coli containing vanillic acid O-demethylase LigM from Example 1 was inoculated into LB medium containing 50 mg / L kanamycin and cultured at 37°C and 180 rpm until mid-log growth to obtain freshly cultured seed culture.

[0054] Freshly cultured seed culture was inoculated at a volume concentration of 2% into 1 L of LB medium containing 50 mg / L kanamycin, and cultured at 37ºC and 180 rpm until OD500. 600nm Approximately 0.6 mg of the solution was added with 1 mM IPTG and incubated overnight at 23°C and 160 rpm to induce protein expression. Collect bacterial cells by centrifugation, wash twice with PBS buffer, and resuspend at OD200. 600nm Add 1 mM PMSF and 10% glycerol to a concentration of 20, then autoclave the bacterial cells and centrifuge at 12,000 rpm and 4ºC for 50 minutes to remove cell debris to obtain the crude extract. Catalytic activity analysis of vanillic acid O-demethylase on tetrahydrofolate: Using 100 mM Tris-HCl as buffer, 10 mM vanillic acid, 15 mM tetrahydrofolate, 6 mM DTT, and 6 mL of crude vanillic acid O-demethylase were added sequentially. The reaction solution was placed in a 50 mL anaerobic flask and reacted at 30 °C and 220 rpm for 3 h. A 200 μL sample of the reaction solution was boiled in a metal bath at 105 °C for 10 min, centrifuged at 10000 × g for 5 min, and the supernatant was diluted for liquid chromatography analysis.

[0055] Liquid chromatography analysis methods: Analytical method for methyl donor vanillic acid: Column: C18 column; Detection wavelength: 254 nm; Mobile phase: methanol (34.5%), acetonitrile (5.4%), formic acid (0.1%); Flow rate: 0.8 mL / min; Column temperature: 30℃; Injection volume: 10 μL. The HPLC chromatogram of vanillic acid standard is shown below. Figure 1 As shown, the retention time is 9.951 min; Analytical methods for the substrate tetrahydrofolate and the product L-5-methyltetrahydrofolate: Column: C18 column; Detection wavelength: Fluorescence detector Ex=295nm, Em=356nm; Mobile phase: 30mM potassium dihydrogen phosphate (pH 2.3)-acetonitrile (93:7); Flow rate: 0.5mL / min; Column temperature: 30℃; Injection volume: 10μL. The HPLC chromatogram of the substrate tetrahydrofolate standard is shown below. Figure 3 As shown, the retention time was 9.285 min; the HPLC chromatogram of the product L-5-methyltetrahydrofolate standard is shown below. Figure 4 As shown, the retention time is 16.071 min.

[0056] Example 3: Construction of recombinant Escherichia coli engineered strains containing aminomethyltransferase Retrieved from the GenBank database were Rhizobium intercalation nodules of Amorpha fruticosa (… Mesorhizobium amorphae The aminomethyltransferase Dmt of *E. coli*, namely GenBank accession WP_006204620, has an amino acid sequence shown in SEQ ID NO. 3. After codon optimization, the coding sequence of the Dmt gene was obtained, as shown in SEQ ID NO. 4. This gene was submitted to BGI Genomics for gene synthesis and cloned into the pET28a plasmid to obtain the recombinant expression plasmid pET28a-Dmt. This plasmid was transformed into the expression host *E. coli* (…). Escherichia coli Recombinant strains were obtained from BL21(DE3) strain. E .coli BL21(DE3) / pET28a-Dmt.

[0057] Example 4: Induced expression and activity analysis of aminomethyltransferase The recombinant Escherichia coli containing aminomethyltransferase Dmt from Example 3 was inoculated into LB medium containing 50 mg / L kanamycin and cultured at 37°C and 180 rpm until mid-log growth to obtain freshly cultured seed culture.

[0058] Freshly cultured seed culture was inoculated at a volume concentration of 2% into 1 L of LB medium containing 50 mg / L kanamycin, and cultured at 37ºC and 180 rpm until OD500. 600nm Approximately 0.6 mg of the solution was added with 1 mM IPTG and incubated overnight at 23°C and 160 rpm to induce protein expression. Collect bacterial cells by centrifugation, wash twice with PBS buffer, and resuspend at OD200. 600nm Add 1 mM PMSF and 10% glycerol to a concentration of 20, then autoclave the bacterial cells and centrifuge at 12,000 rpm and 4ºC for 50 minutes to remove cell debris to obtain the crude extract. Catalytic activity analysis of aminomethyltransferase on the substrate tetrahydrofolate: Using 100 mM Tris-HCl as buffer, 10 mM dicamba, 15 mM tetrahydrofolate, 6 mM DTT, and 6 mL of crude aminomethyltransferase were added sequentially. The reaction solution was placed in a 50 mL anaerobic flask and reacted at 30 °C and 220 rpm for 3 h. A 200 μL sample of the reaction solution was boiled in a metal bath at 105 °C for 10 min, centrifuged at 10000 × g for 5 min, and the supernatant was diluted for liquid chromatography analysis.

[0059] Liquid chromatography analysis methods: Analytical method for methyl donor dicamba: Column: C18 column; Detection wavelength: 275 nm; Mobile phase: ultrapure water (58.4%), acetonitrile (31.7%), methanol (7.5%), and acetic acid (2.4%); Flow rate: 1.0 mL / min; Column temperature: 30℃; Injection volume: 10 μL. The HPLC chromatogram of dicamba standard is shown below. Figure 2 As shown, the retention time is 20.788 min; Analytical methods for the substrate tetrahydrofolate and the product L-5-methyltetrahydrofolate: Column: C18 column; Detection wavelength: Fluorescence detector Ex=295nm, Em=356nm; Mobile phase: 30mM potassium dihydrogen phosphate (pH 2.3)-acetonitrile (93:7); Flow rate: 0.5mL / min; Column temperature: 30℃; Injection volume: 10μL. The HPLC chromatogram of the substrate tetrahydrofolate standard is shown below. Figure 3 As shown, the retention time was 9.285 min; the HPLC chromatogram of the product L-5-methyltetrahydrofolate standard is shown below. Figure 4 As shown, the retention time is 16.071 min.

[0060] Example 5: Construction of recombinant Escherichia coli engineered strain of dihydrofolate reductase Retrieved from the GenBank database from Lactobacillus bulgaricus ( Lactobacillus delbrueckiisubsp.bulgaricus The dihydrofolate reductase LbuDHFR from ATCC 11842, also known as GenBank accession CAI97615, has the amino acid sequence shown in SEQ ID NO. 5. After codon optimization, the coding sequence of the LbuDHFR gene was obtained, as shown in SEQ ID NO. 6. This gene was submitted to BGI Genomics for gene synthesis and cloned into the pET28a plasmid to obtain the recombinant expression plasmid pET28a-LbuDHFR. This plasmid was transformed into the expression host *Escherichia coli* (E. coli). Escherichia coli Recombinant strains were obtained from BL21(DE3) strain. E. coli BL21(DE3) / pET28a-LbuDHFR.

[0061] Example 6: Induced expression and activity analysis of dihydrofolate reductase The recombinant Escherichia coli containing dihydrofolate reductase from Example 5 was inoculated into LB medium containing 50 mg / L kanamycin and cultured at 37°C and 180 rpm until mid-log growth to obtain freshly cultured seed culture.

[0062] Freshly cultured seed culture was inoculated at a volume concentration of 2% into 1 L of LB medium containing 50 mg / L kanamycin, and cultured at 37ºC and 180 rpm until OD500. 600nmApproximately 0.6 mg of the solution was added with 1 mM IPTG and incubated overnight at 23°C and 160 rpm to induce protein expression. Collect bacterial cells by centrifugation, wash twice with PBS buffer, and resuspend at OD200. 600nm Add 1 mM PMSF and 10% glycerol to a concentration of 20, then autoclave the bacterial cells and centrifuge at 12,000 rpm and 4ºC for 50 minutes to remove cell debris to obtain the crude extract. Analysis of the catalytic activity of dihydrofolate reductase on the substrate folic acid: 0.1 mM folic acid, 0.25 mM NADPH and 20 μL of the crude enzyme solution of dihydrofolate reductase were added to 100 mM Tris-HCl buffer, and the change in absorbance at 340 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0063] Example 7: Construction of recombinant Escherichia coli engineered strains for coenzyme cycling system According to the catalytic mechanism of dihydrofolate reductase, the coenzyme NADPH is required. To improve the catalytic efficiency of the preparation of L-5-methyltetrahydrofolate, an NADP+ / NADPH coenzyme cycle system was constructed for this catalytic reaction. PCR amplification was performed using Bacillus subtilis (…). Bacillus subtilis The gene encoding glucose dehydrogenase (GDH) at level 168. gdh The gene fragment, namely GenBank accession CAB12201, has the amino acid sequence shown in SEQ ID NO.7. The gene fragment was digested with EcoRI / HindIII and ligated with the pET-28a(+) plasmid using T4 DNA ligase to obtain the recombinant plasmid pET28a-BsGDH, which was then transformed into the expression host *Escherichia coli*. Escherichia coli In the BL21(DE3) strain, the successfully constructed strains were screened by plating them on LB agar plates containing kanamycin. E. coli BL21(DE3) / pET28a-BsGDH. Single colonies were picked, and the strain was preserved after successful verification by PCR and sequencing.

[0064] Amplification of Bacillus subtilis 168-derived bacteria by PCR gdh The gene and primer design are as follows: Upstream primer 5'-CCGC AAGCTT ttaaccgcggcctgcctggaatgaa-3', carrying a HindIII site; Downstream primer 5'-ATCC GAATTC atgtatccggatttaaaaggaaaag-3' carries an EcoRI site.

[0065] Example 8: Induced expression and activity analysis of glucose dehydrogenase The recombinant Escherichia coli containing glucose dehydrogenase from Example 7 was inoculated into LB medium containing 50 mg / L kanamycin and cultured at 37°C and 180 rpm until mid-log growth to obtain freshly cultured seed culture.

[0066] Freshly cultured seed culture was inoculated at a volume concentration of 2% into 1 L of LB medium containing 50 mg / L kanamycin, and cultured at 37ºC and 180 rpm until OD500. 600nm Approximately 0.6 mg of the solution was added with 1 mM IPTG and incubated overnight at 23°C and 160 rpm to induce protein expression. Collect bacterial cells by centrifugation, wash twice with PBS buffer, and resuspend at OD200. 600nm Add 1 mM PMSF and 10% glycerol to a concentration of 20, then autoclave the bacterial cells and centrifuge at 12,000 rpm and 4ºC for 50 minutes to remove cell debris to obtain the crude extract. Analysis of the catalytic activity of glucose dehydrogenase on glucose substrate: using 100 mM Tris-HCl as buffer, 50 mM glucose, 0.2 mM NADP... + 20 μL of the crude glucose dehydrogenase solution was used to detect the change in absorbance at 340 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0067] Example 9: Construction of a recombinant Escherichia coli engineered strain for the multi-enzyme cascade catalytic synthesis of L-5-methyltetrahydrofolate PCR amplification of the dihydrofolate reductase LbuDHFR derived from Lactobacillus bulgaricus ATCC 11842 was performed. The gene fragment was double-digested with EcoRI / HindIII and ligated with the pACYCDuet-1 plasmid using T4 DNA ligase to obtain the recombinant plasmid pACYCDuet-LbuDHFR. This plasmid was then transformed into the cloning host strain, Escherichia coli DH5α. Successfully constructed strains (DH5α / pACYCDuet-LbuDHFR) were screened on LB agar plates containing chloramphenicol. Single colonies were picked, and after successful verification by PCR and sequencing, the strain was preserved.

[0068] The LbuDHFR gene from Lactobacillus bulgaricus ATCC 11842 was amplified by PCR. The primers were designed as follows: Upstream primer 5'-ATCC GAATTC GATGCTGTCTTACGTTTGGGCTGAAG-3' carries an EcoRI site; Downstream primer 5'-CCGC AAGCTT CTTCTTACGCAGGTAGGTTTCGAAC-3' carries a HindIII site.

[0069] PCR amplification was performed on glucose dehydrogenase GDH derived from Bacillus subtilis 168. The gene fragment was double-digested with KpnI / XhoI and ligated with the pACYCDuet-LbuDHFR plasmid using T4 DNA ligase to obtain the recombinant plasmid pACYCDuet-LbuDHFR-BsGDH. This plasmid was transformed into the cloning host strain *Escherichia coli* DH5α. Successfully constructed strains (DH5α / pACYCDuet-LbuDHFR-BsGDH) were screened on LB agar plates containing chloramphenicol. Single colonies were picked, and after successful verification by PCR and sequencing, the strain was preserved.

[0070] Amplification of Bacillus subtilis 168-derived bacteria by PCR gdh The gene and primer design are as follows: Upstream primer 5'-cgtc ggtacC atgtatccggatttaaaaggaaaag-3' carries a KpnI site; Downstream primer 5'- caga ctcgag ttaaccgcggcctgcctggaatgaa-3' carries an XhoI site.

[0071] The plasmid pACYCDuet-LbuDHFR-BsGDH and the plasmid pET28a-LigM from Example 1 were transformed into the expression host *E. coli*, respectively. Escherichia coli The Nissle 1917(DE3) strain was plated on LB agar plates containing kanamycin and chloramphenicol to screen and successfully construct recombinant Escherichia coli Nissle 1917(DE3) / pET28a-LigM / pACYCDuet-LbuDHFR-BsGDH. Single colonies were picked, and the strain was preserved after successful verification by PCR and sequencing.

[0072] The plasmid pACYCDuet-LbuDHFR-BsGDH and the plasmid pET28a-Dmt from Example 3 were respectively transformed into the expression host *E. coli*. Escherichia coli The Nissle 1917(DE3) strain was plated on LB agar plates containing kanamycin and chloramphenicol to screen and successfully construct recombinant Escherichia coli Nissle 1917(DE3) / pET28a-Dmt / pACYCDuet-LbuDHFR-BsGDH. Single colonies were picked, and the strain was preserved after successful verification by PCR and sequencing.

[0073] Example 10: Multi-enzyme cascade catalysis of folic acid synthesis of L-5-methyltetrahydrofolate Two recombinant Escherichia coli strains that synthesized L-5-methyltetrahydrofolate via multi-enzyme cascade catalysis in Example 9 were inoculated into LB medium containing 50 mg / L kanamycin and 50 mg / L chloramphenicol and cultured at 37°C and 180 rpm until mid-logarithmic growth to obtain freshly cultured seed culture.

[0074] Freshly cultured seed culture was inoculated at a volume concentration of 2% into 1 L of LB medium containing 50 mg / L kanamycin and 50 mg / L chloramphenicol, and cultured at 37ºC and 180 rpm until OD. 600nm Add approximately 0.6 mg of 1 mM IPTG and incubate overnight at 23°C and 160 rpm to induce protein expression.

[0075] Collect bacterial cells by centrifugation, wash twice with PBS buffer, and resuspend at OD200. 600nm Add 1 mM PMSF and 10% glycerol to a concentration of 20, then autoclave the bacterial cells. Centrifuge at 12,000 rpm and 4ºC for 50 minutes to remove cell debris and obtain the crude extract.

[0076] Using 100 mM Tris-HCl as buffer, 50 mM FA, 150 mM vanillic acid or dicamba, 50 μM NADP+, 6 mM DTT, 50 mM glucose, and 6 mL of the above crude enzyme solution were added sequentially. The reaction solution was placed in a 50 mL anaerobic flask and reacted at 30℃ and 220 rpm for 24 h. A 200 μL sample of the reaction solution was boiled in a metal bath at 105℃ for 10 min, centrifuged at 10000×g for 5 min, and the supernatant was diluted 50 or 100 times for liquid chromatography analysis. Liquid chromatography analysis method: Column: C18 column; Detection wavelength: Fluorescence detector Ex=295nm, Em=356nm; Mobile phase: 30 mM potassium dihydrogen phosphate (pH 2.3)-acetonitrile (93:7); Flow rate: 0.5 mL / min; Column temperature: 30℃; Injection volume: 10 μL. The HPLC chromatogram of the sample is shown below. Figure 6 As shown.

[0077] The amino acid / nucleotide sequences involved in this invention Vanillic acid O-demethylase LigM amino acid sequence MSTPTNLEQVLAAGGNTVEMLRNSQIGAYVYPVVAPEFSNWRTEQWAWRNSAVLFDQTHHMVDLYIRGKDALKLLSDTMINSPKGWEPNKAKQYVPVTPYGHVIGDGIIFYLAEEEFVYVGRAPAANWLMYHAQTGGYNVDIVHDDRSPSRPMGKPVQRISWRFQIQGPKAWDVIEKLHGGTLEKLKFFNMAEMNIAGMKIRTLRHGMAGAPGLEIWGPYETQEKARNAILEAGKEFGLIPVGSRAYPSNTLESGWIPSPLPAIYTGDKLKAYREWLPANSYEASGAIGGSFVSSNIEDYYVNPYEIGYGPFVKFDHDFIGRDALEAIDPATQRKKVTLAWNGDDMAKIYASLFDTEADAHYKFFDLPLANYANTNADAVLDAAGNVVGMSMFTGYSYNEKRALSLATIDHEIPVGTELTVLWGEENGGTRKTTVEPHKQMAVRAVVSPVPYSVTARETYEGGWRKAAVTA* (SEQ ID NO.1) Vanillic acid O - demethylase LigM nucleotide sequence aminomethyltransferase Dmt amino acid sequence MVKANGEKSLQTLIDEKRDLVEYFYNDTLAPHYRARTGLTAAYIPPEFTNWRDEQRAWRKSAILFDQSHHMPEMLLKGPDALRLLEKIGINSLANFSTDRAKQLVGCTPRGHVIGDCV VYRLEEETFELISSMPLLNWVQFNAEKGGYDVTIERDDPTPYNPAGKRWFYRFQLEGPNAGKIFNDAVEGDAPEIPFFRTAKVKIGGCKVLVLRHGMAGHLGAELSGPYEEMDKVRSAL VAAGEKHGLKQGGTKTYFSTIFEFGWMPYPLPGIYTGAELRDYREWLSGNGWEANAQLGGSFLSDNIEDYYVTPWDLGYGHIIKFDHDFIGRAALEALPEGKRRKKVTLVWNREDVAKI FGSQFGDGPRFKALDFPVAYYGWPQFDEVRAPDGSLVGHSCHCGYSANEGEMLSLAMLDAACATPGTQIVLTWGEPGGGSRKPHVERHEQLKVRATVAPAPYAQSVQKLKRAAIG (SEQ ID NO.3) aminomethyltransferase Dmt nucleotide sequence Amino acid sequence of dihydrofolate reductase LbuDHFR MLSYVWAEDEKGAIGYQGRLPWHLPADLAHFKAKTMGHPMLMGRKTFESLPGLLPGRQHVVLSTRKLDLPAGVLQLKSEEEVSAWLKEQAGEVCVIGGSSLFALLADQVDKLEVTRIKGIFSADTYMPNLDWAAFALVKSEAHRADGKNKYDYVFETYLRKK (SEQ ID NO.5) Nucleotide sequence of dihydrofolate reductase LbuDHFR ATGCTGTCTTACGTTTGGGCTGAAGATGAGAAAGGTGCGATTGGTTACCAGGGTCGTTTGCCGTGGCATCTGCCAGCGGACCTGGCGCATTTCAAAGCGAAAACTATGGGTCATCCGATGCTGATGGGTCGTAAGACCTTCGAATCTCTGCCGGGTCTGCTGCCGGGTCGTCAGCACGTAGTTCTGTCTACTCGTAAACTGGATCTGCCGGCTGGTGTGTTGCAGCTGAAATCCGAAGAAGAAGTGTCTGCTTGGCTGAAAGAACAGGCTGGTGAAGTTTGCGTTATCGGTGGTTCTAGCCTGTTCGCACTGCTGGCTGATCAGGTTGACAAACTGGAAGTTACTCGTATCAAAGGCATCTTCTCTGCTGACACTTACATGCCGAACCTGGACTGGGCTGCTTTCGCACTGGTGAAATCTGAAGCGCACCGTGCTGATGGTAAGAACAAATACGACTACGTGTTCGAAACCTACCTGCGTAAGAAG (SEQ ID NO.6) Amino acid sequence of glucose dehydrogenase GDH MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKYFVE NDIKGNVINMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG* (SEQ ID NO.7) Matters not covered in this invention are common knowledge.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., 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 protein having tetrahydrofolate-dependent demethylase activity, characterized in that, The protein has any of the amino acid sequences (a1)-(a3): (a1) The amino acid sequence shown in either SEQ ID NO. 1 or 3; (a2) A protein derived from the amino acid sequence shown in SEQ ID NO.1 or 3 by substitution and / or deletion and / or addition of one or more amino acid residues, and having the same function as the amino acid sequence shown in SEQ ID NO.1 or 3; (a3) Other genes encode proteins that have a similarity of more than 90% to the amino acid sequence composition shown in either SEQ ID NO.1 or 3 and have the enzyme activity shown in SEQ ID NO.1 or 3.

2. A gene characterized in that, The gene encodes the protein of claim 1 that has tetrahydrofolate-dependent demethylase activity.

3. The gene as described in claim 2, characterized in that, The gene has any of the nucleotide sequences described in (b1)-(b4): (b1) A nucleotide sequence as shown in either SEQ ID NO. 2 or 4; (b2) A sequence formed by substitution, deletion and / or addition of one or more nucleotides as shown in (b1); (b3) has 80% or more identity with the nucleotide sequence defined in (b1) or (b2) and is a nucleic acid molecule encoding the protein; (b4) A nucleotide sequence that, under stringent conditions, can hybridize with any of the nucleotide sequences described in (b1)-(b3) and encode a protein with the same function.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the gene as described in claim 2 or 3.

5. A host cell, characterized in that, The host cell contains the recombinant expression vector of claim 4 or a chromosome integrated with the gene of any one of claims 2-3 or capable of expressing the protein of claim 1 with tetrahydrofolate-dependent demethylase activity. Furthermore, the host cell is a bacterial cell or a fungal cell.

6. The use of the gene of claim 2 or 3, the recombinant expression vector of claim 4, or the host cell of claim 5 in the preparation of the protein with tetrahydrofolate-dependent demethylase activity as described in claim 1.

7. A method for preparing the protein with tetrahydrofolate-dependent demethylase activity as described in claim 1, characterized in that, include: The host cells of claim 5 are cultured to express the protein having tetrahydrofolate-dependent demethylase activity. The protein with tetrahydrofolate-dependent demethylase activity was isolated and purified.

8. The use of the protein with tetrahydrofolate-dependent demethylase activity as described in claim 1, the gene as described in claim 2 or 3, the recombinant expression vector as described in claim 4, or the host cell as described in claim 5 in the synthesis of L-5-methyltetrahydrofolate or its derivatives.

9. A method for synthesizing L-5-methyltetrahydrofolate, characterized in that, The synthetic route of the method is as follows: 。 10. The method as described in claim 9, characterized in that, The method includes constructing a host bacterium containing the protein-coding gene with tetrahydrofolate-dependent demethylase activity, using wet bacterial cells obtained through fermentation culture of the host bacterium or enzyme-containing liquid obtained by cell disruption as a catalyst to catalyze the production of l-5 MTHF; Furthermore, the method also includes other small molecule compounds as required, including but not limited to the substrate folic acid, hydrogen donors, antioxidants, and methyl donors; The hydrogen donor is NADPH; further, the NADPH is derived from NADP. + It is obtained by converting the hydrogen removed from glucose; The antioxidant is at least one of sodium ascorbate, dithiothreitol, and β-mercaptoethanol; The reaction is carried out at a pH of 5.0-8.0; The reaction temperature is 30℃-40℃, and the reaction time is 2-24h. The method also includes the addition of methyl donor vanillic acid or dicamba.

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