CN116676283A - Formate dehydrogenase mutant, recombinant genetically engineered bacterium and application thereof - Google Patents
Formate dehydrogenase mutant, recombinant genetically engineered bacterium and application thereof Download PDFInfo
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- 108090000698 Formate Dehydrogenases Proteins 0.000 title claims abstract description 75
- 241000894006 Bacteria Species 0.000 title claims abstract description 40
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- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000001569 carbon dioxide Substances 0.000 claims abstract description 24
- 229910002092 carbon dioxide Inorganic materials 0.000 claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 claims abstract description 19
- 230000035772 mutation Effects 0.000 claims abstract description 19
- 239000004471 Glycine Substances 0.000 claims abstract description 10
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims abstract description 10
- 235000004279 alanine Nutrition 0.000 claims abstract description 10
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims abstract description 7
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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Abstract
The invention relates to the technical field of bioengineering, in particular to a formate dehydrogenase mutant, recombinant genetically engineered bacteria and application thereof. The formate dehydrogenase mutant of the present invention contains a mutation of glycine 173 to alanine, a mutation of glycine 297 to alanine and a mutation of aspartic acid 408 to valine, as compared with Paraclostridium bifermentans wild-type formate dehydrogenase. The formate dehydrogenase mutant has higher catalytic activity and can efficiently catalyze the conversion of carbon dioxide into formate. The recombinant engineering bacteria for expressing the formate dehydrogenase mutant can be used for converting whole-cell biocatalysis carbon dioxide into formate, and can realize high-efficiency bioconversion under mild conditions without adding coenzyme, thereby greatly reducing the production cost and having good industrialization prospect.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a formate dehydrogenase mutant, recombinant genetically engineered bacteria and application thereof.
Background
Since the industrial revolution, various industries such as thermal power generation, transportation and the like have discharged a large amount of CO 2 Thereby causing climate change and global warming. To reduce atmospheric CO 2 At the level, not only global regulations such as Paris climate Agreement are implemented, but also CO is developed 2 New technologies including capture, storage and utilization. On the other hand, CO 2 Is a cheap, abundant and renewable carbon raw material (the carbon content in the atmosphere is 7500 hundred million tons), and can be used for synthesizing fuel and chemicals (such as formic acid, methanol, ethanol and the like). Thus, CO is converted into 2 Conversion to fuels and chemicals provides a win-win strategy for global warming mitigation and renewable energy utilization promotion.
In the past decades, people have been on CO 2 Is subjected to intensive studies including chemical conversion, electrochemical conversion and photochemical conversion. However, these methods have respective drawbacks, which prevent large-scale application. Chemical conversions are typically carried out at high temperatures and pressures, requiring the use of expensive catalysts, which in turn result in high energy consumption and high costs. Photochemical conversion requires long-time illumination of a photosensitizer, and has the problems of instability and difficult separation of downstream products. Electrochemical conversion generally has the problems of low selectivity, multiple product types, low current density and low efficiency. In comparison with the above method, the enzymatic conversion of CO 2 Is a promising solution, which can realize high selectivity and high efficiency of CO under mild and environment-friendly conditions 2 And (5) reduction. Enzymatic CO 2 The conversion is to produce important chemicals and fuels, CO, by utilizing multi-enzyme cascade reactions such as Formate Dehydrogenase (FDH), formaldehyde dehydrogenase (FaldDH) and Alcohol Dehydrogenase (ADH) 2 With the oxidation of NADH to formic acid, formaldehyde and methanol, respectively. However, formate dehydrogenase reported so far still has problems of low enzyme activity, low conversion efficiency, and the like.
Disclosure of Invention
The invention provides a formate dehydrogenase mutant, a gene for encoding the formate dehydrogenase mutant, recombinant genetically engineered bacteria containing the gene and application of the genetically engineered bacteria in whole cell catalysis of carbon dioxide to formate.
According to the invention, a novel formate dehydrogenase gene (PbFDH) is excavated from each genome data according to a formate dehydrogenase probe ClFDH from Clostridium ljungdahlii DSM 13528 strain by means of genetic engineering technology, three key amino acid residues in a substrate binding pocket region of the formate dehydrogenase are mutated into nonpolar amino acid residues with smaller side chain groups by means of rational design, and a formate dehydrogenase mutant with high catalytic activity is obtained by screening.
Specifically, the invention provides the following technical scheme:
the present invention provides a formate dehydrogenase mutant comprising a mutation of glycine 173 to alanine, a mutation of glycine 297 to alanine, and a mutation of aspartic acid 408 to valine, as compared with a wild-type formate dehydrogenase of Clostridium bifidum (Paraclostridium bifermentans).
Preferably, the amino acid sequence of the formate dehydrogenase mutant is shown as SEQ ID NO. 1.
SEQ ID NO.1：
The formate dehydrogenase mutant has high catalytic activity and can efficiently catalyze the conversion of carbon dioxide into formate.
The present invention provides genes encoding the formate dehydrogenase mutants described above.
Based on the amino acid sequence and codon usage of the formate dehydrogenase mutant described above, a person skilled in the art can obtain a nucleotide sequence encoding the formate dehydrogenase mutant gene. Based on the degeneracy of the codons, the nucleotide sequence of the gene is not unique and all genes capable of encoding the formate dehydrogenase mutants are within the scope of the invention.
Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO. 2.
The present invention provides a biological material comprising the gene described above, which is an expression cassette, a vector or a host cell.
Wherein the expression cassette is a recombinant gene obtained by operably linking the gene with transcriptional and/or translational regulatory elements.
Such vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, and the like. The plasmid vector comprises an expression vector and a cloning vector.
The host cell includes a microbial cell, preferably an escherichia bacterium, more preferably escherichia coli.
In some embodiments of the invention, the biological material is an expression vector pET32a-PbFDH containing a gene encoding the formate dehydrogenase mutant M 。
In some embodiments of the invention, the host cell is a host cell comprising the expression vector pET32a-PbFDH M Coli BL21 (DE 3).
The invention successfully constructs the genetic engineering bacteria capable of expressing the formate dehydrogenase mutant by transferring the expression vector containing the gene for encoding the formate dehydrogenase mutant into bacteria, and further realizes the bioconversion of carbon dioxide by using whole cell catalysis.
The invention provides a recombinant genetically engineered bacterium which expresses the formate dehydrogenase mutant, comprises the gene or carries a vector comprising the gene.
Preferably, the recombinant genetically engineered bacterium is a recombinant escherichia coli genetically engineered bacterium.
The invention provides the application of any one of the following formate dehydrogenase mutant, the gene, the biological material or the recombinant genetically engineered bacterium:
(1) The application in constructing engineering bacteria for producing formate by converting carbon dioxide;
(2) Use in biocatalytic conversion of carbon dioxide to formate;
(3) Use in the production of formate using carbon dioxide.
In the above (1), the engineering bacteria are preferably E.coli engineering bacteria.
The present invention provides a method for biocatalytically converting carbon dioxide into formate, the method comprising: and (3) incubating bicarbonate with the recombinant genetically engineered bacteria in the presence of hydrogen.
Preferably, the incubation is carried out at 30-37℃with stirring.
Preferably, the above method for biocatalytically converting carbon dioxide into formate is a whole-cell biocatalytic method for converting carbon dioxide into formate.
In some embodiments of the invention, the method comprises: re-suspending the wet bacterial cells of the recombinant genetically engineered bacteria with phosphate buffer until the bacterial cell concentration is 0.3-0.7g/mL, mixing the re-suspended recombinant genetically engineered bacteria with sodium bicarbonate with concentration of 0.1-0.4M as carbon dioxide source, and mixing H 2 Purge into the above mixture, incubate at 35-37 ℃ and stir at the bottom of the mixture using a magnetic stirrer, react for 1-24h.
The invention has the beneficial effects that: the formate dehydrogenase mutant provided by the invention has higher catalytic activity (after being expressed by an escherichia coli expression system, the specific enzyme activity of the formate dehydrogenase mutant reaches 974mU/mg, which is 55.6 times of the specific enzyme activity of Clostridium ljungdahlii formate dehydrogenase and 2.2 times of the specific enzyme activity of pre-mutation formate dehydrogenase PbFDH), and can efficiently catalyze the conversion of carbon dioxide into formate.
The recombinant engineering bacteria for expressing the formate dehydrogenase mutant can be used for carrying out whole-cell biocatalysis on carbon dioxide to convert into formate, does not need to add coenzyme, can efficiently catalyze the conversion of carbon dioxide to convert into formate under mild conditions (the concentration of sodium formate can reach 47mM after the reaction is carried out for 5 hours), greatly reduces the production cost, has good industrialization prospect, and provides an effective method for relieving global warming and promoting renewable energy utilization.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a evolutionary tree of putative formate dehydrogenase involved in example 1 of the present invention; wherein CdFDH-formate dehydrogenase derived from Clostridium drakei (NCBI accession number WP_ 084572129.1), csFDH-formate dehydrogenase derived from Clostridium scatologenes (NCBI accession number WP_ 082085143.1), csp001 FDH-formate dehydrogenase derived from Clostridium sp.001 (NCBI accession number WP_ 217302185.1), clFDH-formate dehydrogenase derived from Clostridium ljungdahlii DSM 13528 (NCBI accession number ADK 13769.1), caFDH-formate dehydrogenase derived from Clostridium aciditolerans (NCBI accession number WP_ 211141100.1), rlFDH-formate dehydrogenase derived from Romboutsia lituseburensis (NCBI accession number WP_ 092726785.1), pbeFDH-formate dehydrogenase derived from Paraclostridium benzoelyticum (NCBI accession number WP_ 084822034.1), pbFDH-formate dehydrogenase derived from Paraclostridium bifermentans (NCBI accession number WP_ 082435282.1), csFDDH-formate dehydrogenase derived from Clostridium sp.PL3 (NCBI accession number WP_ 218320114.1), csFDDH-formate dehydrogenase derived from Clostridium senegalense (NCBI accession number 081496306.1), csFDDH-formate dehydrogenase derived from Clostridium senegalense (NCFDH_89), csFDDH-formate dehydrogenase derived from the probe of the invention (NCFDH-accession number of the invention, and CsFDH-probe of the invention (PbFDH-accession number of the probe).
FIG. 2 shows formate dehydrogenase mutant (PbFDH) of example 4 of the present invention M ) SDS-PAGE patterns of (C); wherein M: pageRuler pre-dye proteinMolecular weight standard, 1: BL21/pET32a,2: BL21/pET32a-PbFDH M Lysate precipitation, 3: BL21/pET32a-PbFDH M Lysate supernatant, 4: BL21/pET32a-PbFDH M And (5) purifying the protein.
FIG. 3 shows a recombinant engineering bacterium BL21/pET32a-PbFDH according to the invention in example 5 of the invention M A curve of the progress of whole cell biocatalysis of carbon dioxide into sodium formate.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The general development process of the invention is as follows: the invention uses the genome cloning technology, takes the protein sequence of formate dehydrogenase ClFDH from Clostridium ljungdahlii which is well studied at present as a probe, carries out BLAST analysis in each genome data, and finds a series of formate dehydrogenase proteins which are from genome information and are not expressed, identified and assumed from the query results. The sequences were subjected to tree construction and analysis, and then representative gene sequences were selected. The selected genes are subjected to codon optimization, complete gene synthesis and expression screening. Wherein a novel high-activity formate dehydrogenase (PbFDH) is excavated from the genome of Paraclostridium bifermentans, and the formate dehydrogenase (PbFDH) is used as a parent for computer-aided rational design. Firstly predicting three-dimensional structure of parent formate dehydrogenase (PbFDH) by alpha Fold 2, performing semi-flexible molecular docking simulation on the protein (PbFDH), substrate and coenzyme by Autodock4.2 software, analyzing three-dimensional structure of the obtained complex by using Pymol and Discovery Studio software to find out amino acid residue of the interaction of the enzyme with the substrate and the coenzyme, and determining 173 th glycine (Gly), 297 th glycine (Gly) and 408 th radix asparagiThe amino acid (Asp) is mutated into alanine (Ala), alanine (Ala) and valine (Val), respectively, and the mutation is achieved by improved whole plasmid amplification technique, and finally the mutant (PbFDH) is successfully obtained M ) Recombinant plasmid pET32a-PbFDH of (E) M And the recombinant plasmid is used for successfully constructing a mutant (PbFDH) capable of efficiently expressing formate dehydrogenase M ) The application of whole cell catalysis of carbon dioxide to formate is further performed.
The construction of the recombinant genetically engineered bacterium comprises the following steps: (1) Connecting the screened high-activity formate dehydrogenase PbFDH with a pET32a expression vector to obtain pET32a-PbFDH; (2) The recombinant vector pET32a-PbFDH for expressing the formate dehydrogenase mutant is obtained by taking the recombinant vector pET32a-PbFDH as a template and adopting an improved full plasmid amplification technology M The method comprises the steps of carrying out a first treatment on the surface of the (3) Recombinant vector pET32a-PbFDH for expressing formate dehydrogenase mutant M The method comprises the steps of performing heat shock transformation into competent cells of escherichia coli BL21 (DE 3), adding 500 mu L of LB liquid for culture based on slow shake culture at 37 ℃ and 200rpm for 1h, coating a proper amount of bacterial liquid into an LB plate corresponding to the resistance of a target plasmid, and performing inversion culture at 37 ℃ for 12-16h to obtain monoclonal recombinant genetically engineered bacteria; (4) And 5-10 single colonies are selected from the transformation plate, bacterial liquid PCR verification is carried out, positive colonies are transferred into 5mL of LB liquid medium containing 50 mug/mL ampicillin, the culture is carried out at 37 ℃ and 200rpm for overnight, plasmids are extracted the next day, bamH I and Xho I are used for carrying out double enzyme digestion identification, and the successful acquisition of the target recombinant genetically engineered bacteria is determined.
The fermentation liquor of the recombinant genetically engineered bacterium is prepared by the following method: (1) Inoculating the recombinant genetic engineering bacteria into 5mL of LB liquid medium containing 50 mug/mL ampicillin, and culturing for 12-16h at 37 ℃ and 200 rpm; (2) Inoculating the bacterial liquid obtained in the step (1) into 100mL of LB liquid culture medium according to the inoculum size of 2%, and culturing for 2.5h at 37 ℃ and 200 rpm; (3) Adding IPTG with the final concentration of 0.1mmol/L, and carrying out induction culture for 20h at the temperature of 16 ℃ and the speed of 220rpm to obtain recombinant genetically engineered bacteria fermentation liquor.
Example 1: gene excavation of formate dehydrogenase encoding gene and construction of recombinant vector
BLAST analysis is carried out by taking the protein sequence of formate dehydrogenase ClFDH from Clostridium ljungdahlii which is thoroughly studied at present as a probe and taking various genome sequences as search objects, and a series of formate dehydrogenase proteins which are from genome information and are not expressed, identified and assumed are found out from the query results. The sequences are subjected to the construction and analysis of a evolutionary tree (shown in figure 1), then 3 representative gene sequences are selected, and complete gene synthesis is carried out after codon optimization. The 3 potential formate dehydrogenases from Paraclostridium bifermentans, clostridium scatologenes and Desulfosporosinus acididurans were selected for further investigation by comparative analysis and named PbFDH, csFDH and DaFDH, respectively.
The codon usage frequency of E.coli K12 strain is used as reference, the potential formate dehydrogenase gene sequence is subjected to codon optimization by utilizing an OPTIMIZER server, and the optimized sequences of PbFDH, csFDH and DaFDH are respectively shown as SEQ ID NO.3, 4 and 5. After BamH I and Xho I cleavage sites were added to the upstream and downstream of the gene, the whole gene was synthesized by the same company as the Kyowa Biotech Co., ltd. The synthesized formate dehydrogenase encoding gene is connected to pET32a plasmid to obtain recombinant vectors pET32a-PbFDH, pET32a-CsFDH and pET32a-DaFDH.
Example 2: rational modification of formate dehydrogenase and construction of mutant recombinant vector
And (3) taking PbFDH with highest catalytic activity in the three screened formate dehydrogenases as a parent to carry out computer-aided rational design. Firstly, predicting the three-dimensional structure of a parent formate dehydrogenase (PbFDH) through alpha Fold 2, sequentially carrying out semi-flexible molecular docking analysis on the three-dimensional structure of the PbFDH and a substrate sodium bicarbonate and a coenzyme NADH by using Autodock4.2 software, then carrying out observation analysis by using PyMol and Discovery studio software to determine a substrate channel of the formate dehydrogenase and key amino acid residues and metal ions thereof which act with the substrate and the coenzyme, and respectively mutating the 173 th glycine (Gly), 297 th glycine (Gly) and 408 th aspartic acid (Asp) of the key amino acid into alanine (Ala), alanine (Ala) and valine (Val), wherein the mutated formate dehydrogenase gene is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 1.The mutation adopts an improved full plasmid amplification technology, a recombinant vector pET32a-PbFDH is used as a template, reverse universal primers are designed at a 3.2kb position at the downstream of a cloning site of the vector, forward mutation primers F1, F2 and F3 are respectively designed on fragments of about 15bp at the upstream and downstream of three mutation sites, a pair of forward and reverse primers are used for carrying out first round PCR amplification, then a product of the first round is used as a large primer for carrying out full plasmid amplification, and after two rounds of PCR amplification, the template is digested by Dpn I, so that a recombinant vector with one mutation site is obtained; then the recombinant vector with one mutation site is used as a template, and the other two pairs of forward and reverse primers are used for carrying out the second amplification and the third amplification according to the method, thus obtaining the recombinant vector pET32a-PbFDH with three mutation sites M . The primer sequences are shown in Table 1.
TABLE 1 primers for obtaining formate dehydrogenase mutants by full plasmid amplification
| Primer(s) | Base sequence |
| F1 | 5’-CTGCTGTTTATTTTCGCTTACAACGGTGCTGAC-3’ |
| F2 | 5’-ATCCTGTACGGCATGGCTGTTTGCCAGTTCGGT-3’ |
| F3 | 5’-GACCCGGTTCAGTCCGTCCCGCATGCATCTGAA-3’ |
| R | 5’-CATACCGCCAGTTGTTTACCC-3’ |
Example 3: construction of recombinant genetically engineered bacterium expressing formate dehydrogenase mutant
1. Recombinant vector pET32a-PbFDH M Transformation into host cells: recombinant vector pET32a-PbFDH M Transferring the strain into competent cells of escherichia coli BL21 (DE 3) by heat shock, adding 0.5mL of LB liquid culture medium, incubating for 1h at 37 ℃ and 200rpm, coating the strain on an LB solid plate containing 50 mug/mL of ampicillin, and culturing for 12-16h at 37 ℃ to obtain monoclonal colonies;
2. screening and identifying recombinant bacteria: selecting a monoclonal colony to 5mL of LB liquid medium containing 50 mug/mL ampicillin, culturing overnight at 37 ℃ and 220rpm, carrying out PCR identification on a secondary bacterial liquid, extracting plasmids, carrying out double enzyme digestion identification by utilizing BamH I and Xho I, judging that the plasmids contain gene fragments consistent with the target gene in size according to electrophoresis results, and preliminarily identifying that the clones are positive clones; then the positive clone is sent to Shanghai worker for sequence determination, and the sequencing result further shows that the clone colony is the target genetic engineering bacteria.
Example 4: inducible expression and analysis of formate dehydrogenase mutants
1. Induction of expression: single colonies of the identified recombinant genetically engineered bacteria obtained in example 3 were selected and inoculated into 5mL of LB liquid medium containing 50. Mu.g/mL ampicillin, and cultured overnight at 37℃and 220 rpm; inoculating the overnight cultured product into 100mL of LB liquid medium with a pipette according to an inoculum size of 2%; then, the cells were incubated at 37℃and 200rpm for 2.5 hours, and finally, IPTG was added to the cells to induce the cells to a final concentration of 0.1mmol/L, and the cells were incubated at 16℃and 220rpm for 20 hours to obtain an induced-expression bacterial liquid (fermentation liquid).
The bacterial liquid after induced expression is subpackaged into 50mL centrifuge tubes, centrifuged at 8000rpm and 4 ℃ for 5min, bacterial cells are collected, and each bacterial cell is washed twice with 50mL deionized water under the same conditions. Each of the above-mentioned cells was suspended in 100mL of lysis buffer (PBS buffer, pH 7.0), and the cells were disrupted at 4℃and 900bar for 3 minutes using a high-pressure homogenizer. Centrifuging at 12000rpm and 4deg.C for 15min, collecting supernatant and precipitate, purifying the supernatant by Ni-Agarose medium affinity chromatography, and analyzing the expression form of target protein by SDS-PAGE (shown in figure 2).
2. Enzyme performance measurement:
(1) Enzyme activity determination: the activity of formate dehydrogenase for reducing carbon dioxide was measured on the lysate, and the enzyme activity was measured as follows: the change in absorbance of NADH was determined by UV spectrophotometry at 340nm (εNADH,340 nm=6.22 mM) - 1 cm -1 ). The reaction was initiated by adding formate dehydrogenase and monitored for 1 minute. The reaction system for enzyme activity assay was 1mL: the reaction system was incubated at 30℃for 2min with 100mM sodium bicarbonate, 0.1mM NADH,100mM PBS buffer, pH 7.0, and 100. Mu.L of the enzyme solution diluted by an appropriate factor. The change in absorbance was measured at 340nm over 1 min. Under this condition, the amount of enzyme required to consume 1. Mu. Mol NADH per minute was defined as 1U. Enzyme activity: u=ew·v·1000/6220·l=ew/6.22. EW: OD at 1min 340 Is a variable of (1), V: volume of reaction solution (mL), 6220: molar extinction coefficient (L. Mol) -1 *cm -1 ) L: optical path distance (cm).
(2) Purification of enzyme: the recombinant formate dehydrogenase was subjected to affinity chromatography using a nickel column, and then the specific activity of the pure enzyme was determined. Formate dehydrogenase mutant PbFDH M The specific activity of the pure enzyme reaches 974mU/mg, which is 55.6 times that of the probe (17.5 mU/mg).
Meanwhile, by taking the formate dehydrogenase PbFDH before mutation (the coding gene sequence is shown as SEQ ID NO.3, and the construction of the recombinant vector pET32a-PbFDH is shown as in example 1) as a control, the result shows that the specific activity of the formate dehydrogenase PbFDH before mutation is 435mU/mg, and the formate dehydrogenase mutant PbFDH M The specific activity (974 mU/mg) was 2.2 times that of the mutant pre-formate dehydrogenase PbFDH.
Example 5: biosynthesis of sodium formate
The wet cell of the recombinant engineering bacterium obtained in example 3 was weighed using 0.25M sodium bicarbonate as a source of carbon dioxide, and resuspended in 50mM phosphate buffer pH 7.0 to give a final cell concentration of 0.5g/mL. Will H 2 Purging into the above mixture, incubating at 37deg.C, and gently stirring at the bottom of the mixture with magnetic stirrer, reacting for 1-5H, wherein H is used during the reactionThe amount of sodium formate produced was measured by the PLC method, and after 5 hours of reaction, the concentration of sodium formate could reach 47mM (as shown in FIG. 3).
The recombinant engineering bacterium disclosed by the invention is used for converting the biocatalytic carbon dioxide into formate, has a good application prospect, and provides a win-win strategy for relieving global warming and promoting renewable energy utilization.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A formate dehydrogenase mutant comprising a mutation from glycine 173 to alanine, a mutation from glycine 297 to alanine, and a mutation from aspartic acid 408 to valine, as compared to a wild-type formate dehydrogenase of clostridium bifidum (Paraclostridium bifermentans).
2. The formate dehydrogenase mutant according to claim 1, characterized in that the amino acid sequence of the formate dehydrogenase mutant is shown in SEQ ID No. 1.
3. A gene encoding the formate dehydrogenase mutant according to claim 1 or 2.
4. A gene according to claim 3, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 2.
5. A biological material, characterized in that it comprises the gene according to claim 3 or 4; the biological material is an expression cassette, a vector or a host cell.
6. A recombinant genetically engineered bacterium expressing the formate dehydrogenase mutant of claim 1 or 2, or comprising the gene of claim 3 or 4, or carrying a vector comprising the gene of claim 3 or 4.
7. The recombinant genetically engineered bacterium of claim 6, wherein the recombinant genetically engineered bacterium is a recombinant escherichia coli genetically engineered bacterium.
8. Use of the formate dehydrogenase mutant according to claim 1 or 2 or the gene according to claim 3 or 4 or the biomaterial according to claim 5 or any of the following recombinant genetically engineered bacteria according to claim 6 or 7:
(1) The application in constructing engineering bacteria for producing formate by converting carbon dioxide;
(2) Use in biocatalytic conversion of carbon dioxide to formate;
(3) Use in the production of formate using carbon dioxide.
9. A method of biocatalytically converting carbon dioxide to formate, the method comprising: incubating bicarbonate with the recombinant genetically engineered bacterium of claim 6 or 7 in the presence of hydrogen.
10. The method of claim 9, wherein the incubating is under stirring.
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