Formate dehydrogenase mutants and their use in catalyzing carbon dioxide reduction

Abstract

The application discloses a formic acid dehydrogenase mutant and application thereof in catalyzing carbon dioxide reduction. Paracoccus The formic acid dehydrogenase from sp.MKU1 is mutated by a site-directed mutation technology Ps The arginine (Arg) at the 223th position of the FDH48 amino acid sequence is mutated into proline (Pro) or the proline (Pro) at the 242th position is mutated into phenylalanine (Phe), and the obtained mutants R223P and P242F have higher catalytic activity, can more effectively catalyze CO2 to generate formic acid in vitro, and have potential application values in efficient activation of CO2 and further conversion into other one-carbon compounds through cascade reactions.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to a formate dehydrogenase mutant and application thereof in catalyzing carbon dioxide reduction. Background Art

[0002] Formate dehydrogenase (FDH, EC 1.17.1.9) is the first rate-limiting enzyme that catalyzes the reduction of CO2 to C1 compounds. Its affinity and stability to CO2 directly limit the conversion efficiency. Candida boidini i Cb FDH, from Thiobacillus sp. KNK65MA Ts FDH, from Chaetomium thermophilum of Ct FDH and from Candida methylica of Cm FDH and others have been identified as being useful for CO2 reduction, but most formate dehydrogenases exhibit lower CO2 reduction activity than formate oxidation. Cb FDH, Ts FDH, Ct FDH and Cm The formate oxidation activities of FDH at the optimal pH were 6.1, 10.9, 3.1, and 13.2 U / mg, respectively, while the corresponding CO2 reduction activities were 1.6, 12.2, 34.6, and 4.7 mU / mg, respectively, representing an order of magnitude difference (Protein Engineering, Design and Selection, 2017, 30(1): 47-55). Therefore, using formate dehydrogenase to efficiently convert CO2 into formate or other products presents certain challenges.

[0003] Improving the catalytic activity of formate dehydrogenase towards CO2 through molecular modification has become an important research approach. Cb For example, FDH, a mutation at a specific site (V120S) can significantly increase its ability to capture CO2 and its catalytic speed ( k cat )and k cat / K m The activity of formate dehydrogenase in the wild type was 3.48 times and 1.60 times that of the wild type, respectively (Applied Microbiology and Biotechnology, 2016, 100(19): 8425-8437). Therefore, it is of great significance to develop new formate dehydrogenase mutants with high CO2 reduction activity for artificial carbon cycle. Summary of the Invention

[0004] The present invention aims to provide a formate dehydrogenase mutant for CO2 reduction, including an amino acid sequence of the formate dehydrogenase mutant, a nucleotide sequence encoding the formate dehydrogenase mutant, a gene expression vector, and a recombinant engineered bacterium thereof. The present invention employs semi-rational design of a self-developed, highly efficient formate dehydrogenase, resulting in a formate dehydrogenase mutant with further enhanced CO2 catalytic efficiency, providing potential technical support for efficient CO2 conversion.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides a formate dehydrogenase mutant, wherein the formate dehydrogenase mutant is obtained by synthesizing a formate dehydrogenase mutant derived from Paracoccus sp. as shown in SEQ ID NO. Paracoccus Formate dehydrogenase from sp.MKU1 Ps The FDH48 amino acid sequence is obtained by mutating the 223rd arginine (Arg) to proline (Pro) or the 242nd proline (Pro) to phenylalanine (Phe).

[0007] Preferably, formate dehydrogenase Ps The amino acid sequence of the mutant obtained by mutating arginine (Arg) at position 223 of the FDH48 amino acid sequence to proline (Pro) is SEQ ID NO.2.

[0008] Preferably, formate dehydrogenase Ps The amino acid sequence of the mutant obtained by mutating proline (Pro) at position 242 of the FDH48 amino acid sequence to phenylalanine (Phe) is SEQ ID NO.3.

[0009] Preferably, the amino acid sequence of the formate dehydrogenase mutant is SEQ ID NO.2 and SEQ ID NO.3.

[0010] In a second aspect, the present invention provides a nucleotide sequence encoding the formate dehydrogenase mutant described above.

[0011] Preferably, the encoding is a formate dehydrogenase as shown in SEQ ID NO.1 Ps The nucleotide sequence of FDH48 is SEQ ID NO.4; the nucleotide sequences encoding the formate dehydrogenase mutants described in SEQ ID NO.2 and SEQ ID NO.3 are both obtained by site-directed mutagenesis based on the nucleotide sequence shown in SEQ ID NO.4.

[0012] In a third aspect, the present invention provides a recombinant expression vector comprising the gene described above.

[0013] The recombinant expression vector can be any of various expression vectors commonly used in the art for expressing target genes in E. coli. Preferably, the gene expression vector is a pET plasmid, more preferably a pETDuet plasmid.

[0014] In a fourth aspect, the present invention also provides a host cell comprising the gene or recombinant expression vector as described above.

[0015] Preferably, the host cell is a prokaryotic cell, more preferably Escherichia coli E. coli. BL21(DE3).

[0016] In a fifth aspect, the present invention provides a method for constructing a formate dehydrogenase mutant as described above, which specifically comprises the following steps: designing homology arm primers based on the single site described in the first aspect, using pETDuet- Ps FDH48 is used as a template, and a sequence is reversely amplified by PCR to obtain a mutant expression vector; the obtained mutant expression vector is transformed into host cells by a heat shock method, and the cells are cultured at a suitable temperature to induce expression of a formate dehydrogenase mutant; the host cells are collected, crushed under high pressure and centrifuged, and the formate dehydrogenase mutant is separated and purified from the supernatant.

[0017] In a sixth aspect, the present invention provides the use of the host cell as described in the fifth aspect in catalyzing the conversion of CO2 hydrate NaHCO3 into formate, and the application method specifically includes the following steps: culturing the host cells, inducing expression, and then centrifuging to collect the bacteria, resuspending them in phosphate buffer and then crushing them with a high-pressure crusher, purifying the protein to obtain a pure enzyme of the formate dehydrogenase mutant, and mixing the pure enzyme with a reaction solution to prepare formate, wherein the reaction solution includes a coenzyme NADH, a substrate NaHCO3 and a certain phosphate buffer.

[0018] In a seventh aspect, the present invention provides a use of the formate dehydrogenase mutant, gene, recombinant expression vector and host cell as described above in catalyzing CO2 reduction, specifically in catalyzing the conversion of CO2 into formate.

[0019] The beneficial effects of the present invention are:

[0020] The present invention is to Paracoccus Formate dehydrogenase from sp.MKU1 Ps Site-directed mutagenesis of FDH48 yielded mutants R223P and P242F with further enhanced enzyme activity. These mutants can efficiently catalyze the conversion of NaHCO3 to formate, and have potential application value in the efficient activation of CO2 and its further conversion into other one-carbon compounds through cascade reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Formate dehydrogenase obtained by homology modeling Ps FDH48 structural model and selected mutated amino acid residues;

[0022] Figure 2 Formate dehydrogenase Ps SDS-PAGE protein electrophoresis of FDH48 and its two mutants R223P and P242F;

[0023] Figure 3 is the standard curve of the formate dehydrogenase activity detection method used in the present invention;

[0024] Figure 4 Formate dehydrogenase Ps Comparison of enzyme specific activities between FDH48 and its 11 mutants;

[0025] Figure 5 Formate dehydrogenase Ps Comparative results of the production of formic acid from NaHCO3 catalyzed by FDH48 and mutants R223P and P242F. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0027] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Materials and reagents used in the following examples are commercially available unless otherwise specified. In the following examples, where specific conditions are not specified, the experiments were conducted under conventional conditions or those recommended by the manufacturer. Reagents and instruments used, where the manufacturer is not specified, are commercially available or can be prepared by known methods.

[0029] Example 1: Formate dehydrogenase Ps Construction of FDH48 mutants

[0030] Formate dehydrogenase Ps The amino acid sequence of FDH48 was uploaded to SWISS-MODEL, and the sequence with the highest homology was selected. Moraxella NAD from sp.C2 +-dependent formate dehydrogenase (2gsd.1) was used as a template for homology modeling. The model file was then uploaded to the HotSpot Wizard v3.1 interactive platform to predict the mutated residues. The parameter conditions were set to default. After the calculation was completed, the calculation results were viewed. Five sites with high hot spots in the catalytic pocket were selected for mutation. According to the predicted possible mutation amino acids, a total of 11 mutation sites were set, namely H224L, H224Y, T221H, T221Y, R223P, P242F, P242L, C256A, C256L, C256T, and C256V. Homologous primers were designed with the mutation site as the center, as shown in Table 1, using pETDuet- Ps FDH48 was used as template and the plasmid was amplified by whole plasmid PCR. Dpn After digestion with I, homologous recombination kit was used for recombination transformation, and mutant plasmids were successfully constructed and transformed into Escherichia coli E. coli. BL21 (DE3) to obtain recombinant engineering bacteria.

[0031] Table 1 Primers for obtaining formic acid mutants by PCR amplification of the whole plasmid

[0032]

[0033] Example 2: Formate Dehydrogenase Ps Expression and purification of FDH48 mutants

[0034] The recombinant engineered bacteria obtained in Example 1 were cultured in LB medium containing 100 mg / L ampicillin sodium resistance at 37° C. overnight to obtain a seed solution;

[0035] The seed solution was inoculated into 1 L of fresh LB medium containing 100 mg / L ampicillin sodium resistance at a 1% inoculum volume and cultured for 2-3 h until the OD 600 The expression was induced by adding IPTG at a final concentration of 0.1 mM. After overnight culture at 20°C for 16-20 h, the cells were collected by centrifugation at 5000 r / min for 20 min.

[0036] Resuspend in 40 mL of binding buffer (20 mM phosphate buffer, 500 mM sodium chloride, 30 mM imidazole, pH = 7.4) and crush using a high-pressure crusher at 800-1000 bar for 5 min; then centrifuge the crushed liquid at 12000 rpm for 30 min, remove the supernatant, filter it with a 0.22 μm filter membrane, and place it on ice until used;

[0037] Purification was performed using a nickel column. The column was first washed with water for 3 column volumes, then with binding buffer for 3 column volumes. The filtered supernatant was then loaded twice, and the column was washed three times with binding buffer for 10 column volumes. Finally, the column was washed once with elution buffer (20 mM phosphate buffer, 500 mM sodium chloride, 500 mM imidazole, pH = 7.4) for 2 column volumes. The eluate was desalted using a 5 mL HiTrap Desalting column and exchanged for storage buffer (50 mM phosphate buffer, 150 mM sodium chloride, 10% glycerol, pH = 7.4) in an AKTA protein purifier. 2-2.5 mL of sample was loaded at a time. The enzyme solution was collected and concentrated using a 10 kDa ultrafiltration tube. The concentration of the purified protein was determined by BCA assay and aliquoted and stored at -80°C.

[0038] Example 3: Formate dehydrogenase Ps Enzyme activity assay of FDH48 mutants

[0039] The wild-type and mutant pure enzyme solutions obtained in Example 2 were diluted to a certain concentration for enzyme activity determination. The reaction volume was 100 μL, containing 10 mM NaHCO3, 200 μM NADH, 50 mM PBS buffer (pH = 7.4), and the enzyme solution diluted a certain multiple. The enzyme activity was detected based on the decrease in the absorbance value of NADH at 340 nm. The amount of enzyme required to consume 1 μmol NADH per minute was defined as 1 U.

[0040] The test results are as follows Figure 4 As shown, the results show that Ps The specific activity of FDH48 was defined as 100%, the specific activity of mutant R223P increased by 20%, the specific activity of mutant P242F increased by 28.35%, and the specific activities of the other mutants decreased.

[0041] Example 4: Formate Dehydrogenase Ps Analysis of formate production in wild-type and optimal mutant FDH48

[0042] The wild type obtained in Example 2 Ps Pure enzyme solutions of FDH48 and mutants R223P and P242F were diluted to 2 mg / mL and added to a 1 mL reaction system. The remaining components included 10 mM NaHCO3, 1 mM NADH, and 50 mM PBS (pH = 7.4). After reacting at 37°C for 1 h, 10% 8 M urea was added to terminate the reaction. After centrifugation at 12,000 r / min for 1 min, the supernatant was collected and the formic acid production was determined by colorimetric analysis.

[0043] The test results are as follows Figure 5The results showed that the formate production of mutant R223P was 0.31 mg / mL, and the formate production of mutant P242F was 0.35 mg / mL, which was higher than that of mutant R223P. Ps FDH48 increased by 34.8% and 52.2%, respectively.

[0044] From the above results, it can be seen that compared with formate dehydrogenase Ps FDH48 wild type, formate dehydrogenase mutant constructed by the present invention Ps FDH48-R223P and Ps FDH48-P242F has higher catalytic activity and is expected to be better used for the efficient conversion of CO2 and the production of other high-value-added products through multi-enzyme cascade reactions.

[0045] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A formate dehydrogenase mutant, characterized in that The formate dehydrogenase mutant is obtained by Ps The FDH48 amino acid sequence is obtained by mutating the arginine at position 223 to proline or the proline at position 242 to phenylalanine.

2. The formate dehydrogenase mutant according to claim 1, characterized in that Formate dehydrogenase Ps The amino acid sequence of the mutant obtained by mutating the arginine at position 223 of the FDH48 amino acid sequence to proline is SEQ ID NO.

2.

3. The formate dehydrogenase mutant according to claim 1, characterized in that Formate dehydrogenase Ps The amino acid sequence of the mutant obtained by mutating proline at position 242 of the FDH48 amino acid sequence to phenylalanine is SEQ ID NO.

3. A gene encoding the formate dehydrogenase mutant according to any one of claims 1 to 3.

5. The gene according to claim 4, characterized in that Encoding formate dehydrogenase as shown in SEQ ID NO.1 Ps The nucleotide sequence of FDH48 is SEQ ID NO.4, and the genes encoding the formate dehydrogenase mutants are all obtained by site-directed mutagenesis based on the nucleotide sequence shown in SEQ ID NO.

4.

6. A recombinant expression vector comprising the gene according to claim 4 or 5.

7. The recombinant expression vector according to claim 6, characterized in that The recombinant expression vector is pETDuet plasmid.

8. A host cell comprising the gene according to claim 4 or 5 or the recombinant expression vector according to claim 6 or 7.

9. The host cell according to claim 8, characterized in that The host cell is Escherichia coli containing the recombinant expression vector according to claim 6 or 7 E. coli. BL21.

10. Use of the formate dehydrogenase mutant according to any one of claims 1 to 3, the gene according to claim 4 or 5, the recombinant expression vector according to claim 6 or 7, or the host cell according to claim 8 or 9 in catalyzing the reduction of CO2 or the conversion of NaHCO3, a hydrate of CO2, into formate.

Images