TETRAHYDROFOLATE METHYLTRANSFERASE MUTANT, ENCODING GENE AND APPLICATIONS THEREOF

Abstract

A tetrahydrofolate methyltransferase mutant, its encoding gene, and applications thereof are provided. The amino acid sequence of a tetrahydrofolate methyltransferase RcoDmdA mutant is as shown in SEQ ID NO: 3. The RcoDmdA mutant possesses higher catalytic activity, allows the substrate THF feeding amount to be significantly increased to 15-20 g/L, increases the product L-5-MTHF yield to 15-17 g/L, and has high substrate conversion rate. The tetrahydrofolate methyltransferase mutant and its encoding gene have important industrial application value.

Claims

1. A tetrahydrofolate methyltransferase RcoDmdA mutant, wherein the amino acid sequence of the tetrahydrofolate methyltransferase RcoDmdA mutant is as shown in SEQ ID NO: 3.

2. A gene encoding the tetrahydrofolate methyltransferase RcoDmdA mutant according to claim 1.

3. The gene according to claim 2, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO: 10.

4. A method for preparing L-5-methyltetrahydrofolate (L-5-MTHF) by enzymatic catalysis of tetrahydrofolate (THF), comprising using the tetrahydrofolate methyltransferase RcoDmdA mutant according to claim 1.

5. The method according to claim 4, wherein the method comprises: constructing a genetically engineered bacterium containing a gene of the tetrahydrofolate methyltransferase RcoDmdA mutant, using wet cells obtained by fermentation culture of the genetically engineered bacterium or enzyme-containing cells obtained by cell disruption as a catalyst, to methylate THF, thereby obtaining L-5-MTHF.

6. The method according to claim 5, wherein a pET28a plasmid and E. coli BL21 host cells are used to construct a recombinant bacterium E. coli BL21.

7. The method according to claim 5, wherein a catalytic action is carried out in a presence of methyl donor dimethylsulfonium chloride.

8. The method according to claim 5, wherein a catalytic reaction is carried out at a pH of 6.0-9.0 and a temperature of 25 C.-50 C.

9. The method according to claim 6, wherein a catalytic action is carried out in a presence of methyl donor dimethylsulfonium chloride.

10. The method according to claim 6, wherein a catalytic reaction is carried out at a pH of 6.0-9.0 and a temperature of 25 C.-50 C.

Description

BRIEF DESCRIPTION OF THF DRAWINGS

[0035] FIG. 1 is a schematic diagram of the structure of the wild-type tetrahydrofolate methyltransferase RcoDmdA.

[0036] FIG. 2 is a schematic diagram showing amino acid residue sites around the THF and methyl donor DMSP binding pockets of the wild-type tetrahydrofolate methyltransferase RcoDmdA.

[0037] FIG. 3 is a schematic diagram showing 13 unsaturated amino acid sites within 5 of THF around the wild-type tetrahydrofolate methyltransferase RcoDmdA.

[0038] FIG. 4 is a schematic diagram showing the grouping and construction of small but smart libraries for trinucleotide saturation mutagenesis.

[0039] FIG. 5 is a schematic diagram showing the initial screening results of the Group A mutant library in the first-generation trinucleotide saturation mutagenesis library.

[0040] FIG. 6 is a schematic diagram showing the initial screening results of the Group B mutant library in the first-generation trinucleotide saturation mutagenesis library.

[0041] FIG. 7 is a schematic diagram showing the initial screening results of the Group C mutant library in the first-generation trinucleotide saturation mutagenesis library.

[0042] FIG. 8 is a schematic diagram showing the initial screening results of the Group D mutant library in the first-generation trinucleotide saturation mutagenesis library.

[0043] FIG. 9 is a comparative plot of product synthesis curves for single-point mutants in the catalytic preparation of L-5-MTHF from THF.

[0044] FIG. 10 is an HPLC chromatogram of THF and L-5-MTHF standard samples.

DETAILED DESCRIPTION OF THF EMBODIMENTS

[0045] The following specific embodiments are used to illustrate the implementation modes of the present invention. Skilled persons in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention may also be implemented or applied in different specific ways, and various modifications or changes may be made to the details of this specification based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the methods used in the examples are conventional methods, and the reagents used can be commercially obtained.

[0046] The following will further describe the present invention in detail with reference to specific examples, which are only used to explain the present invention and do not limit the protection scope of the present invention.

[0047] LB medium: yeast extract 5.0 g/L, peptone 10.0 g/L, NaCl 10.0 g/L, solvent is distilled water.

[0048] Fermentation medium: yeast extract 12.0 g/L, peptone 15.0 g/L, NazHPO.sub.4.Math.12H.sub.2O 8.9 g/L, KH.sub.2PO.sub.4 3.4 g/L, NH.sub.4Cl 2.67 g/L, Na.sub.2SO.sub.4 0.71 g/L, MgSO.sub.4 7H.sub.2O 0.49 g/L, kanamycin 50 g/L, pH 7.0, solvent is distilled water.

[0049] 400 mmol/L HEPES buffer solution (pH 7.5): HEPES 104.1 g, solvent is distilled water.

Example 1 Construction of Recombinant Tetrahydrofolate Methyltransferase RcoDmdA Expression Strain and its Induced Expression

[0050] The tetrahydrofolate methyltransferase RcoDmdA derived from Ruegeria conchae was retrieved from the GenBank database, i.e., GenBank accession No. UWR04622, with its amino acid sequence as shown in SEQ ID NO: 2. After codon optimization, the RcoDmdA encoding gene sequence was obtained, as shown in SEQ ID NO: 1. The gene was submitted to Genewiz for gene synthesis and cloned into the pET28a plasmid to obtain the recombinant expression plasmid pET28a-RcoDmdA. The plasmid was transformed into the expression host Escherichia coli BL21 (DE3) strain to obtain the recombinant strain E. coli BL21 (DE3) (pET28a-RcoDmdA).

[0051] The recombinant strain was streaked from the preserved glycerol stock onto an LB plate and cultured overnight. A single colony was inoculated into LB liquid medium containing 50 g/mL kanamycin and cultured overnight at 37 C., 200 rpm. It was then inoculated into the fermentation medium at 1-2% (v/v) inoculation amount, cultured at 37 C. for 2 h; IPTG was added to a final concentration of 0.5 mmol/L, the cultivation temperature was adjusted to 24 C., and fermentation was continued for 10 h to obtain a bacterial agent overexpressing RcoDmdA.

Example 2 Design of RcoDmdA Single-Point Mutations and Construction of Mutant Sites

[0052] To improve the catalytic reaction rate and product yield of RcoDmdA, mutation sites were screened using a homologous sequence alignment method. The three-dimensional structure was compared and analyzed with known homologous enzymes, specifically the crystal structures of PubDmdA from Pelagibacter ubique, including PDB IDs 3TFH, 3TFI, and 3TFJ. Through comparative analysis of the structural domains of the two enzymes, nine differing amino acid residues were identified around the THF and DMSP binding pockets, including positions 29, 60, 121, 150, 196, 245, 246, 249, and 263, and site-directed mutagenesis was performed at these sites. Based on the wild-type RcoDmdA gene sequence shown in SEQ ID NO: 1, primers for site-directed mutagenesis were designed, as shown in Table 1.

[0053] Each site on RcoDmdA was mutated to the corresponding amino acid residue of PubDmdA. The site-directed mutagenesis was constructed as follows: using the plasmid pET28a-RcoDmdA as a template, PCR amplification was performed with primers introducing the desired mutation site. The amplified product was purified using a PCR purification kit, digested with DpnI, and the digested product was subjected to a ligation reaction using the one-step cloning kit ClonExpress II from Vazyme. The ligation product was transformed into E. coli BL21 (DE3) cells, plated on LB plates containing 50 g/mL kanamycin, and verified by colony PCR and sequencing, thereby obtaining site-directed mutants for the 9 sites.

TABLE-US-00001 TABLE1 PrimersforSite-DirectedReplacementMutation SEQID Mutants Primers(5-3) NO: V29I-F GGAAGGCGTGAAAGGCTATACCATTTATAACCACA 17 V29I-R ATAGCCTTTCACGCCTTCCGCTTCCAC 18 C60A-F ATGTGCAAGTGTGGGATGTGAGCGCGGAACGCCAAGTG 19 C60A-R ATCCCACACTTGCACATGTTTTTTCAGA 20 S247N-F AGCGGCCTGCTGAGCTTTGGCAACGATATGCGC 21 S247N-R GCTCAGCAGGCCGCTTTCAATGCGT 22 V121I-F CTGGCGGATGATCATTATTGGCTGAGCATTGCGGATGGCGATC 23 V121I-R AATAATGATCATCCGCCAGTTTAATCGCCACCGGATC 24 P150I-F GTGAGCGAACCGGATGTGAGCATTCTGGCGGTGCAAGGC 25 P150I-R ACATCCGGTTCGCTCACTTCCACATCCAGTT 26 S196Q-F TGTGATTGCGCGCAGCGGCTGGCAAAAACAAGGCGGC 27 S196R-R CGCTGCGCGCAATCACAAAGCTCGTATCT 28 F245Y-F GCATTGAAAGCGGCCTGCTGAGCTATGGCAGCGAT 29 F245Y-R CAGCAGGCCGCTTTCAATGCGTTCAATGCCGTTCGGGCA 30 M249P-F AGCGGCCTGCTGAGCTTTGGCAGCGATTTTCGCCGCGAAA 31 M249P-R TGCCAAAGCTCAGCAGGCCGCTTTCAATGCGTTCAATG 32 F263Y-F CCCCGTATGAATGCGGCCTGGAACGCTATTGCAACAGCC 33 F263Y-R CCAGGCCGCATTCATACGGGGTGTTTTCGCGGCGC 34

Example 3 Screening and Activity Comparison of Single-Point Mutants

[0054] The transformants from the plates obtained in Example 2 were transferred to LB liquid medium containing 50 g/mL kanamycin and cultured in a shaker until mid-logarithmic phase. They were then inoculated into fermentation medium at 1-2% (v/v), cultured at 37 C. for 2 h, induced with 0.5 mmol/L IPTG, and fermentation was continued at 24 C. for 10 h to obtain the induced bacterial agent.

[0055] Following the method described in Example 1, bacterial agents containing the RcoDmdA mutants were prepared, 50 mL for each agent. Cells were collected by centrifugation at 10000g for 10 min, and the cells were resuspended in 10 mL of 0.4 mol/L HEPES buffer (pH 7.5) for ultrasonic disruption, and the supernatant was collected by centrifugation. THF was added to a final concentration of 10.0 g/L, dimethylsulfonium chloride (MSDS) to 100.0 mmol/L, and DTT to 1.0 g/L. The catalytic reaction was carried out in a 37 C. magnetic stirrer water bath for 10 h, and the catalytic reaction solution was used for HPLC analysis.

[0056] The initial screening results of the single-point mutants at various time points are shown in FIG. 9.

[0057] The catalytic results of the nine single-point mutants are shown in Table 2. By comparing the substrate conversion rates, the preferred mutants F245Y, V121I, and F263Y were obtained.

TABLE-US-00002 TABLE 2 Screening Results of Single-Point Mutants Mutants Conv. (%) WT 61.0 F263Y 96.1 F245Y 78.7 V121I 72.3 C60A 60.6 V29I 3.4 S247N 4.3 P150I 4.7 S196Q 4.6 M249P 53.7

Example 4 Design of RcoDmdA Mutation Sites and Construction of Small but Smart Mutagenesis Libraries

[0058] To further optimize the catalytic pocket of RcoDmdA and improve its catalytic reaction rate and product yield, trinucleotide saturation mutagenesis was performed on the THF binding domain of RcoDmdA. A computer-aided design method was used to screen mutation sites. Based on the reported crystal structure of PubDmdA from Pelagibacter ubique (PDB ID: 3TFH), homology modeling was performed on RcoDmdA, and molecular docking software was used. Considering the docking results of RcoDmdA with substrate THF, the characteristics of the enzyme's substrate binding pocket, and the catalytic mechanism, 13 amino acid residue sites were ultimately determined: positions 29, 93, 107, 121, 122, 150, 175, 176, 193, 194, 242, 243, and 263 in the amino acid sequence of SEQ ID NO: 2. The specific positions are shown in FIG. 3, and the grouping is shown in FIG. 4 (different colors indicate different groups: green: Group A; orange: Group B; blue: Group C; purple: Group D). They were divided into Group A (Y29, Y93, P107), Group B (V121, A122, F175, F176), Group C (P150, S193, G194), and Group D (L242, L243, F263).

[0059] Based on the wild-type RcoDmdA gene sequence shown in SEQ ID NO: 1, primers for trinucleotide saturation mutagenesis of Groups A, B, C, and D were designed, as shown in Table 3. The three codons correspond to serine, isoleucine, and tyrosine. The construction method for the trinucleotide saturation mutagenesis library is as follows: first, PCR amplification of the fragment at the mutation site was performed, with random mutations introduced via primers, followed by gel purification; then, the purified PCR product was used as one primer, and the recombinant plasmid pET28a-RcoDmdA as template, to further amplify the entire plasmid. After 1% agarose gel electrophoresis, the correctly sized band was obtained. The PCR product was digested with DpnI, subjected to a one-step cloning ligation reaction, and then transformed into E. coli BL21 (DE3) cells, plated on LB plates containing 50 g/mL kanamycin, to obtain the trinucleotide saturation mutagenesis library.

TABLE-US-00003 TABLE3 PrimersforTrinucleotideSaturationMutagenesis SEQID Mutants Primers(5-3) NO: 121122WT-F TGATCATTATTGGCTGAGCGTGGCGGATGGCGATCTG 35 121AKT-F TGATCATTATTGGCTGAGCAKTGCGGATGGCGATCTG 36 121TAT-F TGATCATTATTGGCTGAGCTATGCGGATGGCGATCTG 37 122AKT-F TGATCATTATTGGCTGAGCGTGAKTGATGGCGATCTG 38 122TAT-F TGATCATTATTGGCTGAGCGTGTATGATGGCGATCTG 39 121122AKT-F TGATCATTATTGGCTGAGCAKTAKTGATGGCGATCTG 40 121122TAT-F TGATCATTATTGGCTGAGCTATTATGATGGCGATCTG 41 121AKT122TAT-F TGATCATTATTGGCTGAGCAKTTATGATGGCGATCTG 42 121TAT122AKT-F TGATCATTATTGGCTGAGCTATAKTGATGGCGATCTG 43 175176WT-R CGTTTATAGCGAAAAAATTTAATATCGCGCA 44 175TMA-R CGTTTATAGCGAAATMATTTAATATCGCGCA 45 175ATA-R CGTTTATAGCGAAAATATTTAATATCGCGCA 46 176TMA-R CGTTTATAGCGTMAAAATTTAATATCGCGCA 47 175176TMA-R CGTTTATAGCGTMATMATTTAATATCGCGCA 48 175176ATA-R CGTTTATAGCGATAATATTTAATATCGCGCA 49 175TMA176ATA-R CGTTTATAGCGTMAATATTTAATATCGCGCA 50 175ATA176TMA-R CGTTTATAGCGATATMATTTAATATCGCGCA 51 CZ121-R CAGCCAATAATGATCATCCGCCAGTTTAAT 52 150/WT-F GTGAGCGAACCGGATGTGAGCCCGCTGGCGGTGCAA 53 G 150AKT-F GTGAGCGAACCGGATGTGAGCAKTCTGGCGGTGCAA 54 G 150TAT-F GTGAGCGAACCGGATGTGAGCTATCTGGCGGTGCAA 55 G 193194WT-R CTTGTTTGCTCCAGCCGCTGCGCGCAATCACA 56 193ATA-R CTTGTTTGCTCCAGCCATAGCGCGCAATCACA 57 193TAA-R CTTGTTTGCTCCAGCCAATGCGCGCAATCACA 58 194TMA-R CTTGTTTGCTCCAAMTGCTGCGCGCAATCACA 59 194ATA-R CTTGTTTGCTCCAATAGCTGCGCGCAATCACA 60 193ATA194TMA-R CTTGTTTGCTCCAAMTATAGCGCGCAATCACA 61 193TAA194ATA-R CTTGTTTGCTCCAATAAATGCGCGCAATCACA 62 193194ATA-R CTTGTTTGCTCCAATAATAGCGCGCAATCACA 63 193194TMA-R CTTGTTTGCTCCAAMTAATGCGCGCAATCACA 64 CZ150-R TCCGGTTCGCTCACTTCCACATCCAGTTCCA 65 242243WT-F CATTGAACGCATTGAAAGCGGCCTGCTGAGCTTTGGC 66 AGC 242AKT-F CATTGAACGCATTGAAAGCGGCAKTCTGAGCTTTGGC 67 AGC 242TAT-F CATTGAACGCATTGAAAGCGGCTATCTGAGCTTTGGC 68 AGC 243AKT-F CATTGAACGCATTGAAAGCGGCCTGAKTAGCTTTGGC 69 AGC 243TAT-F CATTGAACGCATTGAAAGCGGCCTGTATAGCTTTGGC 70 AGC 242AKT243TAT-F CATTGAACGCATTGAAAGCGGCAKTTATAGCTTTGGC 71 AGC 242TAT243AKT-F CATTGAACGCATTGAAAGCGGCTATAKTAGCTTTGGC 72 AGC 242243TAT-F CATTGAACGCATTGAAAGCGGCTATTATAGCTTTGGC 73 AGC 242243AKT-F CATTGAACGCATTGAAAGCGGCAKTAKTAGCTTTGGC 74 AGC 263WT-R CGGGCTGTTGCAAAAGCGTTCCAGGCCGC 75 263TMA-R CGGGCTGTTGCAAMTGCGTTCCAGGCCGC 76 263ATA-R CGGGCTGTTGCAATAGCGTTCCAGGCCGC 77 CZ242-R GCTTTCAATGCGTTCAATGCCGTTCGGGCA 78 29AKT-F GGAAGGCGTGAAAGGCTATACCGTGAKTAACCACAT 79 GC 93107WT-R GTTTAATCGCCACCGGATCGTTCAGCATGCCGCCGTT 80 CTGATCCACAATCGGCACATAATAGCACTGATC 93107TMA-R GTTTAATCGCCACAMTATCGTTCAGCATGCCGCCGTT 81 CTGATCCACAATCGGCACAMTATAGCACTGATC 107ATA-R GTTTAATCGCCACATAATCGTTCAGCATGCCGCCGTT 82 CTGATCCACAATCGGCACATAATAGCACTGATC 93TMA107ATA-R GTTTAATCGCCACATAATCGTTCAGCATGCCGCCGTT 83 CTGATCCACAATCGGCACAMTATAGCACTGATC 93TMA-R GTTTAATCGCCACCGGATCGTTCAGCATGCCGCCGTT 84 CTGATCCACAATCGGCACAMTATAGCACTGATC 107TMA-R GTTTAATCGCCACAMTATCGTTCAGCATGCCGCCGTT 85 CTGATCCACAATCGGCACATAATAGCACTGATC CZ29-R AGCCTTTCACGCCTTCCGCTTCCACGCCCG 86

Example 5 Screening of the Four Small but Smart Mutant Libraries

[0060] The trinucleotide saturation mutagenesis libraries obtained in Example 4 were used. Antimicrobial transformants growing on the plates were transferred to LB liquid medium containing 50 g/mL kanamycin, cultured until mid-logarithmic phase. They were then inoculated into fermentation medium at 1-2% (v/v), cultured at 37 C. for 2 h, induced with 0.5 mmol/L IPTG, and fermentation was continued at 24 C. for 10 h to obtain the induced bacterial agent.

[0061] 2 mL of the cultured transformants were centrifuged at 10,000g for 5 min to collect cells. The cells were resuspended in 1 mL of 0.4 mol/L HEPES buffer (pH 7.5), and 5.0 g/L THF and 50 mmol/L MSDS were added. The reaction was carried out at 37 C., 1000 rpm in a metal bath for 2 h, and the reaction mixture was analyzed by HPLC.

[0062] The enzyme activity screening results of the mutants in libraries LibA, LibB, LibC, and LibD are shown in FIGS. 5, 6, 7, and 8, respectively.

[0063] By comparing the substrate conversion rates, superior mutants were selected for sequencing analysis, and the mutants F263Y, V121I, P107I, V121S, A122S, and F263I were obtained as the preferred mutants from the first-generation library screening.

Example 6 Activity Analysis of First-Generation Preferred Mutants in Catalyzing THF to Prepare L-5-MTHF

[0064] The bacterial agents of RcoDmdA mutants F263Y, V121I, V121S, A122S, F263I, and P107I were prepared as described in Example 1. Each 30 mL bacterial agent was centrifuged at 10,000g for 10 min to collect cells. The cells were resuspended in 10 mL of 0.4 mol/L HEPES buffer (pH 7.5). 20.0 g/L THF, 100.0 mmol/L MSDS, and 1.0 g/L DTT were added. The reaction was carried out at 37 C. in a magnetic stirrer water bath for 10 h, and the reaction mixture was analyzed by HPLC.

[0065] The re-screening results of the first-generation preferred mutants are shown in Table 4. Among them, the mutant F263Y exhibited the best catalytic activity toward substrate THF and was identified as the first-generation optimal mutant.

TABLE-US-00004 TABLE 4 Re-screening Results of First-Generation Preferred Mutants Mutants Conv. (%) WT 29.4 F263Y 72.5 F263I 63.0 V121I 38.0 V121S 29.6 A122S 33.5 P107I 46.4

Example 7 Application of the Superior Mutant RcoDmdA-F263Y in Catalyzing THF to Prepare Z-5-MTHF

[0066] Induced expression of RcoDmdA mutants. The recombinant strain E. coli BL21 (DE3) (pET28a-RcoDmdA-F263Y) was streaked onto an LB plate from the preserved glycerol stock and cultured overnight. A single colony was inoculated into LB liquid medium containing 50 g/mL kanamycin and cultured overnight at 37 C., 200 rpm. The seed culture was inoculated at 2% into LB medium containing kanamycin sulfate (50 g/mL), cultured at 37 C., 200 rpm for 4-6 h, and then inoculated at 2% into a 3 L fermenter. Fermentation was carried out at 37 C. When the OD600 reached about 10, 10 g/L lactose was added to induce the expression of the target protein. The induction temperature was 22 C., and induction was continued for 8 h before ending fermentation, yielding the bacterial agent for catalytic reaction.

[0067] Catalytic reaction with 1 g/L THF feeding. Cells were collected by centrifugation, resuspended in 0.4 mol/L HEPES buffer (pH 7.5) to an OD600 of 40, and subjected to high-pressure homogenization to obtain cell lysate. 30 mL was added to a round-bottom flask, pre-incubated at 37 C. in a water bath for 10 min, and then 1 g/L THF, 20 mmol/L MSDS, and 1.0 g/L DTT were added sequentially. Magnetic stirring was started immediately to mix uniformly, and the reaction start time was recorded. After 1 h, the L-5-MTHF yield was 1.052 g/L, with a conversion rate of 102%.

[0068] Catalytic reaction with 10 g/L THF feeding. Cells were collected by centrifugation, resuspended in 0.4 mol/L HEPES buffer (pH 7.5) to an OD600 of 40, and subjected to high-pressure homogenization to obtain cell lysate. 30 mL was added to a round-bottom flask, pre-incubated at 37 C. in a water bath for 10 min, and then 10 g/L THF, 100 mmol/L MSDS, and 1.0 g/L DTT were added sequentially. Magnetic stirring was started immediately to mix uniformly, and the reaction start time was recorded. After 5 h, the L-5-MTHF yield was 9.9 g/L, with a conversion rate of 99%.

[0069] Catalytic reaction with 15 g/L THF feeding. Cells were collected by centrifugation, resuspended in 0.4 mol/L HEPES buffer (pH 7.5) to an OD600 of 40, and subjected to high-pressure homogenization to obtain cell lysate. 30 mL was added to a round-bottom flask, pre-incubated at 37 C. in a water bath for 10 min, and then 15 g/L THF, 150 mmol/L MSDS, and 1.0 g/L DTT were added sequentially. Magnetic stirring was started immediately to mix uniformly, and the reaction start time was recorded. After 10 h, the L-5-MTHF yield was 14.9 g/L, with a conversion rate of 99.4%.

[0070] Catalytic reaction with 20 g/L THF feeding. Cells were collected by centrifugation, resuspended in 0.4 mol/L HEPES buffer (pH 7.5) to an OD600 of 40, and subjected to high-pressure homogenization to obtain cell lysate. 30 mL was added to a round-bottom flask, pre-incubated at 37 C. in a water bath for 10 min, and then 20 g/L THF, 150 mmol/L MSDS, and 1.0 g/L DTT were added sequentially. Magnetic stirring was started immediately to mix uniformly, and the reaction start time was recorded. After 12 h, the L-5-MTHF yield was 17.1 g/L, with a conversion rate of 85%.

[0071] Catalytic reaction with 30 g/L THF feeding. Cells were collected by centrifugation, resuspended in 0.4 mol/L HEPES buffer (pH 7.5) to an OD600 of 40, and subjected to high-pressure homogenization to obtain cell lysate. 30 mL was added to a round-bottom flask, pre-incubated at 37 C. in a water bath for 10 min, and then 30 g/L THF, 300 mmol/L MSDS, and 1.0 g/L DTT were added sequentially. Magnetic stirring was started immediately to mix uniformly, and the reaction start time was recorded. The conversion rates at 8 h and 13 h were 55.2% and 63.3%, respectively.