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

Abstract

The invention discloses a construction method and application of recombinant Escherichia coli GDH-FR-MDH, and the method comprises the following steps: by taking a mannitol dehydrogenase gene and a glucose dehydrogenase gene as templates, connecting the two enzyme genes to an expression vector through a homologous recombination method by using a connecting peptide to construct a recombinant plasmid; the recombinant plasmid is transformed into competent Escherichia coli, and the recombinant Escherichia coli GDH-FR-MDH is obtained after the competent Escherichia coli is screened and verified. The recombinant Escherichia coli GDH-FR-MDH is used for synthesizing the mannitol. The mannitol dehydrogenase and the glucose dehydrogenase are subjected to fusion expression through the linker peptide, and the linker peptide is used as a biocatalyst for mannitol, so that the cost required by reaction is reduced.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a construction method and application of recombinant Escherichia coli GDH-FR-MDH. Background Art

[0002] Mannitol, also known as D-mannitol, is a natural hexavalent sugar alcohol. Natural mannitol usually exists in the roots, stems and leaves of plants. It is extracted from plants to become valuable refined alcohol. Mannitol is the earliest functional sugar alcohol to be used in human life. At the same time, mannitol is also a safe chemical additive used in food, medicine and chemical industries. Generally speaking, polyols are not carcinogenic and have a low glycemic index reaction. Therefore, mannitol is widely used to treat specific health problems that require calorie control, such as symptoms such as diabetes.

[0003] There are several main methods for the synthesis of mannitol: natural extraction, chemical synthesis and biotransformation. The natural extraction method is mainly extracted from plants, but the operation is complicated and requires high temperature and high pressure conditions during the extraction process, which greatly increases the cost; the chemical synthesis method synthesizes mannitol by catalytic hydrogenation of fructose and glucose. The reaction needs to be carried out under high temperature and strong acid conditions and requires the addition of metal catalysts in the reaction; and the biotransformation method is greener and more economical than these two methods. The biotransformation method mainly includes microbial fermentation, enzymatic conversion and whole cell conversion.

[0004] The most studied method at present is the enzymatic conversion method, which heterologously expresses the mannitol dehydrogenase gene in Escherichia coli. However, in the enzyme-catalyzed reaction, a large amount of coenzymes need to be added exogenously to achieve a certain conversion rate, so glucose dehydrogenase or formate dehydrogenase is often expressed to realize the coenzyme cycle, such as co-expressing the mannitol dehydrogenase gene and the glucose dehydrogenase gene or the formate dehydrogenase gene in the co-expression vector, and constructing a fusion enzyme to achieve coenzyme regeneration. However, expressing two enzyme genes through a co-expression vector has no effect on improving the enzyme stability, so the method of constructing a fusion enzyme to synthesize mannitol is more suitable for industrial production. Summary of the invention

[0005] The purpose of the present invention is to provide a construction method and application of recombinant Escherichia coli GDH-FR-MDH, which uses a connecting peptide to fuse mannitol dehydrogenase (MDH) and glucose dehydrogenase (GDH) for expression, and is used as a biocatalyst for synthesizing mannitol, thereby reducing the cost required for the reaction, solving the problem of coenzyme consumption in the reaction to a certain extent, and establishing a biosynthesis route with mild reaction conditions and green environmental protection.

[0006] In one aspect of the present invention, the present invention proposes a method for constructing a recombinant Escherichia coli GDH-FR-MDH. According to an embodiment of the present invention, the method comprises the following steps: using mannitol dehydrogenase gene and glucose dehydrogenase gene as templates, connecting the two enzyme genes to an expression vector by homologous recombination using a connecting peptide to construct a recombinant plasmid, transforming the recombinant plasmid into competent Escherichia coli, and obtaining the recombinant Escherichia coli GDH-FR-MDH after screening and verification.

[0007] In addition, the method for constructing a recombinant Escherichia coli GDH-FR-MDH according to the above embodiment 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 construction method of the recombinant Escherichia coli GDH-FR-MDH.

[0011] In addition, the recombinant Escherichia coli GDH-FR-MDH according to the above embodiment 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 the sequence listing SEQ ID NO.1.

[0013] In some embodiments of the present invention, the recombinant Escherichia coli GDH-FR-MDH was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on December 3, 2024, with a deposit number of CGMCC No.32888, and a classification name of Escherichia coli. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0014] In another aspect of the present invention, the present invention provides that the recombinant Escherichia coli GDH-FR-MDH is used to synthesize mannitol.

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

[0016] (1) inoculating the genetically engineered bacteria into a culture medium to obtain a bacterial liquid;

[0017] (2) aspirating the bacterial solution and adding it to the culture medium for cultivation, adding the inducer, culturing, and centrifuging to obtain the bacterial cells;

[0018] (3) washing the cells with a washing solution, disrupting them with low-temperature ultrasonication, centrifuging, and taking the supernatant to obtain a crude enzyme solution;

[0019] (4) Glucose and fructose are used as substrates, detergent and the prepared crude enzyme solution are added to obtain the mannitol.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention forms a fusion enzyme by connecting mannitol dehydrogenase and glucose dehydrogenase through a connecting peptide, which not only solves the problem of low yield and need for a large amount of exogenous NADH when only mannitol dehydrogenase is added, but also improves the conversion rate of the substrate to a certain extent compared with the free double enzyme due to the substrate channel formed between the fusion enzymes, and also improves the stability of the enzyme by adding the connecting peptide. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0023] Figure 2 is an agar gel electrophoresis diagram of the recombinant expression plasmid in Example 1 of the present invention;

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

[0025] Figure 4 is a mechanism diagram of 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 phase diagram of the reaction solution catalyzed by the standard product in Example 3 of the present invention and the recombinant Escherichia coli GDH-FR-MDH;

[0027] Figure 6 is a comparison chart of the optimum temperature and temperature stability of the recombinant Escherichia coli GDH-FR-MDH and the free enzyme in Example 4 of the present invention, wherein: Figure 6 -a is the optimum temperature diagram of free enzyme MDH and MDH in fusion enzyme, Figure 6 -b is the optimum temperature diagram of free enzyme GDH and GDH in fusion enzyme, Figure 6 -c is the temperature stability diagram of free enzyme MDH and fusion enzyme MDH, Figure 6-d is the temperature stability diagram of free GDH and fusion enzyme GDH. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

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

[0031] Example 1

[0032] A method for constructing recombinant Escherichia coli GDH-FR-MDH, using the mannitol dehydrogenase gene MDH and the glucose dehydrogenase GDH gene as templates, using GGGGSEAAAK as a connecting peptide to sequentially homologously recombine the two enzyme genes and transform them into BL21 (DE3) Escherichia coli to obtain recombinant Escherichia coli GDH-FR-MDH. The deposit number is CGMCC No. 32888, and it is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the postal code is 100101. The plasmid map of the recombinant Escherichia coli is as follows Figure 1 shown.

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

[0034] (1) Primer design

[0035] According to the GDH glucose dehydrogenase and vector pET-28a-(+) and mannitol dehydrogenase MDH sequences, the mutant primers were designed with the help of SnapGene software as follows:

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

[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) Recombinant plasmid construction

[0047] Based on the MDH mannitol dehydrogenase gene and vector pET-28a-(+) and glucose dehydrogenase GDH sequence as templates, the above primers were used to obtain the recombinant expression plasmid pET-28a-(+)-GDH-FR-MDH by PCR amplification technology and seamless cloning technology, and the original template was digested by DMT. The PCR electrophoresis diagram is shown in Figure 2 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), and after kanamycin resistance screening, enzyme digestion, bacterial liquid PCR and DNA sequencing verification, recombinant Escherichia coli GDH-FR-MDH suitable for directional synthesis of mannitol was obtained.

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

[0051] ①Take 100 μL of Escherichia coli BL21 (DE3) competent cell suspension (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) from the -80℃ refrigerator 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 minutes;

[0053] ③ After ice bath, heat shock the competent cells in a 42℃ water bath for 45 seconds and then continue ice bathing for 2 minutes;

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

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

[0056] The amino acid sequence of the 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 genetic engineering 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 hours; the seed solution was inoculated into liquid medium A at a volume concentration of 1%, cultured at 37°C and 220 rpm for 4 hours, IPTG (final concentration 0.25 mM) was added, induced at 15°C and 150 rpm for 20 hours, and then the fermentation liquid was centrifuged at 4°C (8000 rpm, 15 min) to collect the bacterial precipitate. 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 per liter.

[0105] (2) Add an appropriate amount of 100 mM, pH 8.0 tris-HCl buffer to the bacteria collected in step (1) to resuspend the bacteria. Ultrasonic disruption was performed for 25 min, and the disrupted liquid was centrifuged for 15 min (8000 rpm) to collect the supernatant, which was the crude enzyme solution of the fusion enzyme GDH-FR-MDH, with an enzyme activity of 4 U / ml.

[0106] (3) The crude enzyme solution obtained in step (2) was filtered through a 0.45 μm filter membrane to remove impurities and then added to a NI-IDA column. The column was placed in a 15°C shaker overnight to allow the His tag in the enzyme gene to specifically bind to the nickel column filler. The protein was eluted using an elution solution of different concentrations of imidazole. The effluent was analyzed by SDS-PAGE and compared with the Protein Marker. The effluent of the appropriate size was collected and ultrafiltered to remove the imidazole in the effluent to obtain the pure protein.

[0107] (4) The same method was used to obtain pure GDH and MDH single enzymes. 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 3 shown.

[0108] Experimental Example 3

[0109] Comparison of catalytic activity between fusion enzyme and free dual enzyme:

[0110] (1) A mixed solution of fructose and glucose with a final concentration of 100 g / L was prepared using a tris-HCl buffer solution at pH 8, and 20 mM NADH and the fusion enzyme or free dual enzyme prepared in Example 2 were added to make the enzyme activity of the enzyme catalyzing the production of mannitol in the reaction system to be 20 U. The reaction was catalytically synthesized in a shaking table at 30° C. and 220 rpm (the catalytic synthesis process is shown in FIG. 2 ). Figure 4 ).

[0111] (2) The sample after the reaction in step (1) was boiled in boiling water for 5 min to inactivate the protein to terminate the reaction, the precipitate was removed by centrifugation, and the supernatant was filtered using a 0.22 μm filter membrane. The filtered supernatant was detected by high performance liquid chromatography (detection conditions: the chromatographic column was an amino column (4.6 mm×250 mm, 5 μm); the mobile phase was acetonitrile: water = 80:20; the flow rate was 1 mL / min, and the column temperature was 40°C). The liquid chromatographic peak diagram of the reaction solution is as shown below. Figure 5 The standard product was also tested by high performance liquid chromatography. The liquid chromatogram of the mannitol standard product is shown in Figure 5 The winning bid is shown.

[0112] The conversion rate of the fusion enzyme G-FR-M in the present invention is higher than that of the unfused double enzyme. After fusion, the conversion rate can reach 79.88%, while the conversion rate of the unfused free double enzyme is 67.9%. The double enzymes are connected by a connecting peptide to form a fusion protein, and the substrate channel is formed between the enzymes, which is beneficial to the conversion of the enzyme to the substrate. That is, the conversion rate of the fusion enzyme in the present invention is improved.

[0113] Example 4

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

[0115] 1. MDH optimum temperature and temperature stability

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

[0117] (2) Incubate the fusion enzyme FR and free MDH at 40°C, take samples at regular intervals to measure the enzyme activity, and compare with the unincubated enzyme to calculate the remaining enzyme activity (the unincubated enzyme activity is 100%). The optimal temperature of the fusion enzyme and free MDH is shown in Figure 6 -a, Temperature stability of fusion enzyme and free MDH see Figure 6 -c.

[0118] 2. GDH optimum temperature and temperature stability

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

[0120] (2) Incubate the fusion enzyme FR and free GDH at 40°C, take samples at regular intervals and calculate their remaining enzyme activity (the enzyme activity without incubation is 100%). The optimum temperature of the fusion enzyme and free GDH is shown in Figure 6 -b, Temperature stability of fusion enzyme and free GDH see Figure 6 -d.

[0121] Figure 6 The results show that the optimum temperature of free MDH is 30°C, and the optimum temperature of MDH in the fusion enzyme G-FR-M is 5°C higher than that of the free enzyme. The temperature stability of MDH at 40°C is relatively good, whether it is free or fused, but the free MDH only maintains more than 40% of the enzyme activity after incubation at 40°C for 10 hours, while the MDH in the fusion enzyme still has 65% of the enzyme activity after incubation at 40°C for 10 hours. For free GDH and GDH in the fusion enzyme, although their optimum temperatures are both 35°C, and only about 20% of the enzyme activity is left after incubation at 40°C for 1 hour, the fusion enzyme is more stable when the temperature rises compared with the free enzyme. Because a rigid connecting peptide is added, the two protein structures can maintain a relatively fixed distance, which improves the stability of the conformation to a certain extent, that is, in the present invention, the temperature stability of the two free enzymes is improved after they are connected by a connecting peptide.

[0122] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they shall all fall within the protection scope of the present invention.

Claims

1. A method for constructing a recombinant Escherichia coli GDH-FR-MDH, characterized in that: The following steps are involved: With mannitol dehydrogenase gene and glucose dehydrogenase gene as templates, the two enzyme genes are connected to an expression vector by homologous recombination using a connecting 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.

2. The method for constructing a recombinant Escherichia coli GDH-FR-MDH according to claim 1, characterized in that: The expression vector is pET-28a-(+).

3. The method for constructing a recombinant Escherichia coli GDH-FR-MDH according to claim 1, characterized in that: The competent Escherichia coli is competent Escherichia coli BL21 (DE3). 4 . A recombinant Escherichia coli GDH-FR-MDH constructed according to the method for constructing a recombinant Escherichia coli GDH-FR-MDH according to any one of claims 1 to 3 .

5. The recombinant Escherichia coli GDH-FR-MDH according to claim 4, characterized in that: The nucleotide sequence of the recombinant Escherichia coli GDH-FR-MDH is shown in the sequence table SEQ ID NO.

1.

6. The recombinant Escherichia coli GDH-FR-MDH according to claim 4, characterized in that: The recombinant Escherichia coli GDH-FR-MDH was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on December 3, 2024, with a deposit number of CGMCC No.32888 and a classification name of Escherichia coli.

7. The recombinant Escherichia coli GDH-FR-MDH of claim 4 is used to synthesize mannitol.

8. A method for producing mannitol using an enzymatic method, characterized in that: The following steps are involved: (1) inoculating the genetically engineered bacteria according to claim 4 into a culture medium to obtain a bacterial liquid; (2) aspirating the bacterial solution and adding it to the culture medium for cultivation, adding the inducer, culturing, and centrifuging to obtain the bacterial cells; (3) washing the cells with a washing solution, disrupting them with low-temperature ultrasonication, centrifuging, and taking the supernatant to obtain a crude enzyme solution; (4) Glucose and fructose are used as substrates, detergent and the prepared crude enzyme solution are added to obtain the mannitol.

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