Escherichia coli with CO2 methanation function, construction method and application of escherichia coli in CO2 biological methanation
By introducing recombinant MoFe nitrogenase gene cluster into E. coli JM109 and regulating nitrogenase expression with TEV protease, E. coli with CO2 methanation function was constructed, which solved the problems of low biomethane efficiency and harsh culture conditions in the prior art, and achieved an efficient and controllable process of converting CO2 into methane.
Patent Information
- Application Number
- CN202411820027.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-03-07
AI Technical Summary
The existing CO2 biomethanation technology faces the problems of slow growth rate, low metabolic efficiency, harsh culture conditions, long culture cycles, and complex and difficult to regulate the structure of nitrogenase gene clusters.
Escherichia coli JM109 was used as the host, and the recombinant MoFe nitrogenase gene cluster was introduced through genetic engineering technology, and nitrogenase assembly and expression was regulated by TEV protease to construct E. coli with CO2 methanation function.
It has achieved the biomethaneification of CO2, which is characterized by simple operation and low cost, simple reaction conditions and controllable process, and has good commercial application value and practicality.
Smart Images
Figure CN119570828A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of genetic engineering and biogas preparation, and specifically relates to an Escherichia coli with CO2 methanogenesis function, a construction method and application thereof in CO2 biomethanogenesis. Background Art
[0002] CO2 biomethanation is one of the important pathways for methane synthesis in nature. Its mild reaction process and high selectivity make it a promising methanogenization pathway. Most of the methanogenic strains discovered so far are anaerobic archaea and anaerobic bacteria, which use organic waste and biomass as substrates to convert CO2 and H2 into methane under anaerobic conditions. However, current methanogenic strains face limiting factors such as slow growth rate and low metabolic efficiency. In addition, traditional methanogenic archaea and methanogenic bacteria require relatively strict culture conditions and require a complex seven-step enzymatic reaction to reduce CO2 to methane. The cumbersome process leads to the inhibition of methanogenic efficiency and carbon utilization efficiency.
[0003] In recent years, researchers have attempted to improve efficiency by shortening the steps of enzymatic reactions and simplifying the methanogenesis process. Yang et al. reported a method for converting CO2 to methane in one step by catalyzing a single enzyme. The researchers modified the natural nitrogenase-catalyzed N2 reduction reaction by mutating the amino acid residues of the MoFe nitrogenase, achieving the eight-electron reduction of CO2 to CH4. Based on this method, Fixen et al. successfully constructed the first intracellular one-step reduction of CO2 to methane biological system (R. palustris CGA009) by modifying the MoFe nitrogenase system of Rhodopseudomonas palustris, greatly shortening the original metabolic pathway of CO2 reduction to methane. However, the CO2 methanogenesis of this method is still limited. As a photosynthetic bacterium (PSB), Rhodopseudomonas palustris must be under light conditions to achieve carbon reduction. At the same time, the culture cycle is long and the culture conditions are harsh, which makes it difficult to achieve large-scale CO2 biological methanogenesis. In addition, the nitrogenase gene cluster structure of R. palustris is complex, making it difficult to directly genetically modify it, and its reductive methanogenesis conditions and nitrogenase expression process are difficult to regulate.
[0004] Therefore, it is necessary to develop a new genetically engineered bacterium for converting CO2 to produce methane to overcome the current problems of harsh CO2 biomethanation culture conditions, long culture cycles, and complex and difficult to regulate nitrogenase gene clusters. Summary of the invention
[0005] In view of some deficiencies in the prior art, the present invention provides an Escherichia coli with CO2 methanogenesis function, a construction method and an application thereof in CO2 biological methanogenesis; the present invention adopts Escherichia coli JM109 as a host for CO2 biological methanogenesis, introduces a recombinant MoFe nitrogenase gene cluster into the Escherichia coli genome through genetic engineering technology, and obtains an Escherichia coli with CO2 methanogenesis function by regulating nitrogenase assembly and expression through TEV protease; the Escherichia coli with CO2 methanogenesis function can biomethanize CO2, has the characteristics of simple operation and low cost, and in the process of converting CO2 to prepare methane, the reaction conditions are simple and the process is controllable, it has good commercial application value in CO2 resource utilization, and has good practicality.
[0006] In order to achieve the above technical objectives, the present invention adopts the following technical means:
[0007] The present invention first provides a method for constructing Escherichia coli with CO2 methanogenesis function, the construction method comprising:
[0008] (1) Gene synthesis of the MoFe nitrogenase gene cluster nifHDK, nifENB, nifUS, nifJVW, nifFMY, and nifAL; mutating the amino acid at position 71 and the histidine at position 196 of the nifD gene in the nifHDK gene cluster to alanine and glutamic acid, respectively, to obtain the mutant nitrogenase gene cluster nifHDK*;
[0009] (2) splicing and connecting the mutant nitrogenase gene cluster nifHDK* with the MoFe nitrogenase gene cluster nifENB, nifUS, nifJVW, nifFMY, and nifAL by the overlap extension method to obtain a recombinant nitrogenase gene that can be used for heterologous expression;
[0010] (3) connecting the obtained recombinant nitrogenase gene that can be used for heterologous expression with the expression vector through Gibson assembly to obtain the recombinant plasmid PACYC184-MoFe that can be used for heterologous expression of recombinant nitrogenase;
[0011] (4) The recombinant plasmid pACYC184-MoFe and the plasmid pET-MBP-TEV expressing TEV protease were transformed into competent Escherichia coli by heat shock transformation, and evenly spread on LB solid plates containing chloramphenicol, kanamycin, and tetracycline. After culturing at 37°C, it was verified that the Escherichia coli with CO2 methanogenesis function was obtained.
[0012] Preferably, in step (1), the MoFe nitrogenase gene cluster nifHDK, nifENB, nifUS, nifJVW, nifFMY, nifAL is derived from Klebsiella oxytoca M5al;
[0013] The nifHDK gene cluster is obtained by sequentially connecting the genes nifH, nifD, nifK and the TEV protease coding sequence;
[0014] The nifENB gene cluster is obtained by sequentially connecting the genes nifE, nifN, nifB and the TEV protease coding sequence;
[0015] The nifUS gene cluster is obtained by sequentially connecting the genes nifU, nifS, and TEV protease coding sequence;
[0016] The nifJVW gene cluster is obtained by sequentially connecting the genes nifJ, nifV, nifW, and TEV protease coding sequence;
[0017] The nifFMY gene cluster is obtained by sequentially connecting the genes nifF, nifM, nifY and the TEV protease coding sequence;
[0018] nifAL is obtained by sequentially connecting genes nifA and nifL.
[0019] Preferably, in step (1), the sequence of the mutant nitrogenase gene cluster nifHDK* is as shown in SEQ ID NO.1.
[0020] Preferably, in step (4), the nucleotide sequence of the plasmid pET-MBP-TEV expressing TEV protease is as shown in SEQ ID NO.2.
[0021] Preferably, in step (4), the Escherichia coli includes Escherichia coli JM109.
[0022] The present invention also provides an Escherichia coli with CO2 methanogenesis function constructed by the above method, wherein the Escherichia coli with CO2 methanogenesis function is obtained by transferring a recombinant plasmid PACYC184-MoFe and a plasmid pET-MBP-TEV expressing TEV protease into a host bacterium Escherichia coli JM109; the Escherichia coli with CO2 methanogenesis function contains a recombinant MoFe nitrogenase gene and a TEV protease.
[0023] The present invention also provides the use of the above-mentioned Escherichia coli with CO2 methanogenic function in converting CO2 to produce methane.
[0024] The present invention also provides a method for converting CO2 to prepare methane, the method comprising:
[0025] The E. coli with CO2 methanogenic function is inoculated into LB liquid medium for activation and then transferred into KPM reaction medium by centrifugation. IPTG is added into the KPM reaction medium to induce the expression of TEV protease and further anaerobic methanogenic fermentation is performed.
[0026] Preferably, the KPM reaction medium comprises: 10.4 g / L Na2HPO4, 3.4 g / L KH2PO4, 26 mg / L CaCl2·2H2O, 30 mg / L MgSO4, 0.3 mg / L MnSO4, 7.6 mg / L Na2MoO4·2H2O, 36 mg / L ferric citrate, 10 mg / L p-aminobenzoic acid, 5 mg / L biotin, 2% (w / v) glucose, and 10 mM sodium glutamate;
[0027] The concentration of IPTG is 20-500 μM.
[0028] Compared with the prior art, the present invention has the following beneficial effects:
[0029] The present invention uses Escherichia coli JM109 as a host, and based on a TEV protease-induced multi-protein assembly strategy, heterologously expresses recombinant MoFe nitrogenase, thereby realizing the conversion and preparation of CO2 to methane in Escherichia coli. Based on the short growth cycle, simple cultivation, and clear genetic background of Escherichia coli, compared with traditional methanogenic archaea / bacteria, it has better commercial application prospects in the conversion and preparation of methanogenetic CO2. The Escherichia coli element with CO2 methanogenetic function constructed by the present invention includes a Nif nitrogenase element and a TEV protease element, which can realize the regulation of the CO2 conversion and preparation of methane process. In addition, the CO2 biomethanation method provides a basic model and basis for future research on nitrogenase genes, nitrogenase CO2 methanogenesis mechanism, and CO2 biomethanation industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 Schematic diagram of the construction of Escherichia coli with CO2 methanogenesis function.
[0031] Figure 2 Comparison of CO2 methanogenesis performance between wild-type E. coli and E. coli with CO2 methanogenesis function
[0032] Figure 3 This is a schematic diagram of the CO2 methanogenesis performance of the Escherichia coli with CO2 methanogenesis function under the induction of different IPTG concentrations. DETAILED DESCRIPTION
[0033] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.
[0034] In the following examples, the technical means used are conventional means well known to those skilled in the art. Various processes and methods not described in detail are conventional methods known in the art. The sources, trade names and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are the same as those indicated for the first time unless otherwise specified; the reagents, materials, etc. involved are all obtained from commercial sources unless otherwise specified.
[0035] In the present invention, the Escherichia coli (Escherichia coli JM109) was purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.; the expression vector PACYC184 was purchased from Shanghai Kelei Biotechnology Co., Ltd.; the TEV protease plasmid pET-MBP-TEV was provided by the laboratory of Jiangsu University, and its nucleotide sequence is shown in SEQ ID NO.2.
[0036] The culture medium formula involved in the present invention is as follows:
[0037] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH = 7.0;
[0038] KPM reaction medium: 10.4g / L Na2HPO4, 3.4g / L KH2PO4, 26mg / L CaCl2·2H2O, 30mg / LMgSO4, 0.3mg / L MnSO4, 7.6mg / L Na2MoO4·2H2O, 36mg / L ferric citrate, 10mg / L p-aminobenzoic acid, 5mg / L biotin, 2% (w / v) glucose, 10mM sodium glutamate, pH=7.0.
[0039] Example 1: Construction of Escherichia coli with CO2 methanogenesis function
[0040] This embodiment is based on Figure 1 The process shown constructs Escherichia coli with CO2 methanogenesis function, and the construction method is as follows:
[0041] (1) MoFe nitrogenase gene cluster nifHDK, nifENB, nifUS, nifJVW, nifFMY, nifAL were synthesized based on MoFe nitrogenase in Klebsiella oxytoca M5al (full genome sequence accession number GCA_015183075.1). The MoFe nitrogenase gene cluster was obtained by gene synthesis by Sangon Biotech (Shanghai) Co., Ltd.
[0042] Among them, nifHDK is composed of nifH (accession number ARI09454.1), nifD (accession number ARI09455.1), nifK gene (accession number ARI09456.1) and TEV protease coding sequence (sequence ENLYFQS);
[0043] nifENB is composed of the genes nifE (accession number ARI09459.1), nifN (accession number ARI09460.1), nifB and TEV protease coding sequence;
[0044] nifUS is composed of the genes nifU (accession number ARI09462.1), nifS (accession number ARI09463.1) and the TEV protease coding sequence;
[0045] nifJVW is composed of the genes nifJ (accession number ARI09453.1), nifV (accession number ARI09464.1), nifW (accession number ARI09465.1) and the TEV protease coding sequence;
[0046] nifFMY is composed of the genes nifF (accession number ARI09468.1), nifM (accession number ARI09467.1), nifY (accession number ARI09458.1) and the TEV protease coding sequence;
[0047] nifAL is composed of the nifA (accession number ARI09470.1) and nifL (accession number ARI09469.1) genes.
[0048] (2) The gene cluster nifHDK was amplified by PCR using the three pairs of primers nifHDK-F and V71A-R, V71A-F and H196Q-R, and H196Q-F and nifHDK-R (SEQ ID NOs. 3 to 8) in Table 1, and then the overlapping extension method was used to obtain the recombinant nifHDK* gene cluster containing the nifD mutation (V71A, H196Q). The nucleotide sequence of the recombinant nifHDK* gene cluster is shown in SEQ ID NO. 1.
[0049] Table 1. PCR amplification primer sequences
[0050]
[0051] (3) Using the primers nifHDK-F and nifHDK-R, nifENB-F and nifENB-R, nifUS-F and nifUS-R, nifJVW-F and nifJVW-R, nifFMY-F and nifFMY-R, nifAL-F and nifAL-R (SEQ ID NO.3, NO.8, NO.9-18) in Table 1, PCR amplification is performed on the recombinant gene cluster nifHDK* in step (2) and the other MoFe nitrogenase gene clusters nifENB, nifUS, nifJVW, nifFMY, and nifAL in step (1), respectively, and then the gene clusters are spliced and connected by overlapping extension to obtain a recombinant nitrogenase gene that can be used for heterologous expression.
[0052] Using primers nifHDK-F and nifHDK-R, PCR amplification is performed on the recombinant gene cluster nifHDK* in step (3) to obtain the gene fragment nifHDK*;
[0053] Using primers nifENB-F and nifENB-R, PCR amplification is performed on the MoFe nitrogenase gene cluster nifENB in step (1) to obtain a gene fragment nifENB;
[0054] Using primers nifUS-F and nifUS-R, PCR amplification is performed on the MoFe nitrogenase gene cluster nifUS in step (1) to obtain a gene fragment nifUS;
[0055] Using primers nifJVW-F and nifJVW-R, PCR amplification is performed on the MoFe nitrogenase gene cluster nifJVW in step (1) to obtain a gene fragment nifJVW;
[0056] Using primers nifFMY-F and nifFMY-R, PCR amplification is performed on the MoFe nitrogenase gene cluster nifFMY in step (1) to obtain a gene fragment nifFMY;
[0057] The MoFe nitrogenase gene cluster nifAL in step (1) is amplified by PCR using primers nifAL-F and nifAL-R to obtain the gene fragment nifAL.
[0058] The PCR product gene fragments nifHDK*, nifENB, nifUS, nifJVW, nifFMY and nifAL were spliced and connected in sequence by overlapping extension method to obtain a recombinant nitrogenase gene that can be used for heterologous expression.
[0059] (4) The obtained recombinant nitrogenase gene that can be used for heterologous expression was amplified by PCR using primers nifUS-F (SEQ ID NO.11) and nifFMY-R (SEQ ID NO.16) in Table 1 to obtain the complete recombinant nitrogenase gene fragment MoFe.
[0060] The expression vector PACYC184 was amplified by PCR using primers PACYC184-F (SEQ ID NO. 19) and PACYC184-R (SEQ ID NO. 20) to obtain the complete vector fragment PACYC184.
[0061] The complete recombinant nitrogenase gene fragment MoFe of the PCR amplification product and the complete vector fragment PACYC184 were connected by Gibson assembly to obtain a recombinant plasmid that can be used for heterologous expression of recombinant nitrogenase, which was recorded as PACYC184-MoFe.
[0062] (5) The recombinant plasmid pACYC184-MoFe and the TEV protease plasmid pET-MBP-TEV, which can be used for heterologous expression of recombinant nitrogenase, were transformed into competent E. coli JM109 by heat shock transformation. The E. coli JM109 was then evenly spread on an LB solid plate containing 25 μg / mL tetracycline, 25 μg / mL chloramphenicol, and 50 μg / mL kanamycin. After overnight culture at 37°C, single clones expressing the above two plasmids were picked and cultured to obtain E. coli with CO2 methanogenesis ability.
[0063] Example 2: Verification of CO2 methanogenesis function of Escherichia coli with CO2 methanogenesis capability
[0064] In this example, the E. coli with CO2 methanogenic ability obtained in Example 1 was subjected to anaerobic fermentation to verify its ability to convert CO2 to produce methane. The specific steps are as follows:
[0065] (1) The Escherichia coli with CO2 methanogenic ability obtained in Example 1 was inoculated into LB liquid culture medium, and cultured for 14 h at 37° C. and 180 rpm for activation. The activated bacterial solution was then centrifuged at 5000 rpm, and the obtained bacterial solution was resuspended in KPM reaction medium to obtain a resuspended bacterial solution. The concentration of the Escherichia coli with CO2 methanogenic ability in the resuspended bacterial solution was controlled to be OD 600 =0.5.
[0066] (2) Add 100 μM IPTG and 10 mM sodium bicarbonate to the above resuspended bacterial solution, and continuously introduce sterile nitrogen for 30 min to remove oxygen in the bottle, and seal the bottle with a rubber stopper and an aluminum cap to obtain a deoxygenated bacterial solution. Place the deoxygenated bacterial solution in a shaker and culture it at 30°C and 180 rpm for 20 h to induce nitrogenase expression. Finally, transfer the bacterial solution to a 37°C constant temperature shaker and culture it at 180 rpm for 72 h to convert CO2 into methane.
[0067] (3) A gas chromatograph GC-7920 (Beijing Zhongjiao Jinyuan Technology Co., Ltd.) was used to detect the methane content in the headspace gas of the bacterial liquid after 72 hours of culture in step (2) to verify the CO2 methanogenic function of the strain. The gas chromatograph detector was a FID hydrogen flame ionization detector, the carrier gas was argon, the column temperature was 70°C, and the injection port temperature was 150°C. 1 ml of the headspace gas of the above-mentioned Escherichia coli with CO2 methanogenic ability and the wild Escherichia coli culture liquid obtained by the same culture conditions was injected into the gas chromatograph injection port, and the methane peak area was recorded. The results are as follows: Figure 2 shown.
[0068] from Figure 2 It can be seen that no methane is produced in the wild Escherichia coli. In contrast, the Escherichia coli with CO2 methanogenesis ability constructed by the method of the present invention detected a methane content of 182.18 nmol after 72 hours of reaction, indicating that the reconstructed Escherichia coli has CO2 methanogenesis ability.
[0069] Example 3: Investigation of CO2 methanogenic performance of Escherichia coli under induction of different concentrations of IPTG
[0070] This example investigates the effect of adding different concentrations of IPTG to induce the expression of recombinant nitrogenase on the CO2 methanogenesis ability of Escherichia coli with CO2 methanogenesis ability. The investigation steps are basically the same as those in Example 2, with only the following differences: In step (2), 0 μM, 20 μM, 100 μM, and 500 μM IPTG are added to the resuspended bacterial solution to induce heterologous expression of the recombinant nitrogenase in Escherichia coli.
[0071] The method in step (3) is used to detect the methane gas content by gas chromatography to optimize the efficiency of the CO2 methanogenic Escherichia coli in converting CO2 to produce methane. The detection result is as follows: Figure 3 shown.
[0072] As can be seen from the figure, under the induction condition of 500 μM IPTG, Escherichia coli with CO2 methanogenesis ability showed a CO2 to methane conversion efficiency of 1.82%.
[0073] In summary, the present invention adopts Escherichia coli JM109 as the host for CO2 biomethanization, introduces the recombinant MoFe nitrogenase gene cluster into the Escherichia coli genome through genetic engineering technology, and obtains an Escherichia coli with CO2 methanogenesis function by regulating the assembly and expression of nitrogenase through TEV protease; the Escherichia coli with CO2 methanogenesis function can biomethanize CO2, has the characteristics of simple operation and low cost, and its reaction conditions are simple and the process is controllable in the process of CO2 conversion to prepare methane, has good commercial application value in CO2 resource utilization, and has good practicality.
[0074] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.
Claims
1. A method for constructing an Escherichia coli with CO2 methanogenic function, characterized in that: The construction method comprises: (1) Gene synthesis of the MoFe nitrogenase gene cluster nifHDK, nifENB, nifUS, nifJVW, nifFMY, and nifAL; mutating the amino acid at position 71 and the histidine at position 196 of the nifD gene in the nifHDK gene cluster to alanine and glutamic acid, respectively, to obtain the mutant nitrogenase gene cluster nifHDK*; (2) splicing and connecting the mutant nitrogenase gene cluster nifHDK* with the MoFe nitrogenase gene cluster nifENB, nifUS, nifJVW, nifFMY, and nifAL by the overlap extension method to obtain a recombinant nitrogenase gene that can be used for heterologous expression; (3) connecting the obtained recombinant nitrogenase gene that can be used for heterologous expression with the expression vector through Gibson assembly to obtain the recombinant plasmid PACYC184-MoFe that can be used for heterologous expression of recombinant nitrogenase; (4) The recombinant plasmid pACYC184-MoFe and the plasmid pET-MBP-TEV expressing TEV protease were transformed into competent Escherichia coli by heat shock transformation, and evenly spread on LB solid plates containing chloramphenicol, kanamycin, and tetracycline. After culturing at 37°C, it was verified that the Escherichia coli with CO2 methanogenesis function was obtained.
2. The method for constructing Escherichia coli with CO2 methanogenesis function according to claim 1, characterized in that: In step (1), the MoFe nitrogenase gene cluster nifHDK, nifENB, nifUS, nifJVW, nifFMY, and nifAL are derived from Klebsiella oxytoca M5al; The nifHDK gene cluster is obtained by sequentially connecting the genes nifH, nifD, nifK and the TEV protease coding sequence; The nifENB gene cluster is obtained by sequentially connecting the genes nifE, nifN, nifB and the TEV protease coding sequence; The nifUS gene cluster is obtained by sequentially connecting the genes nifU, nifS, and TEV protease coding sequence; The nifJVW gene cluster is obtained by sequentially connecting the genes nifJ, nifV, nifW, and TEV protease coding sequence; The nifFMY gene cluster is obtained by sequentially connecting the genes nifF, nifM, nifY and the TEV protease coding sequence; nifAL is obtained by sequentially connecting genes nifA and nifL.
3. The method for constructing Escherichia coli with CO2 methanogenesis function according to claim 1, characterized in that: In step (1), the sequence of the mutant nitrogenase gene cluster nifHDK* is shown in SEQ ID NO.
1.
4. The method for constructing Escherichia coli with CO2 methanogenesis function according to claim 1, characterized in that: In step (4), the nucleotide sequence of the plasmid pET-MBP-TEV expressing TEV protease is shown in SEQ ID NO.
2.
5. The method for constructing Escherichia coli with CO2 methanogenesis function according to claim 1, characterized in that: In step (4), the Escherichia coli includes Escherichia coli JM109.
6. The Escherichia coli with CO2 methanogenesis function constructed by the method according to any one of claims 1 to 5, wherein the Escherichia coli with CO2 methanogenesis function is obtained by transferring the recombinant plasmid PACYC184-MoFe and the plasmid pET-MBP-TEV expressing TEV protease into the host bacterium Escherichia coli JM109; the Escherichia coli with CO2 methanogenesis function contains the recombinant MoFe nitrogenase gene and TEV protease.
7. Use of the Escherichia coli with CO2 methanogenic function as claimed in claim 6 in converting CO2 to produce methane.
8. A method for converting CO2 to produce methane, characterized in that: The method comprises: The E. coli with CO2 methanogenic function is inoculated into LB liquid medium for activation and then transferred into KPM reaction medium by centrifugation. IPTG is added into the KPM reaction medium to induce the expression of TEV protease and further anaerobic methanogenic fermentation is performed.
9. The method for preparing methane by converting CO2 according to claim 8, characterized in that: The KPM reaction culture medium includes: 10.4g / L Na2HPO4, 3.4g / L KH2PO4, 26mg / L CaCl2·2H2O, 30mg / L MgSO4, 0.3mg / LMnSO4, 7.6mg / L Na2MoO4·2H2O, 36mg / L ferric citrate, 10mg / L p-aminobenzoic acid, 5mg / L biotin, 2% (w / v) glucose, and 10mM sodium glutamate.
10. The method for preparing methane by converting CO2 according to claim 8, characterized in that: The concentration of IPTG is 20-500 μM.