Construction method and application of recombinant escherichia coli GDH-FR-MDH

Abstract

The application discloses a construction method and application of recombinant escherichia coli GDH-FR-MDH, and the method comprises the following steps: taking mannitol dehydrogenase gene and glucose dehydrogenase gene as templates, using a connecting peptide to connect the two enzyme genes to an expression vector by a homologous recombination method to construct a recombinant plasmid, transforming the recombinant plasmid into competent escherichia coli, and after screening and verification, obtaining the recombinant escherichia coli GDH-FR-MDH. The recombinant escherichia coli GDH-FR-MDH is used for synthesizing mannitol. The mannitol dehydrogenase and the glucose dehydrogenase are fused and expressed by the connecting peptide, and are used as a biological catalyst for mannitol, so that the cost required by the reaction is reduced.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for constructing and applying recombinant Escherichia coli GDH-FR-MDH. Background Technology

[0002] Mannitol, also known as D-mannitol, is a natural hexa-ol. It is typically found in the roots, stems, and leaves of plants. Extracted from these plants, it becomes a valuable refined alcohol. Mannitol was the first of all functional sugar alcohols to be used by humans. It is also a safe chemical additive used in the food, pharmaceutical, and chemical industries. Generally, polyols are not carcinogenic and have a low glycemic index. Therefore, mannitol is widely used to treat specific health problems requiring calorie control, such as diabetes.

[0003] Mannitol can be synthesized through several methods: natural extraction, chemical synthesis, and biotransformation. Natural extraction primarily involves extracting mannitol from plants, but this method is complex and requires high temperature and pressure, significantly increasing costs. Chemical synthesis involves the catalytic hydrogenation of fructose and glucose, a reaction that requires high temperature and strong acid conditions, and necessitates the addition of a metal catalyst. Biotransformation, on the other hand, is more environmentally friendly and economical than these two methods. Biotransformation methods mainly include microbial fermentation, enzymatic conversion, and whole-cell conversion.

[0004] Currently, the most researched method is enzymatic conversion, which involves heterologous expression of the mannitol dehydrogenase gene in *Escherichia coli*. However, a large amount of exogenous coenzyme is required to achieve a certain conversion rate in the enzymatic catalytic reaction. Therefore, glucose dehydrogenase or formate dehydrogenase is often expressed to achieve coenzyme cycling. This can be achieved by co-expressing the mannitol dehydrogenase gene and glucose dehydrogenase gene or formate dehydrogenase gene in a co-expression vector, or by constructing fusion enzymes to achieve coenzyme regeneration. However, expressing two enzyme genes in a co-expression vector does not improve enzyme stability. Therefore, constructing fusion enzymes is more suitable for industrial production when synthesizing mannitol. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing and applying recombinant Escherichia coli GDH-FR-MDH, which uses a linker peptide to fuse mannitol dehydrogenase (MDH) with glucose dehydrogenase (GDH) for expression, and uses it as a biocatalyst for the synthesis of mannitol. This reduces the cost of the reaction, solves the problem of coenzyme consumption in the reaction to a certain extent, and establishes a biosynthetic route with mild reaction conditions and environmental protection.

[0006] In one aspect of the present invention, a method for constructing recombinant Escherichia coli GDH-FR-MDH is provided. According to an embodiment of the present invention, the method includes the following steps: using mannitol dehydrogenase gene and glucose dehydrogenase gene as templates, the two enzyme genes are ligated to an expression vector via homologous recombination using a linker peptide to construct a recombinant plasmid; the recombinant plasmid is transformed into competent Escherichia coli; and after screening and verification, the recombinant Escherichia coli GDH-FR-MDH is obtained.

[0007] In addition, the method for constructing recombinant Escherichia coli GDH-FR-MDH according to the above embodiments of the present invention may also have the following additional technical features:

[0008] In some embodiments of the present invention, the expression vector is pET-28a-(+).

[0009] In some embodiments of the present invention, the competent Escherichia coli is competent Escherichia coli BL21(DE3).

[0010] In another aspect of the present invention, the present invention provides a recombinant Escherichia coli GDH-FR-MDH constructed according to the method for constructing the recombinant Escherichia coli GDH-FR-MDH described above.

[0011] In addition, the recombinant Escherichia coli GDH-FR-MDH according to the above embodiments of the present invention may also have the following additional technical features:

[0012] In some embodiments of the present invention, the nucleotide sequence of the recombinant Escherichia coli GDH-FR-MDH is shown in SEQ ID NO.1.

[0013] In some embodiments of the present invention, the recombinant Escherichia coli GDH-FR-MDH was deposited on December 3, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32888, classified as Escherichia coli, and located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0014] In another aspect of the invention, the present invention proposes the use of the recombinant Escherichia coli GDH-FR-MDH for the synthesis of mannitol.

[0015] In another aspect, the present invention provides a method for producing mannitol using an enzymatic process. According to an embodiment of the present invention, the method includes the following steps:

[0016] (1) The genetically engineered bacteria are inoculated into a culture medium and cultured to obtain a bacterial solution;

[0017] (2) Take the bacterial solution and add it to the culture medium for culture. Add the inducing agent, culture, and centrifuge to obtain the bacterial cells;

[0018] (3) Wash the bacterial cells with washing solution, sonicate them at low temperature, centrifuge, and take the supernatant to obtain crude enzyme solution;

[0019] (4) Mannitol is obtained by adding detergent and the prepared crude enzyme solution to glucose and fructose as substrates.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] This invention solves the problem of low yield and the need for large amounts of exogenous NADH when only mannitol dehydrogenase is added by linking mannitol dehydrogenase and glucose dehydrogenase together to form a fusion enzyme. It also improves the substrate conversion rate to a certain extent compared with free dual enzymes due to the substrate channels formed between the fusion enzymes. At the same time, the addition of linking peptides also improves the stability of the enzyme. Attached Figure Description

[0022] Figure 1 This is a plasmid map of recombinant Escherichia coli GDH-FR-MDH in Example 1 of this invention;

[0023] Figure 2 This is an agarose gel electrophoresis image of the recombinant expression plasmid in Example 1 of this invention;

[0024] Figure 3 This is a protein electrophoresis diagram of recombinant Escherichia coli GDH-FR-MDH, free enzyme MDH, and purified GDH enzyme in Example 2 of this invention;

[0025] Figure 4 This is a schematic diagram of the mechanism for one-step production of mannitol using recombinant Escherichia coli GDH-FR-MDH in Example 3 of the present invention;

[0026] Figure 5 This is a liquid phase diagram of the reaction solution generated by the standard sample and recombinant Escherichia coli GDH-FR-MDH catalysis in Example 3 of this invention;

[0027] Figure 6 This is a comparison diagram of the optimal temperature and temperature stability of recombinant Escherichia coli GDH-FR-MDH and free enzyme in Example 4 of this invention. Figure 6 -a is a graph showing the optimal temperature for free MDH and the optimal temperature for MDH in the fusion enzyme. Figure 6 -b shows the optimal temperature for free GDH and GDH in the fusion enzyme. Figure 6 -c shows the temperature stability of free MDH and fusion MDH. Figure 6-d represents the temperature stability of free GDH and the fusion enzyme GDH. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] In the following examples or comparative examples: Escherichia coli DH5α and Escherichia coli BL21(DE3) were purchased from TransGen Biotech; the vector pET-28a-(+) was purchased from Novagen.

[0031] Example 1

[0032] A method for constructing recombinant Escherichia coli GDH-FR-MDH involves using the mannitol dehydrogenase gene (MDH) and the glucose dehydrogenase gene (GDH) as templates, and using GGGGSEAAAK as a linker peptide to sequentially perform homologous recombination of the two enzyme genes, followed by transformation into BL21(DE3) Escherichia coli to obtain recombinant Escherichia coli GDH-FR-MDH. The deposit number is CGMCC No. 32888, and it is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The plasmid map of the recombinant Escherichia coli is shown below. Figure 1 As shown.

[0033] The construction method specifically includes the following steps:

[0034] (1) Primer design

[0035] Based on the sequences of GDH glucose dehydrogenase, the vector pET-28a-(+), and mannitol dehydrogenase MDH, the following mutant primers were designed using SnapGene software:

[0036] pET-28a-(+)-GDH primers:

[0037] Upstream primer:

[0038] 5'-AGAGGCATAAGTCGACAAGCTTGCGGCCGCACTCGAGCA-3'

[0039] Downstream primer:

[0040] 5'-CTCCTCCTCCCCTTAGTAAATAGCCGCCATCAACAGGGATTG-3'

[0041] MDH primers:

[0042] Upstream primer:

[0043] 5'-TTTACTAAGGGGAGGAGGAGGATCCGAAGCAGCAGCAAAA-3'

[0044] Downstream primer:

[0045] 5'-GCTTGTCGACTTATGCCTCTTCGCCGCCAACCTTCACAA-3'

[0046] (2) Construction of recombinant plasmids

[0047] Using the MDH mannitol dehydrogenase gene, the vector pET-28a-(+), and the glucose dehydrogenase GDH sequence as templates, and employing the aforementioned primers, the recombinant expression plasmid pET-28a-(+)-GDH-FR-MDH was obtained through PCR amplification and seamless cloning techniques, followed by DMT digestion of the original template. The PCR electrophoresis image is shown below. Figure 2 As shown.

[0048] (3) Host bacterial transformation

[0049] The recombinant expression plasmid pET-28a-(+)-GDH-FR-MDH was transformed into Escherichia coli competent cells BL21(DE3). After kanamycin resistance screening, enzyme digestion, bacterial PCR and DNA sequencing verification, recombinant Escherichia coli GDH-FR-MDH suitable for mannitol synthesis was obtained.

[0050] Host bacterial transformation specifically includes the following steps:

[0051] ① Take 100 μL of Escherichia coli BL21(DE3) competent cell suspension (purchased from Beijing TransGen Biotech Co., Ltd.) from a -80℃ freezer and thaw it on ice;

[0052] ② Add the prepared recombinant expression plasmid solution, add about 5 μl of plasmid DNA to every 50 μl of competent cells, and place on ice for 30 min;

[0053] ③ After the ice bath, the competent cells were placed in a 42℃ water bath for 45 seconds for heat shock, and then continued to be ice bathed for 2 minutes.

[0054] ④ Add 500 μl of sterilized LB liquid medium (without antibiotics) to the tube, mix well, and incubate at 37°C for 1 hour;

[0055] ⑤ After shaking the above bacterial solution well, take 100 μL and spread it on a screening plate containing antibiotics. Incubate at 37°C for 16-18 hours. Select positive colonies and verify them by bacterial PCR to obtain recombinant Escherichia coli BL21(DE3) / GDH-FR-MDH genetically engineered bacteria.

[0056] The amino acid sequence of recombinant Escherichia coli GDH-FR-MDH is as follows:

[0057] MYTDLKGKVVAITGASSGLGKAMAIRFGQEQAKVVVNYYSNEKDAQTVKEEIQK

[0058] AGGEAVIVQGDVTKEEDVKNIVQTAVKEFGTLDVMINNAGMENPVQSHEMMPLKDWNK

[0059] VINTNLTGAFLGSREAIKYYVENDIQGNVINMSSVHEMIPWPLFVHYAASKGGIKLMTE

[0060] TLALEYAPKRIRVNNIGPGAINTPINAEKFADPVQKKDVESMIPMGYIGEPEEIAAVAVW

[0061] LASKESSYVTGITLFADGGMTKYPSFQAGRGGGGGSEAAAKMEALVLTGTKKLEVKDI

[0062] DRPKVLPNEVLIHTAFAGICGTDHALYAGLPGSADAVPPIVLGHENSGVVAEIGSAVTNV

[0063] KVGDRVTVDPNIYCGQCKYCRTARPELCENLSAVGVTRDGGFEEFFTAPASVVYPIPDN

[0064] VSLKSAAVVEPISCAVHGIQLLKVTPYQKALVIGDGFMGELFVQILQAYGIHQVDLAGIV

[0065] DEKLAMNKEKFGVKNTYNTMKGDKIPEGEYDVIIEAVGLPQTQEAAIEASARGAQVLM

[0066] FGVGGPDAKFQMNTYEVFQKQLTIQGSFINPNAFEDSLALLSSGKLNVEALMSHELDYK

[0067] TVDDFVNGKLGVVSKAVVKVGGEEA

[0068] The nucleotide sequence of recombinant Escherichia coli GDH-FR-MDH is as follows:

[0069] ATGTACACCGACCTGAAAGGTAAAGTTGTGGCGATCACTGGTGCTTCTTCTGGT

[0070] CTGGGCAAAGCTATGGCAATCCGTTTCGGCCAAGAACAGGCCAAAGTTGTGGTCAA

[0071] CTACTACTCCAACGAGAAAGACGCACAGACCGTTAAAGAAGAAATCCAGAAAGCA

[0072] GGTGGTGAAGCAGTGATCGTTCAGGGTGACGTTACTAAAGAAGAAGACGTTAAGAA

[0073] CATCGTTCAGACCGCTGTTAAAGAGTTCGGTACGCTGGATGTTATGATCAACAACGC

[0074] TGGTATGGAGAATCCGGTTCAGTCTCACGAAATGCCGCTGAAAGATTGGAACAAAG

[0075] TTATCAACACCAACCTGACTGGTGCGTTTCTGGGCAGCCGTGAAGCTATCAAATACT

[0076] ACGTAGAGAACGACATCCAGGGTAACGTTATCAACATGTCCAGCGTGCACGAAATG

[0077] ATTCCGTGGCCGTTGTTCGTTCACTATGCTGCGTCTAAAGGTGGCATCAAACTGATG

[0078] ACCGAAACTCTGGCGCTGGAATACGCACCAAAGCGTATCCGTGTTAACAACATCGG

[0079] TCCGGGCGCAATCAACACTCCAATCAACGCGGAGAAATTCGCTGATCCGGTGCAGA

[0080] AAAAAGACGTTGAATCTATGATTCCGATGGGTTACATCGGTGAACCGGAAGAAATC

[0081] GCAGCAGTAGCTGTTTGGCTGGCAAGCAAAGAATCTTCTTACGTTACCGGTATCACC

[0082] TTGTTCGCTGACGGCGGTATGACCAAATATCCGTCTTTCCAGGCTGGTCGTGGTGGA

[0083] GGAGGAGGATCCGAAGCAGCAGCAAAAATGGAAGCACTTGTTCTAACCGGAACTAA

[0084] GAAATTAGAAGTAAAGGACATTGACCGTCCAAAGGTTTTGCCAAATGAAGTTTTGA

[0085] TTCACACAGCGTTTGCTGGTATTTGTGGAACTGATCACGCACTATACGCTGGTTTGC

[0086] CAGGTTCAGCTGATGCTGTTCCTCCAATCGTTCTTGGTCATGAGAACTCTGGTGTTGT

[0087] TGCAGAAATCGGTTCTGCTGTTACTAACGTTAAAGTTGGTGACCGCGTAACTGTTGA

[0088] CCCTAACATTTATTGTGGACAATGCAAGTACTGCCGTACTGCACGCCCTGAATTATG

[0089] CGAAAACTTGTCAGCTGTTGGTGTGACACGTGATGGTGGCTTTGAAGAATTCTTCAC

[0090] TGCACCTGCATCTGTTGTTTATCCAATTCCTGATAACGTTTCATTAAAGTCAGCTGCT

[0091] GTTGTTGAACCAATTTCATGTGCCGTTCACGGTATTCAATTGTTGAAGGTTACCCCTT

[0092] ACCAAAAGGCTTTGGTAATTGGTGATGGATTCATGGGCGAATTATTCGTTCAAATCT

[0093] TGCAAGCATATGGTATTCACCAAGTTGACTTAGCTGGTATTGTTGACGAAAAGTTGG

[0094] CAATGAACAAAGAAAAGTTTGGCGTAAAGAACACGTACAACACAATGAAGGGTGA

[0095] CAAGATCCCTGAAGGTGAATATGATGTCATTATTGAAGCCGTTGGTCTACCACAAAC

[0096] ACAAGAAGCTGCTATTGAAGCATCAGCTCGTGGCGCTCAAGTATTGATGTTTGGTGT

[0097] TGGTGGTCCTGACGCCAAGTTCCAAATGAACACTTATGAAGTATTCCAAAAGCAGTT

[0098] AACTATTCAAGGTTCATTTATCAACCCTAACGCCTTTGAAGATTCACTGGCTCTGCTA

[0099] TCATCTGGTAAGTTGAATGTTGAAGCATTGATGTCACACGAATTAGACTATAAGACT

[0100] GTTGACGACTTTGTTAACGGTAAGTTAGGTGTCGTATCTAAGGCTGTTGTGAAGGTT

[0101] GGCGGCGAAGAGGCA

[0102] Experimental Example 2

[0103] Induction, expression and purification of recombinant Escherichia coli BL21(DE3) / GDH-FR-MDH genetically engineered bacteria:

[0104] (1) The recombinant Escherichia coli GDH-FR-MDH obtained in Example 1 was inoculated into 50 ml of LB liquid medium containing 100 μg / mL kanamycin and cultured overnight at 37°C in a shaker for 16 h. The above seed culture was inoculated into liquid medium A at a volume concentration of 1% and cultured at 37°C and 220 rpm for 4 h. IPTG (final concentration 0.25 mM) was added, and the culture was induced at 15°C and 150 rpm for 20 h. The fermentation broth was then centrifuged at 4°C (8000 rpm, 15 min), and the bacterial precipitate was collected. Each liter of the above-mentioned medium A contained 5 g glycerol, 5 g glucose, 10 g peptone, 10 g potassium nitrate, 17.105 g Na2HPO4·12H2O, 3 g KH2PO4, 1 g NH4Cl, and 0.1 mmol / L MgSO4·7H2O.

[0105] (2) Add an appropriate amount of 100mM, pH 8.0 tris-HCl buffer to the bacterial cells collected in step (1) to resuspend the bacterial cells. Sonicate for 25 min, centrifuge the lysate for 15 min (8000 rpm), and collect the supernatant, which is the crude enzyme solution of the fusion enzyme GDH-FR-MDH, with an enzyme activity of 4 U / ml.

[0106] (3) After filtering the crude enzyme solution obtained in step (2) through a 0.45 μm filter membrane to remove impurities, add it to an NI-IDA column and incubate overnight at 15°C on a shaker to allow the His tag in the enzyme gene to specifically bind to the nickel column packing. Elute the protein using imidazole elution buffers of different concentrations. Perform SDS-PAGE analysis on the eluent, compare it with the Protein Marker, collect the eluents of the correct size, and ultrafilter to remove the imidazole from the eluent to obtain the purified protein.

[0107] (4) GDH and MDH single-enzyme purified enzymes were obtained using the same method. The purity and molecular weight of the fusion enzyme and single enzyme were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results are shown in [Figure 1]. Figure 3 As shown.

[0108] Experimental Example 3

[0109] Comparison of catalytic activities between fusion enzymes and free dual enzymes:

[0110] (1) A mixed solution of fructose and glucose with a final concentration of 100 g / L and 150 g / L was prepared using a tris-HCl buffer solution with pH = 8. 20 mM NADH and the fusion enzyme or free dual enzyme prepared in Example 2 were added to make the enzyme activity for catalyzing the production of mannitol in the reaction system 20 U. The reaction was carried out in a shaker at 30 °C and 220 rpm to catalyze the synthesis of mannitol (see [link to catalytic synthesis process]). Figure 4 ).

[0111] (2) After the reaction in step (1), boil the sample in boiling water for 5 minutes to inactivate the protein and terminate the reaction. Centrifuge to remove the precipitate, and filter the supernatant using a 0.22 μm filter membrane. Detect the filtered supernatant by high performance liquid chromatography (HPLC) (detection conditions: amino column (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile:water = 80:20; flow rate: 1 mL / min; column temperature: 40 °C). The HPLC peak chromatogram of the reaction solution is shown below. Figure 5 The reaction solution with the medium label is shown; the standard was also analyzed by high performance liquid chromatography, and the liquid chromatogram of the mannitol standard is shown below. Figure 5 The winning bid is shown.

[0112] The fusion enzyme G-FR-M in this invention exhibits a higher conversion rate than the unfused dual enzymes, achieving a conversion rate of 79.88% after fusion, compared to 67.9% for the unfused free dual enzymes. The fusion protein is formed by linking the two enzymes with a linker peptide, creating a substrate channel between the enzymes that facilitates substrate conversion. Therefore, the fusion enzyme in this invention demonstrates improved conversion efficiency.

[0113] Example 4

[0114] Comparison of optimal temperature and temperature stability between fusion enzymes and free enzymes:

[0115] 1. Optimal temperature and temperature stability of MDH

[0116] (1) In a 1 ml reaction system, add fructose with a final concentration of 10 mM, NADH with 1 mM, the fusion enzyme FR prepared in Example 2, 200 μL of free MDH, and tris-HCl buffer at pH 8.0. Place the mixture in a water bath at 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C for the reaction. After the reaction, record the absorbance at 340 nm. Calculate the amount of NADH consumed based on the decrease in absorbance to determine the enzyme activity (the highest enzyme activity is 100%).

[0117] (2) The fusion enzyme FR and free MDH were incubated at 40℃, and samples were taken at regular intervals to measure enzyme activity. The remaining enzyme activity was calculated by comparing the results with that of the unincubated enzyme (unincubated enzyme activity was considered as 100%). The optimal temperatures for the fusion enzyme and free MDH are shown in [reference needed]. Figure 6 -a, temperature stability of fusion enzyme and free MDH can be found in [reference needed]. Figure 6 -c.

[0118] 2. Optimal temperature and temperature stability of GDH

[0119] (1) In a 1 ml reaction system, add glucose with a final concentration of 10 mM, NAD+ with a final concentration of 20 mM, the fusion enzyme FR prepared in Example 2, 200 μL of free GDH, and tris-HCl buffer at pH 8.0. Place the mixture in a water bath at 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C for the reaction. After the reaction is completed, record the absorbance value at 340 nm. Calculate the amount of NADH consumed based on the increase in absorbance value to determine the enzyme activity (the highest enzyme activity is 100%).

[0120] (2) The fusion enzyme FR and free GDH were incubated at 40℃, and samples were taken at regular intervals to calculate the remaining enzyme activity (unincubated enzyme activity was counted as 100%). The optimal temperatures for the fusion enzyme and free GDH are shown in [reference]. Figure 6 -b, Temperature stability of fusion enzyme and free GDH can be found in [reference needed]. Figure 6 -d.

[0121] Figure 6 The results showed that the optimal temperature for free MDH was 30℃. The optimal temperature for MDH in the fusion enzyme G-FR-M was 5℃ higher than that of the free enzyme. Both free and fused MDH showed good temperature stability at 40℃. However, after 10 hours of incubation at 40℃, free MDH retained only slightly more than 40% of its activity, while the fusion enzyme retained 65% of its activity. For both free GDH and the fusion enzyme, although their optimal temperature was 35℃ and both retained only about 20% of their activity after 1 hour of incubation at 40℃, the fusion enzyme was more stable at higher temperatures compared to the free enzyme. The addition of a rigid linker peptide maintained a relatively fixed distance between the two protein structures, improving conformational stability to some extent. Therefore, the temperature stability of the two free enzymes was improved after being linked by the linker peptide in this invention.

[0122] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the present invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A recombinant Escherichia coli GDH-FR-MDH, characterized in that: The recombinant Escherichia coli GDH-FR-MDH was deposited on December 3, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32888, and classified as Escherichia coli. Escherichia coli .

2. The application of the recombinant Escherichia coli GDH-FR-MDH according to claim 1, characterized in that: The recombinant Escherichia coli GDH-FR-MDH was used to synthesize mannitol.

3. A method for producing mannitol using an enzymatic process, characterized in that, Includes the following steps: (1) The recombinant Escherichia coli GDH-FR-MDH of claim 1 is inoculated into a culture medium and cultured to obtain a bacterial solution; (2) Add the bacterial solution to the culture medium for culture, add an inducer, culture, and centrifuge to obtain bacterial cells; (3) Wash the bacterial cells with washing solution, sonicate them at low temperature, centrifuge, take the supernatant, and obtain crude enzyme solution; (4) Using glucose and fructose as substrates, the prepared crude enzyme solution is added to obtain the mannitol.

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