A method for one-pot enzymatic preparation of l-5-mthf

Abstract

The application relates to the field of drug synthesis and discloses a one-pot enzyme method for preparing L-5-MTHF. The method provided by the application adds dihydrofolate reductase and tetrahydrofolate methyltransferase into folic acid, so that the folic acid is subjected to asymmetric hydrogenation reaction under the catalysis of dihydrofolate reductase (DHFR), an intermediate compound L-tetrahydrofolic acid is obtained, and then the L-tetrahydrofolic acid is subjected to catalysis by tetrahydrofolate methyltransferase (DmdA) to obtain L-5-MTHF with optical purity. The one-pot enzyme method can avoid the separation step of an intermediate product, meanwhile, the produced intermediate is converted into the product L-5-MTHF at the moment, the product is used immediately, and the problem of product inhibition is avoided. The method for preparing L-5-MTHF has high yield, great application value and market prospect.

Description

Technical Field

[0001] This invention relates to the field of drug synthesis, and more particularly to a one-pot enzymatic method for preparing L-5-MTHF. Background Technology

[0002] L-5-methyltetrahydrofolate (L-5-MTHF) is the main form of naturally occurring folic acid from food sources and the only form of folic acid found in human plasma and blood circulation, where it is used for inter-tissue folic acid transport. L-5-MTHF can be directly absorbed and utilized in the body without additional metabolic steps, exhibiting higher bioavailability than synthetic folic acid. Furthermore, recent studies have shown that L-5-MTHF is the only folic acid drug that can cross the blood-brain barrier, playing a role in the prevention and treatment of Alzheimer's disease, and also serving as an antidote for the broad-spectrum antitumor drug methotrexate. Therefore, L-5-MTHF is the most biologically active and functional form of folic acid. The United States, Japan, and Europe have approved L-5-MTHF as a food additive for various foods, leading to a surge in market demand.

[0003] L-5-MTHF contains two chiral carbon atoms, and when hydrogen is added to the double bond between C5 and C6, it readily forms optical isomers, including 6R-5-MTHF (i.e., D-5-MTHF) and 6S-5-MTHF (i.e., L-5-MTHF). Among them, the (6S) configuration of L-5-MTHF is the only naturally occurring form and has good medicinal effects, while the 6R configuration of D-5-MTHF cannot be absorbed and utilized by the human body and has no therapeutic or health care effects.

[0004] The synthesis of L-5-MTHF mainly uses folic acid as a precursor and includes chemical methods, enzymatic-chemical methods, and microbial fermentation methods.

[0005] Chemical synthesis is the most commonly used method for synthesizing L-5-MTHF in current industrial production. Chemical synthesis methods include catalytic hydrogenation, sodium borohydride reduction, sodium dithionite reduction, and dihydrofolate reductase method. For example, the sodium borohydride reduction method uses NaBH4 as a reducing agent to reduce folic acid to a racemic mixture of THF, which is then separated and methylated to obtain L-5-MTHF. The disadvantages of the sodium borohydride reduction method are: ① NaBH4 is highly irritating and flammable, posing a safety hazard; ② the product is a racemic mixture, making separation difficult; ③ the production process involves many steps and is relatively cumbersome.

[0006] For example, Chinese patent application CN114874216A provides a method for preparing L-5-methyltetrahydrofolate, which also synthesizes L-5-MTHF via chemical methods. The technical solution for synthesizing L-5-methyltetrahydrofolate in this patent is as follows: using folic acid as a raw material, the intermediate 6-(R,S)-tetrahydrofolate is reduced to obtain the intermediate 6-(R,S)-tetrahydrofolate; then, 6-(R,S)-tetrahydrofolate undergoes chiral resolution to obtain the intermediate 6-S-tetrahydrofolate; finally, the intermediate 6-S-tetrahydrofolate is methylated in a buffer solution to obtain L-5-methyltetrahydrofolate. While this patent method advances the chiral resolution to the second step, it still cannot avoid the challenging chiral resolution step. Furthermore, the method is relatively complex, resulting in high production costs for industrial applications.

[0007] In existing technologies, L-5-MTHF can also be prepared through microbial fermentation. This method relies on microorganisms capable of accumulating high concentrations of folic acid and L-5-MTHF intracellularly, and then producing L-5-MTHF through fermentation. High-yielding strains are generally obtained through natural selection, mutagenesis breeding, and genetic engineering breeding. However, because folic acid compounds are coenzyme factors in intracellular metabolism and are subject to multiple dynamic equilibrium regulations, their intracellular accumulation concentration is very limited. The reported levels of L-5-MTHF produced by fermentation by existing microbial strains and genomically modified strains are all below 1.3 mg / L.

[0008] In existing technologies, L-5-MTHF can also be prepared using a combination of enzymatic and chemical methods. The enzymatic-chemical method relies on the activity of dihydrofolate reductase (DHFR) to reduce dihydrofolate (DHF) to tetrahydrofolate (6S-THF), followed by a chemical transmethylation reaction. However, in the enzyme-catalyzed system, the first problem is that the activity of DHFR is inhibited by the product 6S-THF, limiting the conversion rate. Secondly, 6S-THF has poor stability and is easily lost; further separation and extraction for the subsequent chemical methylation reaction results in a low product yield.

[0009] To address the problems in the prior art, our team is dedicated to developing a method for preparing L-5-methyltetrahydrofolate that is high-yield, simple in production steps, and safe and mild. Summary of the Invention

[0010] To address the aforementioned technical problems, this invention provides a one-pot enzymatic method for preparing L-5-MTHF. The method provided by this invention is a total enzymatic catalytic conversion method with mild and safe conditions. Furthermore, the one-pot preparation process of this invention simplifies the steps and avoids the problems of intermediate product separation and product inhibition by the enzyme catalytic system. The one-pot enzymatic process for preparing L-5-methyltetrahydrofolate provided by this invention features high yield and has significant application value and market potential.

[0011] The specific technical solution of this invention is as follows:

[0012] This invention provides a one-pot enzymatic method for preparing L-5-MTHF, comprising the following steps:

[0013] Dihydrofolate reductase and tetrahydrofolate methyltransferase were added to folic acid to catalyze the reaction and obtain L-5-MTHF.

[0014] This invention employs a one-pot enzymatic process to prepare L-5-methyltetrahydrofolate. In this method, the substrate compound (II), i.e., folic acid, first undergoes an asymmetric hydrogenation reaction catalyzed by dihydrofolate reductase (DHFR) to yield the intermediate compound (III), i.e., L-tetrahydrofolate 6S-THF. This intermediate is then catalyzed by tetrahydrofolate methyltransferase (DmdA) to obtain optically pure L-5-MTHF. This one-pot enzymatic process avoids the intermediate product separation step. In this method, the generated intermediate 6S-THF is immediately converted to the product L-5-MTHF, allowing for immediate use and eliminating the disadvantage of product inhibition. Using 10 g / L folic acid as the substrate for 10 hours, the substrate conversion rate is greater than 90%, achieving highly efficient enzymatic preparation of L-5-MTHF. This synthetic process has not been reported in the published literature. The process route is as follows:

[0015] The dihydrofolate reductases mentioned above are XcaDHFR (SEQ ID NO.2, NCBI accession number WP_011036024.1) from Xanthomonas campestris, LasDHFR (SEQ ID NO.4, NCBI accession number WP_014568348.1) from Lactobacillus salivarius, BhaDHFR (SEQ ID NO.6, NCBI accession number WP_010899586.1) from Bacillus halodurans C-125, and the human double mutant hDHFR-35K64F (SEQ ID NO.8, NCBI accession number 3N0H_A). The nucleotide sequences of the dihydrofolate reductase XcaDHFR, LasDHFR, BhaDHFR and hDHFR-35K64F genes are shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7, respectively, and the amino acid sequences encoding them are shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8, respectively.

[0016] The nucleotide sequences of the dihydrofolate reductase gene, SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.7, were cloned into *Escherichia coli* to construct the corresponding recombinant *E. coli* engineered bacteria. Dihydrofolate reductase was then expressed and purified. It should be noted that the amino acid sequence of dihydrofolate reductase has >80% homology with the sequences shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, or SEQ ID NO.8, and all such sequences should be included within the scope of protection of this invention.

[0017] The aforementioned tetrahydrofolate methyltransferase is a dimethylsulfoniopropionate (DMSP)-dependent demethylase DmdA (SEQ ID NO.10, NCBI accession number WP_011281570.1) derived from the marine microorganism Pelagabacter ubique. The nucleotide sequence of the gene encoding the amino acid sequence of this tetrahydrofolate methyltransferase SEQ ID NO.10 is shown in SEQ ID NO.9.

[0018] The nucleotide sequence of the tetrahydrofolate methyltransferase gene, SEQ ID NO.9, was cloned into *Escherichia coli* to construct the corresponding recombinant *E. coli* engineered strain. The tetrahydrofolate methyltransferase was then expressed and purified. It should be noted that the amino acid sequence of the tetrahydrofolate methyltransferase shares >80% homology with the sequence shown in SEQ ID NO.10, and both sequences should be included within the scope of protection of this invention.

[0019] As a preferred embodiment of the present invention, the amino acid sequence of the dihydrofolate reductase used has ≥90% homology with the sequences shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8, more preferably, it has ≥95% homology.

[0020] As a preferred embodiment of the present invention, the amino acid sequence of the tetrahydrofolate methyltransferase used has ≥90% homology with the sequence shown in SEQ ID NO.10, more preferably, it has ≥95% homology.

[0021] As a preferred embodiment of the present invention, dimethyl mercaptopropionic acid (DMSP) is added to the catalytic reaction system as a methyl donor substrate. Preferably, an NAD(P)-dependent dehydrogenase and its substrate are also added to the catalytic reaction system.

[0022] In the one-pot enzymatic catalytic conversion of folic acid to L-5-methyltetrahydrofolate in this invention, NAD+ is required. + or NADP + Using NAD(P)-dependent dehydrogenases and their substrates as coenzyme substrates, the production of L-5-methyltetrahydrofolate can achieve a high yield.

[0023] Preferably, the dehydrogenase is selected from alcohol dehydrogenase, glucose dehydrogenase, and formic acid dehydrogenase. The substrate is selected from isopropanol, glucose, and formic acid.

[0024] As a preferred embodiment of the present invention, the temperature of the catalytic reaction is 25–40°C, and the pH of the catalytic reaction is 7.0–9.0. In a reaction environment with a temperature of 25–40°C and a pH of 7.0–9.0, folic acid can be converted to L-5-methyltetrahydrofolate with a high yield.

[0025] Compared with the prior art, the present invention has the following technical effects:

[0026] (1) The present invention provides a method for synthesizing L-5-methyltetrahydrofolate by catalyzing the conversion of folic acid with a whole enzyme method. The conditions are mild and avoid the safety hazards of the chemical method for synthesizing L-5-methyltetrahydrofolate commonly used in the prior art.

[0027] (2) The method provided by the present invention uses a one-pot cooking process, which avoids the intermediate product separation step in the enzyme-chemical synthesis process. In this method, the generated intermediate is converted into the product L-5-methyltetrahydrofolate in a timely manner, which can be used immediately after production, and solves the problem of product inhibition in the enzyme-chemical method.

[0028] (3) Compared with the existing microbial fermentation method for preparing L-5-methyltetrahydrofolate, the method provided by the present invention is simple and has a high conversion yield. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the structure of a recombinant plasmid pET28a-XcaDHFR according to the present invention;

[0030] Figure 2 This is a schematic diagram of the structure of a recombinant plasmid pET21a-hDHFR-35K64F according to the present invention;

[0031] Figure 3 This is an SDS-PAGE analysis diagram of dihydrofolate reductase expression in Example 2 of the present invention;

[0032] Figure 4 This is the HPLC chromatogram of folic acid, the substrate in Example 2 of the present invention;

[0033] Figure 5 This is the HPLC chromatogram of DHFR-catalyzed folic acid conversion in Example 2 of the present invention;

[0034] Figure 6 This is a schematic diagram of the structure of a recombinant plasmid pET28a-DmdA according to the present invention;

[0035] Figure 7 This is an SDS-PAGE analysis diagram of tetrahydrofolate methyltransferase expression in Example 4 of the present invention;

[0036] Figure 8 This is the HPLC chromatogram of DmdA-catalyzed L-tetrahydrofolate methylation in Example 4 of the present invention;

[0037] Figure 9 This is the HPLC chromatogram of the reaction for preparing L-5-MTHF from folic acid in Example 9 of the present invention after 10 h. Detailed Implementation

[0038] The present invention will be further described below with reference to embodiments.

[0039] In the examples, the LB medium used consisted of 5 g / L yeast extract, 10 g / L peptone, and 10 g / L NaCl, with deionized water as the solvent and a pH of 6.8–7.0. The final concentration of the fermentation medium was: 12 g / L yeast extract, 15 g / L peptone, 10 g / L glycerol, 8.9 g / L Na₂HPO₄·12H₂O, 3.4 g / L KH₂PO₄, 2.67 g / L NH₄Cl, 0.71 g / L Na₂SO₄, and 0.3 g / L MgSO₄·7H₂O, with deionized water as the solvent and a pH of 6.8–7.0.

[0040] Example 1: Construction of recombinant Escherichia coli engineered strain for dihydrofolate reductase

[0041] Literature review and NCBI database search identified 40 candidate dihydrofolate reductases, which may possess asymmetric hydrogenation catalytic activity on the double bond between C5 and C6 of folate substrate. Based on the gene sequences corresponding to the 40 candidate enzymes, upstream and downstream primers were designed as shown in Table 1, and the corresponding source strains are listed in Table 2. pET-28a(+) was selected as the recombinant expression vector, and the amplified gene was inserted between NdeⅠ and BamHI restriction sites to construct recombinant plasmids of dihydrofolate reductase from different strains.

[0042] Furthermore, point mutations were performed on the recombinant plasmid pET28a-EcoDHFR to construct the *E. coli*-derived dihydrofolate reductase mutant *EcoDHFR ANLYF*. The construction of the human dihydrofolate reductase double mutant was as follows: the corresponding amino acid sequence was sent to a gene synthesis company (Suzhou Genewise Biotechnology Co., Ltd.), where the gene (SEQ ID NO.7) was artificially synthesized and cloned into the NdeI and BamHI spaces of the expression vector pET21a to obtain the recombinant plasmid pET21a-hDHFR-35K64F. Figure 1 This is a schematic diagram of the structure of the recombinant plasmid pET28a-XcaDHFR. Figure 2 This is a schematic diagram of the structure of the recombinant plasmid pET21a-hDHFR-35K64F.

[0043] Table 1 Primer Sequence List for DHFR Recombinant Plasmid

[0044]

[0045]

[0046] The recombinant plasmids of the genes from each enzyme were transformed into the expression host *E. coli* BL21(DE3). The specific procedures were as follows: 50 ng of the recombinant plasmid was added to 100 μL of *E. coli* BL21(DE3) competent cells. The mixture was gently tapped against the tube wall several times to mix, and then incubated in an ice-water bath for 30 min. A heat shock at 42℃ for 45 s was performed, followed by incubation in ice water for 3 min. 900 μL of antibiotic-free LB medium was added, and the cells were incubated at 37℃ for 60 min to restore antibiotic resistance. 50 μL of the bacterial culture was evenly spread onto LB agar plates containing the corresponding antibiotics (50 mg / L kanamycin / 100 mg / L ampicillin). The plates were inverted and incubated overnight at 37℃. Clones that showed positive PCR results were picked, purified by streak plasmiding and shake-cultured, and the plasmids were extracted. Enzyme digestion and sequencing verification were performed to obtain the confirmed positive clones, yielding recombinant *E. coli* containing each enzyme for subsequent enzyme expression and catalytic activity analysis.

[0047] Example 2: Induced expression and enzyme activity analysis of dihydrofolate reductase

[0048] The recombinant Escherichia coli engineered bacteria expressing dihydrofolate reductase from different strains prepared in Example 1 were inoculated into LB medium containing the corresponding resistance (50 mg / L kanamycin / 100 mg / L ampicillin) and cultured at 37°C and 200 rpm to mid-logarithmic growth to obtain freshly cultured seed culture.

[0049] Freshly cultured seed culture was inoculated at a volume concentration of 2% into E. coli fermentation medium containing the corresponding resistance (50 mg / L kanamycin / 100 mg / L ampicillin), and incubated at 37°C for 3 hours until the OD of the bacterial cells was observed. 600 When the concentration reaches 0.6–0.8, add IPTG to a final concentration of 0.25 mmol / L, control the fermentation temperature at 24°C, and continue fermentation for 10 hours to obtain a fermentation broth with a wet cell content of 5 g / L.

[0050] The centrifuged fermentation broth was resuspended in pH 7.5, 400mM HEPES buffer, and the cells were disrupted using a high-pressure cell homogenizer to obtain crude enzyme solution. This solution should be used for catalytic reactions as soon as possible to avoid long-term storage.

[0051] The crude enzyme was used for SDS-PAGE electrophoresis analysis to determine the induced expression of dihydrofolate reductase. The results are as follows: Figure 3 As shown.

[0052] Analysis of the catalytic activity of dihydrofolate reductase on the substrate folate: To the crude enzyme solution of dihydrofolate reductase described above, NADP was added sequentially to a final concentration of 1.0 mM. +3% isopropanol, 25mM vitamin C, 6mM DTT, and 1.0 g / L folic acid were added to a 20 mL round-bottom flask and magnetically stirred at 37°C for 8 h. Under magnetic stirring, 500 μL of the reaction solution was added to 500 μL of 5% ammonia water and centrifuged at 10000×g for 5 min. The supernatant was used for liquid chromatography analysis. Liquid chromatography analysis method: Column: C18 column; Detection wavelength: 254 nm (folic acid) and 290 nm (L-tetrahydrofolic acid); Mobile phase: phosphate buffer (pH 6.3); Flow rate: 1.0 mL / min; Column temperature: 40°C; Injection volume: 10 μL. The HPLC chromatogram of the substrate folic acid is shown below. Figure 4 As shown, the retention time was 10.003 min; dihydrofolate reductase catalyzes the asymmetric hydrogenation reaction of folic acid to produce L-tetrahydrofolate, and the HPLC chromatogram of the catalytic process is shown below. Figure 5 As shown, the retention time of the product L-tetrahydrofolate was 5.039 min.

[0053] The catalytic activity results of each recombinant Escherichia coli engineered strain are shown in Table 2. Further comparative analysis was conducted, and based on their enzyme activity, four recombinant strains with the highest dihydrofolate reductase activity were selected as preferred strains, including Escherichia coli XcaDHFR, LasDHFR, BhaDHFR, and hDHFR-35K64F.

[0054] The amino acid sequence of dihydrofolate reductase from XcaDHFR derived from Xanthomonas campestris is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1; the amino acid sequence of dihydrofolate reductase from LasDHFR derived from Lactobacillus salivarius is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3; the amino acid sequence of dihydrofolate reductase from BhaDHFR derived from Bacillushalodurans C-125 is shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5; the amino acid sequence of dihydrofolate reductase from the human double mutant hDHFR-35K64F is shown in SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7.

[0055] Table 2 Comparison of catalytic activities of recombinant dihydrofolate reductase strains

[0056]

[0057]

[0058]

[0059] Example 3: Construction of recombinant Escherichia coli engineered strain of tetrahydrofolate methyltransferase

[0060] Literature review and NCBI database search identified a tetrahydrofolate methyltransferase that may possess transmethylation activity to convert 6S-tetrahydrofolate (compound III) to L-5-methyltetrahydrofolate (compound I). Its gene was artificially synthesized (SEQ ID NO. 9) and cloned into the NcoI and BamHI spaces of the expression vector pET28a to obtain the recombinant plasmid pET28a-DmdA of tetrahydrofolate methyltransferase. A schematic diagram of the structure of the recombinant plasmid pET28a-DmdA is shown below. Figure 6 As shown. The amino acid sequence of the tetrahydrofolate methyltransferase expressed by the gene encoding SEQ ID NO.9 is shown in SEQ ID NO.10.

[0061] The recombinant plasmid containing the enzyme gene was transformed into the expression host *E. coli* BL21(DE3) as follows: 50 ng of the recombinant plasmid was added to 100 μL of *E. coli* BL21(DE3) competent cells. The mixture was gently tapped against the tube wall several times to mix, and then incubated in an ice-water bath for 30 min. The cells were then heat-shocked at 42°C for 45 s and incubated in ice-water for 3 min. 900 μL of antibiotic-free LB medium was added, and the cells were incubated at 37°C for 60 min to restore antibiotic resistance. 50 μL of the bacterial culture was evenly spread onto an LB agar plate containing 50 mg / L kanamycin. The plate was inverted and incubated overnight at 37°C. Clones that showed positive results by colony PCR were selected, purified by streak plasmid and cultured on a shaker, and then the plasmid was extracted. The plasmid was then verified by enzyme digestion and sequencing. Finally, the positive clones that were verified were obtained, and the recombinant E. coli engineered strain E. coli BL21(DE3)(pET28a-DmdA), abbreviated as E. coli DmdA, was obtained for subsequent enzyme expression and enzyme catalytic activity analysis.

[0062] Example 4: Induced expression and enzyme activity analysis of tetrahydrofolate methyltransferase

[0063] The recombinant Escherichia coli engineered strain E. coli DmdA prepared in Example 3 was inoculated into LB medium containing 50 mg / L kanamycin and cultured at 37°C and 200 rpm until mid-logarithmic growth to obtain freshly cultured seed culture.

[0064] Freshly cultured seed culture was inoculated at a volume concentration of 2% into E. coli fermentation medium containing 50 mg / L kanamycin and incubated at 37°C for 3 hours until the OD of the bacterial cells was observed. 600When the concentration reaches 0.6–0.8, add IPTG to a final concentration of 0.25 mmol / L, control the fermentation temperature at 24°C, and continue fermentation for 10 hours to obtain a fermentation broth with a wet cell content of 5 g / L.

[0065] The centrifuged fermentation broth was resuspended in pH 7.5, 400mM HEPES buffer, and the cells were disrupted using a high-pressure cell homogenizer to obtain crude enzyme solution. This solution should be used for catalytic reactions as soon as possible to avoid long-term storage.

[0066] The crude enzyme was used for SDS-PAGE electrophoresis analysis to determine the induced expression of tetrahydrofolate methyltransferase. The results are as follows: Figure 7 As shown.

[0067] Catalytic activity analysis of tetrahydrofolate methyltransferase for L-tetrahydrofolate: To the crude tetrahydrofolate methyltransferase solution, DMSP (10 mM), Vc (25 mM), DTT (6 mM), and L-tetrahydrofolate (1.0 g / L) were added sequentially. The reaction solution was placed in a 20 mL round-bottom flask, magnetically stirred, and reacted at 37 °C for 1 h. Under magnetic stirring, 500 μL of the reaction solution was added to 500 μL of 5% ammonia water, centrifuged at 10000 × g for 5 min, and the supernatant was used for liquid chromatography analysis. Liquid chromatography analysis method: Column: C18 column; Detection wavelength: 290 nm; Mobile phase: phosphate buffer (pH 6.3); Flow rate: 1.0 mL / min; Column temperature: 40 °C; Injection volume: 10 μL. The HPLC chromatogram of the catalytic process is shown below. Figure 8 As shown, the retention time of the product L-5-methyltetrahydrofolate was 16.353 min.

[0068] Example 5: Construction of recombinant Escherichia coli engineered strains for coenzyme cycling system

[0069] Based on the catalytic mechanism of dihydrofolate reductase, the coenzyme NAD(P)H is required. To improve the catalytic efficiency of the one-pot enzymatic preparation of L-5-methyltetrahydrofolate, an NAD(P)+ / NAD(P)H coenzyme cycling system is used in this catalytic reaction system. Three types of NAD(P) can be used in this invention. + The / NAD(P)H coenzyme cycle system is shown in Table 3. The alcohol dehydrogenase lbADH from Lactobacillus brevis was selected as the coenzyme cycle system for subsequent examples.

[0070] The crude enzyme solution of alcohol dehydrogenase lbADH was prepared as follows: Recombinant E. coli engineered bacteria containing lbADH enzyme, stored in a laboratory glycerol tube under cryopreservation, was activated by streaking onto LB agar plates containing 50 mg / L kanamycin. Single colonies were picked and inoculated into LB medium containing 50 mg / L kanamycin, and cultured at 37°C and 200 rpm until mid-logarithmic growth was achieved, yielding a freshly cultured seed culture.

[0071] Table 3 Coenzyme Cyclic System

[0072] enzyme name source NCBI login number Substrate byproducts alcohol dehydrogenase lbADH Lactobacillus brevis WP_011668302 Isopropanol acetone glucose dehydrogenase bsGDH Bacillus subtilis NP_388275.1 glucose gluconic acid Formate dehydrogenase psFDH Pseudomonas P33160.3 Formic acid CO2

[0073] Freshly cultured seed culture was inoculated at a volume concentration of 2% into E. coli fermentation medium containing 50 mg / L kanamycin and incubated at 37°C for 3 hours until the OD of the bacterial cells was observed. 600 When the concentration reaches 0.6–0.8, add IPTG to a final concentration of 0.25 mmol / L, control the fermentation temperature at 24°C, and continue fermentation for 10 hours to obtain a fermentation broth with a wet cell content of 5 g / L.

[0074] The centrifuged fermentation broth was resuspended in pH 7.5, 400mM HEPES buffer, and the cells were disrupted using a high-pressure cell homogenizer to obtain crude enzyme solution. This solution should be used for catalytic reactions as soon as possible to avoid long-term storage.

[0075] Example 6: Preparation of crude enzyme solution of dihydrofolate reductase by fermentation in a 3L tank.

[0076] According to Example 1, activated recombinant Escherichia coli XcaDHFR, LasDHFR, BhaDHFR and hDHFR-35K64F were inoculated into LB medium containing 50 mg / L kanamycin and cultured at 37°C and 200 rpm until mid-logarithmic growth to obtain freshly cultured seed culture.

[0077] Fermentation broths of XcaDHFR, LasDHFR, BhaDHFR, and hDHFR-35K64F were prepared by fermentation in a 3L fermenter: Freshly cultured seed culture was inoculated at a volume concentration of 2% into E. coli fermentation medium containing the corresponding resistance (50 mg / L kanamycin / 100 mg / L ampicillin), and cultured at 37℃ for 4 h. α-lactose was added to a final concentration of 10 g / L, and the fermentation temperature was controlled at 24℃. Dissolved oxygen (DO) was controlled to be greater than 20%, and the fermentation pH was controlled to 6.8 with 25% ammonia. Fermentation was continued for 16 h to obtain a fermentation broth with a wet cell count of 30 g / L, which was designated as DHFR fermentation broth.

[0078] The centrifuged fermentation broth was resuspended in pH 7.5, 400mM HEPES buffer, and the cells were disrupted using a high-pressure cell homogenizer to obtain crude DHFR enzyme solution. This solution should be used for catalytic reactions as soon as possible to avoid long-term storage.

[0079] Example 7: Preparation of crude enzyme solution of tetrahydrofolate methyltransferase by fermentation in a 3L tank.

[0080] According to Example 3, the recombinant Escherichia coli DmdA was activated and inoculated into LB medium containing 50 mg / L kanamycin. It was cultured at 37°C and 200 rpm until the mid-logarithmic growth phase to obtain freshly cultured seed culture.

[0081] Fermentation broth for preparing DmdA in a 3L fermenter: Freshly cultured seed culture was inoculated into E. coli fermentation medium containing 50 mg / L kanamycin at a volume concentration of 2%. The culture was incubated at 37°C for 4 hours. α-lactose was added to a final concentration of 10 g / L. The fermentation temperature was controlled at 24°C, dissolved oxygen (DO) was controlled to be greater than 20%, and the fermentation pH was controlled at 6.8 with 25% ammonia. Fermentation was continued for 16 hours to obtain a fermentation broth with a wet cell count of 30 g / L, which was denoted as DmdA fermentation broth.

[0082] The centrifuged fermentation broth was resuspended in pH 7.5, 400mM HEPES buffer, and the cells were disrupted using a high-pressure cell homogenizer to obtain crude DmdA enzyme solution. This solution should be used for catalytic reactions as soon as possible to avoid long-term storage.

[0083] Example 8: Preparation of crude enzyme solution of 1lbADH by fermentation in a 3L tank

[0084] According to Example 5, recombinant Escherichia coli lbADH was activated and inoculated into LB medium containing 50 mg / L kanamycin. It was cultured at 37°C and 200 rpm until mid-logarithmic growth to obtain freshly cultured seed culture.

[0085] Fermentation broth for preparing lbADH in a 3L fermenter: Freshly activated recombinant E. coli-lbADH was inoculated into LB medium containing 50 mg / L kanamycin and cultured at 37°C and 200 rpm until mid-logarithmic growth to obtain freshly cultured seed culture.

[0086] Fermentation broth for preparing lbADH in a 3L fermenter: Freshly cultured seed culture was inoculated into Escherichia coli fermentation medium containing 50 mg / L kanamycin at a volume concentration of 2%. The culture was incubated at 37℃ for 4 h. α-lactose was added to a final concentration of 10 g / L, and the fermentation temperature was controlled at 24℃. Dissolved oxygen (DO) was controlled to be greater than 20%. The fermentation pH was controlled at 6.8 with 25% ammonia water. Fermentation was continued for 16 h to obtain a fermentation broth with a wet cell content of 30 g / L, which was denoted as lbADH fermentation broth.

[0087] Centrifuged fermentation broth was resuspended in pH 7.5, 400mM HEPES buffer, and cells were disrupted using a high-pressure cell homogenizer to obtain crude enzyme solution for coenzyme cycling. This solution should be used for catalytic reactions as soon as possible to avoid long-term storage.

[0088] Example 9: Preparation of L-5-methyltetrahydrofolate by mixing XcaDHFR, lbADH, and DmdA fermentation broth.

[0089] The catalytic process for the one-pot enzymatic preparation of L-5-MTHF is as follows:

[0090]

[0091] Centrifuge the XcaDHFR fermentation broth prepared by the method in Example 6 and collect 20.0 g of wet cell culture; centrifuge the lbADH fermentation broth prepared by the method in Example 8 and collect 10.0 g of wet cell culture; centrifuge the DmdA fermentation broth prepared by the method in Example 7 and collect 30.0 g of wet cell culture. Resuspend the three types of wet cell culture in 150 mL of pH 7.5, 400 mM HEPES buffer, homogenize the cells using a high-pressure homogenizer, and add NADP to a final concentration of 1.0 mM. + 3% isopropanol, 25mM vitamin C, 6mM DTT, 10mM MDMSP, and 10.0g / L folic acid were added to a final volume of 200ml with deionized water, and the pH was adjusted to 7.5. The reaction system was magnetically stirred and catalyzed in a 37℃ water bath for 12 hours. The reaction solution was then analyzed by HPLC, and the results are as follows: Figure 9 As shown, some of the substrate compounds II (folic acid) and III (L-tetrahydrofolic acid) remained unconverted, with a final conversion rate of 80%.

[0092] The sampling HPLC analysis method is as follows: Under magnetic stirring, 500 μL of the reaction solution was added to 500 μL of 5% ammonia water, centrifuged at 10000×g for 5 min, and the supernatant was used for liquid chromatography analysis. Liquid chromatography analysis method: Column: C18 column; Detection wavelength: 254 nm and 290 nm; Mobile phase: phosphate buffer (pH 6.3); Flow rate: 1.0 mL / min; Column temperature: 40℃; Injection volume: 10 μL.

[0093] Example 10: Preparation of L-5-methyltetrahydrofolate by mixing LasDHFR, lbADH, and DmdA fermentation broth. The LasDHFR fermentation broth prepared in Example 6 was centrifuged, and 20.0 g of wet cell culture was collected. The lbADH fermentation broth prepared in Example 8 was centrifuged, and 10.0 g of wet cell culture was collected. The DmdA fermentation broth prepared in Example 7 was centrifuged, and 30.0 g of wet cell culture was collected. All three types of wet cell culture were resuspended in 150 mL of pH 7.5, 400 mM HEPES buffer. The cells were homogenized using a high-pressure homogenizer, and NADP was added to a final concentration of 1.0 mM. + The reaction mixture consisted of 3% isopropanol, 25 mM vitamin C, 6 mM DTT, 10 mM DMSP, and 10.0 g / L folic acid (compound II), with the volume made up to 200 ml with deionized water. The pH was then adjusted to 7.5. The reaction system was subjected to magnetic stirring in a 37°C water bath for 12 h. The reaction solution was analyzed by HPLC. The results showed that the substrate compound II (folic acid) was completely converted, and compound III (L-tetrahydrofolic acid) was undetectable, indicating a conversion rate greater than 99%.

[0094] The sampling HPLC analysis method is as follows: Under magnetic stirring, 500 μL of the reaction solution was added to 500 μL of 5% ammonia water, centrifuged at 10000×g for 5 min, and the supernatant was used for liquid chromatography analysis. Liquid chromatography analysis method: Column: C18 column; Detection wavelength: 254 nm and 290 nm; Mobile phase: phosphate buffer (pH 6.3); Flow rate: 1.0 mL / min; Column temperature: 40℃; Injection volume: 10 μL.

[0095] Example 11: Preparation of L-5-methyltetrahydrofolate by mixing BhaDHFR, lbADH, and DmdA fermentation broth. The BhaDHFR fermentation broth prepared in Example 6 was centrifuged, and 20.0 g of wet cell culture was collected. The lbADH fermentation broth prepared in Example 8 was centrifuged, and 10.0 g of wet cell culture was collected. The DmdA fermentation broth prepared in Example 7 was centrifuged, and 30.0 g of wet cell culture was collected. All three types of wet cell culture were resuspended in 150 mL of pH 7.5, 400 mM HEPES buffer. The cells were homogenized using a high-pressure homogenizer, and NADP was added to a final concentration of 1.0 mM. + The reaction mixture consisted of 3% isopropanol, 25 mM vitamin C, 6 mM DTT, 10 mM DMSP, and 10.0 g / L folic acid (compound II), with the volume made up to 200 ml with deionized water. The pH was then adjusted to 7.5. The reaction system was subjected to magnetic stirring in a 37°C water bath for 10 h. The reaction solution was analyzed by HPLC. The results showed that some folic acid from substrate II remained unconverted, while compound III, L-tetrahydrofolic acid, was undetectable. The final conversion rate was 85%.

[0096] The sampling HPLC analysis method is as follows: Under magnetic stirring, 500 μL of the reaction solution was added to 500 μL of 5% ammonia water, centrifuged at 10000×g for 5 min, and the supernatant was used for liquid chromatography analysis. Liquid chromatography analysis method: Column: C18 column; Detection wavelength: 254 nm and 290 nm; Mobile phase: phosphate buffer (pH 6.3); Flow rate: 1.0 mL / min; Column temperature: 40℃; Injection volume: 10 μL.

[0097] Example 12: Preparation of L-5-methyltetrahydrofolate by mixing hDHFR-35K64F, lbADH, and DmdA fermentation broth. The hDHFR-35K64F fermentation broth prepared in Example 6 was centrifuged, and 20.0 g of wet cell culture was collected. The lbADH fermentation broth prepared in Example 8 was centrifuged, and 10.0 g of wet cell culture was collected. The DmdA fermentation broth prepared in Example 7 was centrifuged, and 30.0 g of wet cell culture was collected. All three types of wet cell culture were resuspended in 150 mL of pH 7.5, 400 mM HEPES buffer. The cells were homogenized using a high-pressure homogenizer, and NADP was added to a final concentration of 1.0 mM. + The reaction mixture consisted of 3% isopropanol, 25 mM vitamin C, 6 mM DTT, 10 mM DMSP, and 10.0 g / L folic acid (compound II), with the volume made up to 200 ml with deionized water. The pH was then adjusted to 7.5. The reaction system was subjected to magnetic stirring in a 37°C water bath for 10 h. The reaction solution was analyzed by HPLC. The results showed that the substrate compound II (folic acid) was undetectable, and compound III (L-tetrahydrofolic acid) was partially unconverted, with a final conversion rate of 93%.

[0098] The sampling HPLC analysis method is as follows: Under magnetic stirring, 500 μL of the reaction solution was added to 500 μL of 5% ammonia water, centrifuged at 10000×g for 5 min, and the supernatant was used for liquid chromatography analysis. Liquid chromatography analysis method: Column: C18 column; Detection wavelength: 254 nm and 290 nm; Mobile phase: phosphate buffer (pH 6.3); Flow rate: 1.0 mL / min; Column temperature: 40℃; Injection volume: 10 μL.

[0099] Analysis and evaluation by HPLC of the reaction solutions in Examples 9-12 showed that using recombinant Escherichia coli LasDHFR or hDHFR-35K64F, after 10-12 hours of catalysis, the concentration of L-5-methyltetrahydrofolate was greater than 9.0 g / L, i.e., the conversion rate was greater than 90%. The one-pot method of this invention for preparing L-5-methyltetrahydrofolate has a high yield and significant application value and market potential.

[0100] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0101] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A one-pot enzymatic method for preparing L-5-MTHF, characterized in that: Includes the following steps: Dihydrofolate reductase and tetrahydrofolate methyltransferase are added to folic acid to catalyze the reaction, yielding L-5-MTHF; wherein: The amino acid sequence of the dihydrofolate reductase has >80% homology with the sequence shown in SEQ ID NO.2, or >80% homology with the sequence shown in SEQ ID NO.4, or >80% homology with the sequence shown in SEQ ID NO.6, or >80% homology with the sequence shown in SEQ ID NO.8; The amino acid sequence of the tetrahydrofolate methyltransferase has >80% homology with the sequence shown in SEQ ID NO.10; The temperature of the catalytic reaction is 25~40℃, and the pH of the catalytic reaction is 7.0~9.

0.

2. The method for one-pot enzymatic preparation of L-5-MTHF as described in claim 1, characterized in that: The catalytic reaction incorporates one or more of a methyl donor, an NAD or NADP-dependent dehydrogenase, and a coenzyme substrate.

3. The method for one-pot enzymatic preparation of L-5-MTHF as described in claim 2, characterized in that: The methyl donor is dimethyl mercaptopropionic acid.

4. The method for one-pot enzymatic preparation of L-5-MTHF as described in claim 2, characterized in that: The dehydrogenase is selected from alcohol dehydrogenase, glucose dehydrogenase and formate dehydrogenase, and the coenzyme substrate is selected from isopropanol, glucose and formate.

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