Formic acid dehydrogenase mutant and application thereof

Abstract

The application belongs to the technical field of bioengineering, and relates to a formic acid dehydrogenase mutant and application thereof Cl The amino acid sequence of the FDH is shown in SEQ ID NO. 1, and the amino acid mutation is at least one of positions 52, 87, 111, 124, 177 and 243. As The double-enzyme coupling is performed on the strain containing the mannitol dehydrogenase of the MDH plasmid, and thus the efficiency of producing D-mannitol from D-fructose is significantly improved. The double-enzyme system of the recombinant strain containing the L124F mutant has a catalytic conversion rate of 92.3% within 10 hours, which is obviously better than the conversion rate before the improvement, and provides potential for the industrial development of mannitol.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and specifically relates to a formate dehydrogenase mutant and its applications. Background Technology

[0002] To address the cost bottleneck of coenzyme-dependent reactions, constructing efficient coenzyme regeneration systems has become a key technological direction in the field of biocatalysis. Formate dehydrogenase (FDH), a type of oxidoreductase capable of reversibly catalyzing the dehydrogenation of formate to carbon dioxide, can simultaneously oxidize the substrate and reduce the oxidized NAD+ of the coenzyme nicotinamide adenine dinucleotide (NADH) to its reduced state, thus achieving coenzyme recycling. This enzyme-mediated coenzyme regeneration system boasts advantages such as abundant and inexpensive substrate (formate), mild reaction conditions, and easy separation of byproducts (carbon dioxide), and has been widely applied in cofactor cycling systems in fields such as chiral chemical synthesis and energy conversion.

[0003] D-Mannitol, a natural hexaol, exhibits irreplaceable application value in food, pharmaceuticals, and chemical industries due to its low calorie content, high stability, and good physiological compatibility. The mainstream biocatalytic route for the biological synthesis of D-mannitol involves a reduction reaction of D-fructose as a substrate, catalyzed by mannitol dehydrogenase (MDH). This reaction strictly relies on the participation of the reduced coenzyme NADH. However, the reduced coenzyme NADH suffers from high prices (its market value is far higher than its oxidized form), poor chemical stability, and the inability to be reused, directly resulting in high production costs for enzymatic synthesis processes and limiting its large-scale application.

[0004] In the enzymatic synthesis of D-mannitol, constructing a dual-enzyme cascade system with FDH and MDH allows for the continuous supply of NADH through FDH-catalyzed formate dehydrogenation, supporting the efficient catalytic fructose reduction reaction by MDH. This forms a closed-loop reaction system of "substrate conversion-coenzyme regeneration," providing a feasible solution for reducing production costs and improving process economics. Although FDH has significant advantages in coenzyme regeneration, natural FDH and existing mutants generally suffer from low catalytic efficiency and weak coenzyme binding capacity, resulting in mannitol synthesis efficiency and substrate conversion rates in the cascade reaction system they support that are difficult to meet industrial production requirements. For example, the conversion number of some wild-type FDHs is only 6.3 s⁻¹, which cannot provide sufficient reducing power for the MDH-catalyzed reaction. At the same time, the catalytic rates of FDH and MDH are mismatched, easily leading to the problem of "insufficient reducing power supply" where coenzyme regeneration lags behind substrate conversion, directly restricting the overall reaction efficiency. Therefore, optimizing the enzymatic properties of FDH and improving its coenzyme regeneration efficiency through molecular modification technology has become the core breakthrough in overcoming the bottleneck of D-mannitol enzymatic synthesis technology. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a formate dehydrogenase mutant and its application, which overcomes the key shortcomings of current FDH enzyme molecules (including wild type and existing mutants) in the two-enzyme cascade system of fructose to mannitol, such as low catalytic efficiency, poor stability and poor synergy with MDH, and solves the industrial bottleneck problems caused by this, such as slow mannitol synthesis rate, low fructose conversion rate, high process production cost and insufficient stability.

[0006] The present invention is achieved by providing a formate dehydrogenase mutant, wherein the formate dehydrogenase mutant has an amino acid mutation in the amino acid sequence shown in SEQ ID NO.1, and the amino acid mutation site is at least one of the following positions: position 52, position 87, position 111, position 124, position 177, and position 243.

[0007] Furthermore, the nucleotide sequence of the formate dehydrogenase mutant is shown in SEQ ID NO.2.

[0008] Further, the formate dehydrogenase mutant is formed by mutating the amino acid sequence shown in SEQ ID NO.1 to one of the following: (1) valine at position 52 is mutated to isoleucine (V52I); (2) aspartic acid at position 87 is mutated to proline (D87P); (3) serine at position 111 is mutated to aspartic acid (S111D); (4) leucine at position 124 is mutated to phenylalanine (L124F); (5) valine at position 177 is mutated to isoleucine (V177I); (6) alanine at position 243 is mutated to tryptophan (A243W).

[0009] This invention is implemented in such a way that it also provides a gene encoding a formate dehydrogenase mutant as described above.

[0010] This invention is implemented in such a way that a recombinant strain is provided, comprising the formate dehydrogenase mutant as described above, or comprising the gene as described above.

[0011] This invention is implemented in such a way that it also provides the use of the formate dehydrogenase mutant as described above, or the gene as described above, or the recombinant strain as described above in the preparation of D-mannitol.

[0012] This invention is implemented in such a way that it also provides the use of formate dehydrogenase mutants as described above, or genes as described above, or recombinant strains as described above as NADH (reduced nicotinamide adenine dinucleotide) or NADPH (reduced nicotinamide adenine dinucleotide phosphate) regeneration tool enzymes, coupled with oxidoreductases to produce chiral drug intermediates or non-natural amino acids.

[0013] This invention is implemented in such a way that it also provides the application of coupling the formate dehydrogenase mutant, the gene, or the recombinant strain described above with ribitol dehydrogenase (RDH) to produce allool (allicinol) using D-alolostose as a substrate.

[0014] This invention is implemented in such a way that it also provides the use of the formate dehydrogenase mutant as described above, or the gene as described above, or the recombinant strain as described above, as a kit for detecting formate, NAD⁺ (oxidized nicotinamide adenine dinucleotide), NADP⁺ (oxidized nicotinamide adenine dinucleotide phosphate), NADH, NADPH, or related metabolites.

[0015] This invention is implemented as follows, and also provides a method for preparing D-mannitol, comprising the following steps: mixing the recombinant strain as described above with a solution containing... As The mannitol dehydrogenase of the MDH plasmid was coupled with a strain for catalysis, and D-fructose was used as the substrate and 4-hydroxyethylpiperazine ethanesulfonic acid buffer was used as the reaction medium to form a reaction system to obtain D-mannitol by conversion reaction.

[0016] Compared with the prior art, the formate dehydrogenase mutant of the present invention and its application have a significant impact on the discovery of formate dehydrogenase. Cl FDH was subjected to amino acid mutations according to the amino acid sequence shown in SEQ ID NO.1, wherein the mutation sites are at least one of positions 52, 87, 111, 124, 177, and 243. Recombinant strains containing the mutant and those containing... AsThe MDH plasmid-derived mannitol dehydrogenase strain was coupled with a dual-enzyme system, which significantly improved the efficiency of D-mannitol production from D-fructose. The dual-enzyme system of the recombinant strain containing the L124F mutant achieved a catalytic conversion rate of 92.3% within 10 hours, significantly better than the conversion rate before modification, demonstrating potential for the industrialization of mannitol. Attached Figure Description

[0017] Figure 1 This invention relates to the wild-type formate dehydrogenase. Cl A schematic diagram of electrophoresis verification of the target protein elution of FDH and mutants; in the figure, 1 represents the flow buffer, 2 represents the lysate supernatant, 3 represents the 100mM imidazole elution buffer, and 4 represents the 500mM imidazole elution buffer.

[0018] Figure 2 This is a schematic diagram of the NADH standard curve for Embodiment 4 of the present invention;

[0019] Figure 3 This is a schematic diagram showing the growth of different mutant strains and their relative enzyme activities in Example 4 of the present invention;

[0020] Figure 4 This is a schematic diagram showing the enzyme activity stability of different mutant strains in Example 5 of the present invention;

[0021] Figure 5 This is a schematic diagram illustrating the catalytic effects of different mutant strains in Example 6 of the present invention;

[0022] Figure 6 This is a schematic diagram of the liquid chromatography of the fermentation broth of the mutant strain L124F in Example 6 of the present invention after catalysis. Detailed Implementation

[0023] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0024] Unless otherwise specified, the experimental methods used in the examples are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0025] The culture medium formulations used in the following examples are as follows:

[0026] (1) LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, pH adjusted to 7.0, autoclaved at 121℃ for 20 min, and cooled for later use.

[0027] (2) LB solid medium: The solid medium is based on the LB medium formula with the addition of 1.5% agarose. After sterilization, it is poured into plates (LB plates) for later use.

[0028] (3) TB medium: yeast powder 24g / L, peptone 12g / L, glycerol 5g / L, K2HPO4 9.4g / L, KH2PO4 2.2g / L, autoclaved at 121℃ for 20min, and cooled for later use.

[0029] The preparation methods of the reagents used in the following examples are as follows:

[0030] (1) Ampicillin stock solution (100mg / mL): Add 10g of ampicillin to 100mL of ultrapure water, filter sterilize with a 0.22μm filter membrane, dispense into aliquots, store at -20℃, and use at a concentration of 0.1%.

[0031] (2) Isopropyl β-D-thiogalactopyranoside (IPTG) stock solution: Dissolve 1.2g of IPTG in 50mL of sterile water, filter sterilize using a 0.22μm filter membrane, dispense into aliquots, and store at -20℃.

[0032] (3) Phosphate Buffered Solution (PBS): Purchase PBS Buffer premixed powder (1X) from Sangon Biotech (Shanghai) Co., Ltd. and prepare it according to the instructions.

[0033] (4) Lysis buffer: Add 1‰ Triton X-100 (about 50µL), 5mM 2-mercaptoethanol, 200~500µL PMSF and 25mM imidazole to 50mL phosphate buffer.

[0034] (5) Protein gradient elution buffer: Prepare imidazole concentrations of 50, 100, 200 and 500 mM using phosphate buffer and adjust the pH to 8.0.

[0035] (6) Protein replacement solution: Phosphate buffer with 5 mM DTT and 30% glycerol.

[0036] The strains and plasmids used in this invention are as follows:

[0037] The strain used to construct the expression strain is Escherichia coli TOP10 and E. coli Escherichia coli BL21(DE3) and the plasmids carrying the target gene were all pETDuet-1 series plasmids, all purchased from Beijing Qingke Biotechnology Co., Ltd.

[0038] Example 1, Wild Type Ecoli. BL21(DE3)- Cl FDH Construction

[0039] According to data mined from the NCBI database Clostridium Formate dehydrogenase sp., the amino acid sequence of its protein is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2. It was synthesized by Beijing Qingke Biotechnology Co., Ltd., and its length is 1023 bp. Formate dehydrogenase... Cl The FDH gene was inserted under the T7 promoter of pETDuet-1 to obtain the expression plasmid pETDuet- Cl FDH. The expression plasmid was transformed into *E. coli*. E. coli In BL21(DE3), the sample was spread on LB agar plates containing 50 μg / mL ampicillin and incubated at 37°C for 8–12 h. Positive clones were then picked, which were identified as wild-type. E. coli .BL21(DE3)- Cl FDH.

[0040] Example 2: Construction of site-directed mutants

[0041] 2.1. Primer design for site-directed mutagenesis

[0042] Formate dehydrogenase Cl Plasmids were extracted from the original strain of MDH to obtain... Cl The plasmid of the MDH gene (pETDuet-) Cl MDH-WT). Clostridium Formate dehydrogenase from sp. Cl The FDH gene sequence was analyzed using software to predict mutants.

[0043] Formate dehydrogenase Cl The FDH gene mutant is formed by site-directed mutation of the amino acid sequence shown in SEQ ID NO.1 as follows: (1) valine at position 52 is mutated to isoleucine (V52I); (2) aspartic acid at position 87 is mutated to proline (D87P); (3) serine at position 111 is mutated to aspartic acid (S111D); (4) leucine at position 124 is mutated to phenylalanine (L124F); (5) valine at position 177 is mutated to isoleucine (V177I); (6) alanine at position 243 is mutated to tryptophan (A243W).

[0044] The primer designs for site-directed mutagenesis are shown in Table 1.

[0045] Table 1 Primer Design Table

[0046]

[0047] 2.2. Site-directed mutagenesis PCR

[0048] The PCR reaction procedure using the site-directed mutagenesis two-step method is as follows:

[0049] Table 2 Reaction system 1 (10 μL)

[0050]

[0051] Table 3 Reaction system 2 (10 μL)

[0052]

[0053] Table 4. Two-step first-round PCR reaction procedure

[0054]

[0055] The symbol * in the table indicates that the denaturation, annealing, and extension steps are performed sequentially in the same stage, which constitutes one cycle.

[0056] In site-directed mutagenesis reaction system 1, 5 μL of high-fidelity DNA polymerase (2×Phanta Max Master Mix) purchased from Nanjing Novizan Biotechnology Co., Ltd. and 1 μL of pETDuet- were added. Cl MDH-WT was used as the template in reaction system 1, and 2 μL of each of V52I-F / D87P-F / S111D-F / L124F-F / V177I-F / A243W-F were added as upstream primers. ddH2O was added to bring the total volume of reaction system 1 to 10 μL. In reaction system 2, the amounts of high-fidelity polymerase, template, and ddH2O were the same as in reaction system 1, and 2 μL of each of V52I-R / D87P-R / S111D-R / L124F-R / V177I-R / A243W-R were added as downstream primers. Both reaction systems were first subjected to the first round of PCR reaction program shown in Table 4. After the reaction, the PCR products in both reaction systems were mixed and shaken until no air bubbles remained. The second round of PCR reaction program shown in Table 5 was then continued to obtain the final product. Cl Amplification products of MDH-V52I / D87P / S111D / L124F / V177I / A243W.

[0057] Table 5. Two-step second-round PCR reaction procedure

[0058]

[0059] The symbol * in the table indicates that the denaturation, annealing, and extension steps are performed sequentially in the same stage, which constitutes one cycle.

[0060] Take 7.5 μL of amplification product from each sample and add 1.5 μL of [unspecified ingredient]. Dpn Mix 1 μL of the fast digester and 1 μL of CutSmart fast digester buffer, and incubate at 37°C for 30 min. The template in the digestion system will be used for subsequent transformations. Take 10 μL of the template-digested PCR product and inject it into the digester. E. coli Top 10 competent cells. Resuscitate and culture at 37°C on a shaker for 1 hour, then plate onto LB agar plates containing ampicillin and culture overnight for 8-10 hours. Pick single clones and inoculate into 10 mL of LB medium for another 8-10 hours, followed by sequencing to determine if the picked single clones have undergone mutation. Analyze the sequencing results for correctly mutated mutants, and transfer 2.5 μL of each mutant into... E. coli BL21(DE3) competent cells. After culturing on plates for 8-10 hours, single clones were selected for preservation and stored at -80°C for later use.

[0061] Example 3: Mutant Induction, Expression, and Purification

[0062] 3.1. Induction: Selecting different mutants Cl A single colony of FDH engineered bacteria was inoculated into 10 mL of LB liquid medium containing 100 μg / mL ampicillin resistance and incubated at 37°C and 200 rpm for 12–14 h. The activated bacterial culture was then transferred at a 3% inoculation rate to 50 mL of TB liquid medium containing 100 μg / mL ampicillin resistance and incubated at 37°C and 200 rpm for 2–3 h, monitoring the OD600 value during incubation. When the OD600 value reached 0.6–0.8, IPTG was added to a final concentration of 0.2 mM, and the culture was incubated at 20°C and 200 rpm for 20 h.

[0063] 3.2. Crude Enzyme Acquisition: After induction, the bacterial culture was centrifuged to remove the supernatant. The bacterial cell pellet was collected, resuspended in PBS, washed twice, and the supernatant was discarded. The pellet was stored at -20°C. The bacterial resuspension was sonicated for 15 min. The cell lysis buffer was centrifuged at 8000 rpm for 20 min at 4°C to remove bottom cell debris. The supernatant was collected, and the pellet was also retained as a sample.

[0064] 3.3. Obtaining Pure Enzyme: The filtered crude enzyme solution was re-coated onto the column three times. Impurities were eluted with 10 mL of 25 mM, 50 mM, and 100 mM imidazole solutions, respectively. The target protein was then eluted with 5 mL of 200 mM imidazole solution, and finally, residual protein was eluted thoroughly with 500 mM imidazole solution. The eluent containing the target protein was ultrafiltered using an ultrafiltration tube, concentrated by centrifugation at 3500 rpm at 4°C, and diluted more than 50-fold with protein stock solution. SDS-PAGE analysis showed that all mutant strains expressed the target protein normally, with a clear protein band around 45 kDa. Most of the protein was eluted at 500 mM imidazole concentration. Figure 1 As shown.

[0065] exist Figure 1 In this formula, M represents a marker used in protein electrophoresis to determine the molecular weight of proteins. 1 represents the sample obtained directly after ultrasonic disruption of the OD5 bacterial culture (the culture after induced fermentation by a mutant strain), called the throughflow. 2 represents the sample obtained by ultrasonic disruption of the OD5 bacterial culture (the culture after induced fermentation by a mutant strain), followed by centrifugation and collection of the supernatant. 3 represents the eluent used to elute the target protein; specifically, it is prepared by weighing 100 mL of 500 mM imidazole solution, diluting it to approximately 400 mL with 0.05 M PBS (pH=7.5), adjusting the pH to 7.5 with HCl or NaOH, bringing the volume to 500 mL, filtering through a membrane, and sonicating. 4 also represents the eluent used to elute the target protein; specifically, it is prepared by weighing 17.02 g of imidazole, dissolving it in 400 mL of 0.05 M PBS (pH=7.5), adjusting the pH to 7.5 with HCl or NaOH, bringing the volume to 500 mL, filtering through a membrane, and sonicating.

[0066] Example 4: Determination and Screening of Mutant Enzyme Activity

[0067] Different mutants were induced to express enzymes according to the above procedures. OD600 values ​​were measured after induction, followed by purification and enzyme activity assays. 1 mM DTT solution, 100 mM sodium formate solution, and 2.5 mM NAD+ solution were prepared separately. + Solution, 2.0 mg / mL pure enzyme. The total reaction volume for enzyme activity assay was 200 μL, including DTT: 10 μL; sodium formate: 20 μL; NAD: 10 μL. + Solution: 20 μL; Purified enzyme: 10 μL; PBS buffer: 140 μL. Add all liquids except the purified enzyme to a 96-well plate according to the specified ratio. Incubate the reaction solution in a 40°C metal bath for 5 min. Then, use a pipette to add the purified enzyme to the reaction solution, controlling the reaction time to 5 min. Immediately afterwards, measure the absorbance at 340 nm using an ELISA reader. The blank control group consisted of the reaction solution with added buffer, measured under the same conditions.

[0068] Enzyme activity assays using purified enzymes, such as Figure 3 As shown, the relative enzyme activities of different mutant strains revealed that the D87P and L124F mutants had higher relative enzyme activities, with the L124F mutant exhibiting the best activity, better meeting the requirements for screening mutants with higher enzyme activities. Furthermore, the L124F mutant also showed good growth and rapid cell enrichment.

[0069] Example 5: Temperature stability of mutants

[0070] The purified enzymes obtained from different mutants were incubated at 65°C, and enzyme activity was measured at 0, 0.5, 1, 1.5, 2, 2.5, and 3 hours, using the same method as in Example 4. Enzyme activity stability was continuously tested simultaneously with enzyme activity measurement. At 65°C, the D87P and L124F mutants showed relatively better stability. The results of enzyme activity measurements at 0, 0.5, 1, 1.5, 2, 2.5, and 3 hours are shown below. Figure 4 As shown, both D87P and L124F mutant strains exhibited good stability within 0-3 hours, with L124F being the best. The enzyme activity remained at a high level within 3 hours, reaching a maximum of 3.55 U / mL, which was significantly better than that of the wild-type strain.

[0071] Example 6: Application of the superior mutant coupled with mannitol dehydrogenase

[0072] 6.1. Obtaining Mannitol Dehydrogenase (MDH)

[0073] Mannitol dehydrogenase was constructed and expressed using Escherichia coli BL21(DE3). As The strain carrying FDH (NCBI sequence number MBZ0299354.1) is... As The plasmid for MDH was pETDuet-1, and the sequence and vector were synthesized by Beijing Qingke Biotechnology Co., Ltd. This expression plasmid was transformed into *E. coli*. E. coli In BL21(DE3), the sample was plated on LB agar plates containing 50 μg / mL ampicillin and incubated at 37°C for 12 h. Positive clones were then picked to obtain the expression strain. As MDH is used to express mannitol dehydrogenase.

[0074] Pick AsSingle colonies of the MDH-expressing strain were inoculated into 10 mL of LB liquid medium containing 50 μg / mL ampicillin and cultured at 37°C and 200 rpm for 12–14 h. The activated bacterial culture was then transferred at a 3% inoculation rate to 50 mL of TB liquid medium containing 50 μg / mL ampicillin and cultured at 37°C and 200 rpm for 2–3 h, monitoring the OD600 value during the culture. When the OD600 value reached 0.6–0.8, IPTG was added to a final concentration of 0.2 mM, and the culture was incubated at 20°C and 200 rpm for 20 h. The induced bacterial culture was centrifuged, the supernatant was discarded, and the bacterial pellet was collected. The pellet was resuspended in PBS and washed twice, the supernatant was discarded, and the culture was obtained containing… As The enzyme solution of MDH was stored at -20°C.

[0075] 6.2. Obtaining Formate Dehydrogenase

[0076] Pick Cl Single colonies of the FDH-expressing strain were inoculated into 10 mL of LB liquid medium containing 50 μg / mL ampicillin and cultured at 37°C and 200 rpm for 12–14 h. The activated bacterial culture was then transferred at a 3% inoculation rate to 50 mL of TB liquid medium containing 50 μg / mL ampicillin and cultured at 37°C and 200 rpm for 2–3 h, monitoring the OD600 value during the culture. When the OD600 value reached 0.6–0.8, IPTG was added to a final concentration of 0.2 mM, and the culture was incubated at 20°C and 200 rpm for 20 h. The induced bacterial culture was centrifuged, the supernatant was discarded, and the bacterial pellet was collected. The pellet was resuspended in PBS and washed twice, the supernatant was discarded, and the culture was obtained containing… Cl The FDH enzyme solution was stored at -20°C.

[0077] mutant formate dehydrogenase Cl FDH (D87P) and Cl FDH (L124F) fermentation method and wild type Cl The FDH method is the same, only the strain is replaced with the corresponding strain. The fermentation method for other formate dehydrogenase mutants is also the same.

[0078] 6.3. Two-enzyme coupled catalytic reaction

[0079] Using the obtained mannitol dehydrogenase As MDH and formate dehydrogenase mutants Cl The reaction was catalyzed by coupling with FDH (L124F). The reaction system consisted of 10 mL of HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer at 50 mM, with an initial pH of 6.5. 150 g / L fructose, 34 g / L sodium formate, and 5 mM NADH were added, and the temperature was 50 °C. As MDH, ClFDH (L124F) and other mutant bacterial cultures were added to the reaction solution. 600 The reaction temperature was 20°C, and the reaction time was 8 hours. Each sample was analyzed in triplicate. The reaction was terminated by boiling for 5 minutes. After dilution and membrane filtration, the samples were analyzed by high-performance liquid chromatography (HPLC). A RID-20A differential refractive index detector and a Shodex SUGAR SC1011 column were used to analyze the product D-mannitol and the substrate D-fructose. The mobile phase was ultrapure water. Parameters were set as follows: flow rate 0.8 mL / min, column oven temperature 80°C, detector temperature 40°C, and injection volume 10 μL. The conversion rate of D-mannitol was calculated using the external standard method. The conversion results are shown below. Figure 5 As shown, the liquid phase diagram is... Figure 6 As shown. Cl The conversion rate of FDH coupled with mannitol dehydrogenase was determined by dual plasmid coupling. The L124F mutant showed superior conversion, reaching 92.3% at 10 h, a 27.3% improvement over the WT mutant. The D87P mutant also achieved a conversion rate of 82.16%, a 17.16% improvement over the WT mutant. The modified mutants effectively synergized with mannitol dehydrogenase in the conversion of fructose, demonstrating great potential for the industrial production of mannitol.

[0080] It should be noted that, in addition to its application in the preparation of D-mannitol, the formate dehydrogenase mutant, gene, and recombinant strain of the present invention also have the following applications:

[0081] 1. Pharmaceuticals and Fine Chemicals (Coenzyme Regeneration and Chiral Synthesis)

[0082] Core Applications: As a tool enzyme for NAD(P)H regeneration, it is coupled with oxidoreductases to produce chiral drug intermediates and non-natural amino acids. For example, when coupled with leucine dehydrogenase (LeuDH), it catalyzes the reduction and amination of trimethylpyruvate to synthesize L-tert-leucine (a key intermediate for HIV protease inhibitors). The reaction is nearly complete within 200 minutes, with a conversion rate of 99%, a space-time yield of 786 g / (L·d), and an ee value > 99%.

[0083] Application method: Free enzymes or immobilized enzymes (such as those immobilized on magnetic nanocarriers, with an activity loss of <10% after more than 15 reuses) participate in one-pot two-step or multi-enzyme cascade reactions. Formate is an inexpensive co-substrate, and the byproduct CO2 is easily spilled out, reducing separation costs.

[0084] Value: Significantly reduces the cost of coenzymes (NAD(P)H is expensive), improves reaction efficiency and selectivity, and meets the requirements of green pharmaceutical manufacturing.

[0085] 2. Food and Biomanufacturing (Rare Sugar Alcohol Production)

[0086] Core application: Coupled with ribitol dehydrogenase (RDH), it produces rare sugar alcohols such as allool (allotitol) using D-allulose as a substrate. For example, at 30°C and pH 8.0, the substrate is almost completely converted after 48 hours of reaction, yielding 10 g / L allool.

[0087] Application method: The product is added to the reaction system in the form of whole-cell catalysis or enzyme preparation, and the NADH required for RDH regeneration is generated by FDH, so as to achieve efficient and low-cost conversion.

[0088] Value: Improve the utilization rate of low-value-added sugar resources (such as D-allulose mother liquor), produce high-value functional sugar alcohols, and expand the sources of raw materials for food additives and health products.

[0089] 3. Analytical detection (enzymatic sensors and activity detection)

[0090] Core application: Based on the characteristic of FDH-catalyzed formic acid oxidation accompanied by NAD(P)H generation, develop detection kits for formic acid, NAD(P)⁺ / NAD(P)H or related metabolites.

[0091] Application method: Enzyme electrode or enzyme-coupled colorimetric method, quantitative detection is performed by changes in absorbance at 340 nm, with a detection limit as low as 0.01 U / L, suitable for monitoring clinical micro-samples and industrial fermentation processes.

[0092] Value: Easy to operate, highly specific, fast detection, and supports batch sample analysis and real-time process control.

[0093] 4. Carbon capture and conversion (CO2-formic acid-hydrogen closed loop)

[0094] Core application: Utilizing the reversible catalytic properties of FDH, CO2 is reduced to formic acid, or formic acid is oxidized to CO2 and hydrogen is produced, thus constructing a "CO2-formic acid-hydrogen energy" cycle system.

[0095] Application method: The immobilized high-temperature resistant FDH (optimal temperature up to 65℃) is coupled with an electrochemical system. The CO2 conversion efficiency can reach 92% under normal pressure, the formic acid yield is stable at 1.8 mol / (L·h), and the system energy conversion efficiency exceeds 35%.

[0096] Value: It contributes to carbon reduction goals, enables the utilization of CO2 resources, and provides a liquid formic acid carrier solution for hydrogen energy storage and transportation.

[0097] 5. Environmental protection and bioremediation (including formic acid wastewater treatment and formaldehyde degradation)

[0098] Core applications: treating formic acid-containing wastewater from chemical, fermentation and other industries, or coupling with formaldehyde dehydrogenase to degrade formaldehyde and other toxic pollutants.

[0099] Application methods: Immobilized FDH reactors or genetically engineered bacteria (expressing highly stable FDH) biological treatment devices can efficiently decompose formic acid into CO2 or synergistically convert formaldehyde into harmless substances.

[0100] Value: It replaces traditional chemical treatment methods, reduces secondary pollution, and improves wastewater treatment efficiency and economy.

[0101] 6. Energy and Bioelectrochemistry (Biofuel Cells and Coenzyme Regeneration Electrodes)

[0102] Core applications: as a bioanolyte catalyst to catalyze the oxidation of formic acid to release electrons, or to construct coenzyme regeneration electrodes for electro-enzyme coupling synthesis.

[0103] Application method: FDH is modified on the surface of electrodes such as carbon nanotubes to achieve mediator-free direct electron transfer (DET) and improve the catalytic current density.

[0104] Value: To develop efficient and environmentally friendly biofuel cells, or to use them for in-situ regeneration of coenzymes in electro-enzyme synthesis, thereby reducing energy consumption and costs.

[0105] 7. Synthetic Biology and Metabolic Engineering (Construction of Artificial Metabolic Pathways)

[0106] Core applications: As a modular enzyme, it can be used to construct artificial carbon fixation or energy metabolism pathways and enhance the utilization of C1 substrates such as formic acid by microorganisms.

[0107] Application method: The highly stable FDH gene is introduced into microorganisms (such as Chlamydomonas reinhardtii and Escherichia coli) through technologies such as CRISPR-Cas9 to enhance formic acid metabolism and improve carbon conversion efficiency or product synthesis ability.

[0108] Value: Expanding the microbial substrate spectrum enables the efficient synthesis of chemicals, fuels, or proteins from inexpensive raw materials such as formic acid and CO2, thus promoting sustainable biomanufacturing.

[0109] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A formate dehydrogenase mutant, characterized in that, The formate dehydrogenase mutant has an amino acid mutation in the amino acid sequence shown in SEQ ID NO.1, at position 87 or 124. The formate dehydrogenase mutant is formed by mutating the amino acid sequence shown in SEQ ID NO.1 to one of the following: (1) The 87th aspartic acid is mutated to proline; (2) The leucine at position 124 is mutated to phenylalanine.

2. A gene encoding the formate dehydrogenase mutant as described in claim 1.

3. A recombinant bacterial strain, characterized in that, It contains the formate dehydrogenase mutant as described in claim 1, or it contains the gene as described in claim 2.

4. The application of a formate dehydrogenase mutant as described in claim 1, or a gene as described in claim 2, or a recombinant strain as described in claim 3, in the production of D-mannitol by coupling with mannitol dehydrogenase as a NADH regeneration tool enzyme.

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