Novel protein with methane oxidation activity

Abstract

The present invention relates to a protein in which ferritin monomers self-assemble, in which a methane oxidation activity domain and an electron transfer domain containing a FAD (flavin adenine dinucleotide) binding domain are fused. The protein can oxidize methane using NADH in the body as a reducing agent, and therefore can oxidize methane to methanol without the use of a separate reducing agent. The present invention also relates to a microorganism expressing the protein, a composition for producing methanol utilizing the protein, and a method for producing methanol.

Description

[Technical Field]

[0001] This invention relates to a novel protein having methane oxidation activity. [Background technology]

[0002] Methane monooxygenase (MMO) derived from methane-oxidizing bacteria (methanotrophs) is an extremely useful biocatalyst that can catalyze the oxidation reactions of various hydrocarbons (C1-C8), including methane gas, under mild conditions at room temperature and atmospheric pressure, producing high-value-added products. Global interest is focused on the development of bioprocesses that utilize this biocatalyst.

[0003] Furthermore, enzymes in a similar series, such as ammonia monoxygenase (AMO) from Nitrosomonas europaea, are useful biocatalysts that can catalyze oxidation reactions of a wide range of hydrocarbons (C1-C10 chain / halogenated hydrocarbons, monocyclic / polycyclic aromatic hydrocarbons) through a mechanism similar to that of methane oxidase. However, basic research on their 3D structure, active domain, reaction mechanism, and substrate specificity has been extremely insufficient to date.

[0004] Currently, methanol production through chemical processes using methane gas faces numerous technical, environmental, and economic challenges, including complex processes, environmental pollution from by-products (such as carbon dioxide and syngas), low reaction conversion rates, and high energy consumption due to high temperature and pressure reaction conditions. Furthermore, methane gas presents problems such as high transportation and storage costs, resulting in reduced economic viability, and serious greenhouse gas emissions upon release. Therefore, methanol production using small-scale bioplants that can easily connect to local gas fields offers significant technical and economic advantages.

[0005] While bioprocess development efforts have attempted to improve metabolically engineered strains of methane-oxidizing bacteria to produce high-additive products other than methanol, limitations in the use of genetic engineering tools and problems with the difficulty of culturing strains have prevented successful industrial application of heterologous expression of methane oxidase using industrial strains. This is due to the high technical difficulty of expressing water-soluble proteins and the need for precise interactions of enzyme complexes. [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to provide a protein with excellent methane oxidation ability.

[0007] The present invention aims to provide a microorganism that expresses the aforementioned protein.

[0008] The present invention aims to provide a methanol production composition containing the aforementioned protein or microorganism.

[0009] The present invention aims to provide a method for producing methanol using the aforementioned protein or microorganism. [Means for solving the problem]

[0010] 1. A protein in which ferritin monomers, each fused with a methane oxidation active domain and an electron transport domain, self-assemble.

[0011] 2. In item 1 above, the methane oxidation active domain is pmoB1 (Particulate methane monooxygenase alpha subunit domain 1), MMOH (Soluble methane monooxygenase hydroxylase), or amoB1 (Ammonia monooxygenase beta subunit domain 1), which is a protein.

[0012] 3. In item 2 above, the pmoB1 consists of the amino acid sequence of SEQ ID NO: 1, and the MMOH consists of the amino acid sequence of SEQ ID NO: 2 or 3, which is a protein.

[0013] 4. In item 2 above, the amoB1 consists of the amino acid sequence of SEQ ID NO: 4, which is a protein.

[0014] 5. In item 1 above, the electron transfer domain contains a FAD (flavin adenine dinucleotide) binding domain, which is a protein.

[0015] 6. In item 1 above, the electron transfer domain consists of the amino acid sequence of SEQ ID NO: 5 or 6, which is a protein.

[0016] 7. In item 1 above, the protein is such that the ferritin monomer fused with the methane oxidation active domain and the ferritin monomer fused with the electron transfer domain containing the FAD binding domain self-assemble.

[0017] 8. In item 1 above, the ferritin monomer is further fused with MMOB (Methane monooxygenase regulatory protein B), which is a protein.

[0018] 9. In item 8 above, the MMOB consists of the amino acid sequence of SEQ ID NO: 7 or 8, which is a protein.

[0019] 10. In item 8 above, the methane oxidation active domain is MMOH (Soluble methane monooxygenase hydroxylase), and the MMOB is fused to the ferritin monomer fused with MMOH, which is a protein.

[0020] 11. In item 8 above, the methane oxidation active domain is MMOH (Soluble methane monooxygenase hydroxylase), and a protein in which a ferritin monomer fused with the MMOH and the electron transfer domain and a ferritin monomer fused with MMOB self-assemble.

[0021] 12. In item 11 above, the MMOB is a protein consisting of the amino acid sequence of SEQ ID NO: 7 or 8.

[0022] 13. In item 1 above, the ferritin monomer is a human ferritin heavy chain monomer, a protein.

[0023] 14. In item 1 above, each of the domains is fused to any one selected from the group consisting of the inside of the α-helix of the ferritin monomer, between adjacent α-helices, the N-terminus, the C-terminus, the AB loop, the BC loop, the CD loop, the DE loop, between the N-terminus and the A helix, and between the E helix and the C-terminus, a protein.

[0024] 15. A microorganism that expresses the protein according to any one of items 1 to 14.

[0025] 16. In item 15 above, the microorganism is one into which a vector containing a gene encoding a ferritin monomer, a gene encoding a methane oxidation active domain, and a gene encoding an electron transfer domain containing a FAD (flavin adenine dinucleotide) binding domain has been introduced, a microorganism.

[0026] 17. In item 16 above, the gene encoding the methane oxidation active domain and the gene encoding the electron transfer domain containing the FAD binding domain are contained in one vector or each contained in two vectors, a microorganism.

[0027] 18. In item 15 above, the microorganism is Escherichia coli, a microorganism.

[0028] 19. In item 15 above, the microorganism is a microorganism that further expresses FDH (formate dehydrogenase).

[0029] 20. A methanol production composition comprising a protein according to any of items 1 to 14 above, or a microorganism according to any of claims 15 to 19.

[0030] 21. A method for producing methanol, comprising the step of reacting the composition described in item 20 with methane gas. [Effects of the Invention]

[0031] The protein of the present invention has methane oxidation activity and can be used as a reducing agent necessary for methane oxidation of NADH in living organisms.

[0032] The production of methanol in vivo using the protein of the present invention does not require the introduction of another reducing agent. [Brief explanation of the drawing]

[0033] [Figure 1] Figures 1a to 1c are schematic diagrams of the vectors used in each embodiment. [Figure 2] Figure 2 shows the expression rate and cytoplasmic solubility of the recombinant proteins produced in each example. [Figure 3] Figures 3a and 3b confirm that recombinant proteins containing cMMO, cAMO, and sMMO-mimics produced in the examples form self-assemblies. [Figure 4-6] Figures 4a to 6d show the EXAFS (Extended X-ray absorption fine structure), EPR, and XANES analysis results for recombinant proteins including cMMO, cAMO, and sMMO-m3 produced in the examples. [Figure 7]Figure 7 shows the methane gas oxidation activity of recombinant proteins containing cMMO, cAMO, and sMMO-mimics produced in the examples. [Figure 8] Figure 8 shows the 13C-methane gas oxidation activity of cMMO, cAMO, and sMMO-m3 produced in the examples. [Figure 9] Figure 9 shows the methane gas oxidation activity of a recombinant protein in which the methane oxidation active domain is fused with an electron transport domain containing [2Fe-2S] and FAD binding domains. [Figure 10] Figure 10 shows the methane gas oxidation activity of E. coli lysate expressing cAMO and sMMO-m3 proteins. [Figure 11] Figure 11 shows the 13C-methane gas oxidation activity of E. coli lysates expressing cAMO and sMMO-m3 proteins. [Modes for carrying out the invention]

[0034] The present invention will be described in detail below.

[0035] This invention relates to a protein in which ferritin monomers, each fused with a methane oxidation active domain and an electron transport domain, self-assemble.

[0036] The protein of the present invention can exhibit methane oxidation activity and electron transport activity.

[0037] Methane can be oxidized to methanol by the reaction shown in mathematical formula 1 below. However, the protein of the present invention is a self-assembled ferritin monomer in which a methane oxidation active domain and an electron transport domain are fused. It can carry out the methane oxidation reaction using the reducing agent NADH, and in particular, it can utilize NADH in the body during the reaction in vivo. Therefore, the use of another reducing agent is unnecessary.

[0038] [Mathematical formula 1] CH4 + O2 + NAD(P)H + H + → CH3OH+NAD(P) + +H2O

[0039] A methane oxidation active domain is a domain that has the activity to oxidize methane. It can be a domain containing the active site of a methane oxidase, or, even if it is not derived from a methane oxidase, any domain containing the active site of an enzyme with methane oxidation ability due to sequence or structural similarity is acceptable.

[0040] In the present invention, the methane oxidation active domain can be, for example, pmoB1 (Particulate methane monooxygenase alpha subunit domain 1), MMOH (Soluble methane monooxygenase hydroxylase), amoB1 (Ammonia monooxygenase beta subunit domain 1), etc., but is not limited to these. MMOH may also be MMOHα. More specifically, for example, pmoB1 can be one containing the amino acid sequence of SEQ ID NO: 1. MMOH can be one containing the amino acid sequence of SEQ ID NO: 2 or 3. amoB1 can be one containing the amino acid sequence of SEQ ID NO: 4.

[0041] The electron transport domain may include, for example, a FAD (flavin adenine dinucleotide) binding domain.

[0042] The FAD-binding domain may be derived from sMMO (soluble MMO (Methane monooxygenase)), specifically from the FAD-binding domain of MMOR, one of its constituent elements (MMOR F ) is also acceptable.

[0043] The electron transport domain includes an FAD-binding domain, which may consist solely of the FAD-binding domain, or may further include additional portions in addition to the FAD-binding domain in the MMOR, or may further include at least a portion of the 2Fe-2S domain in addition to the FAD-binding domain, or may include both the FAD-binding domain and the 2Fe-2S domain. For example, the electron transport domain may include the amino acid sequence of SEQ ID NO: 5 or 6.

[0044] The protein of the present invention is a protein formed by the self-assembly of ferritin monomers, each of which is fused with a methane oxidation active domain and an electron transport domain. The domains may be fused entirely within a single ferritin monomer, fused individually to each ferritin monomer, or a mixture thereof.

[0045] In other words, the protein of the present invention may be formed by the self-assembly of ferritin monomers fused with a methane oxidation active domain and ferritin monomers fused with an electron transport domain, or by the self-assembly of ferritin monomers fused with all of these domains, or by the self-assembly of ferritin monomers fused with all of these domains and ferritin monomers fused with one of each domain.

[0046] The ferritin monomer according to the present invention may be further fused with MMOB (Methane monooxygenase regulatory protein B).

[0047] The MMOB may be an MMOB that is a component of an sMMO, and for example, the MMOB may contain the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 8, but is not limited to these.

[0048] For example, MMOB can be used together with MMOH (Soluble methane monooxygenase hydroxylase), which is a methane oxidation active domain. In this case, MMOH, MMOB, and the electron transport domain may fuse to one ferritin monomer, or they may be divided and fused to two ferritin monomers. If they are divided to two ferritin monomers, MMOH and MMOB may fuse to one ferritin monomer and the electron transport domain may fuse to the remaining ferritin monomer, or MMOH and the electron transport domain may fuse to one ferritin monomer and MMOB may fuse to the remaining ferritin monomer.

[0049] The protein of the present invention is a protein formed by the self-assembly of ferritin monomers, in which a methane oxidation active domain and an electron transport domain are fused. As the ferritin monomer, ferritin derived from various organisms can be used, and in the case of vertebrates, heavy chain or light chain monomers can be used. For example, human ferritin heavy chain can be used.

[0050] In ferritin monomers, if the self-assembled protein can perform its function, the binding site of each domain is not limited. For example, it may be fused to any one selected from the group consisting of the inside of an α-helix, between adjacent α-helices, the N-terminus, the C-terminus, the AB loop, the BC loop, the CD loop, the DE loop, between the N-terminus and the A-helix, and between the E-helix and the C-terminus. It is preferably fused to the C-terminus because it can be easily expressed externally from the protein and exert its function.

[0051] In the protein of the present invention, a linker may be further included between the ferritin monomer and each domain.

[0052] As a linker, any known in this field can be used without restriction; for example, S1 (G3SG3TG3SG3), S2 (GKLGGG), etc., can be used.

[0053] The protein of the present invention is obtained, for example, by transforming an organism with a vector containing a gene encoding a ferritin monomer, a gene encoding a methane oxidation active domain, and a gene encoding an electron transport domain including a FAD (flavin adenine dinucleotide) binding domain, and from that organism, but is not limited thereto.

[0054] The protein of the present invention exhibits a high expression rate in soluble form within microorganisms and a high production yield during biosynthesis.

[0055] Furthermore, the present invention relates to a microorganism that expresses the aforementioned protein.

[0056] The microorganism of the present invention may be expressed by introducing a vector containing a gene encoding a ferritin monomer, a gene encoding a methane oxidation active domain, and a gene encoding an electron transport domain.

[0057] In the protein of the present invention, each domain may be fused entirely within a single ferritin monomer, fused individually to each ferritin monomer, or a mixture thereof. Therefore, the gene encoding the methane oxidation active domain and the gene encoding the electron transport domain including the FAD binding domain may be contained in one vector or in two separate vectors.

[0058] As vectors, expression vectors known in this field can be used, such as the BLUESCRIPT vector (Stratagene), the T7 expression vector (Invitrogen), and the pET vector (Novagen), but are not limited to these.

[0059] The vector may further include additional components known in the art, such as promoters for protein expression, tags for isolation / purification, and transformation markers.

[0060] As for the microorganism, any type is acceptable as long as it can express the protein after the vector is introduced; for example, E. coli can be used.

[0061] Since the microorganism of the present invention expresses the aforementioned protein, it can be used in the production of methanol by the oxidation of methane. When using the microorganism of the present invention, it is not necessary to add another reducing agent to produce methanol.

[0062] The microorganism of the present invention may further express FDH (formate dehydrogenase). The FDH can reduce intracellular NAD+ to NADH and enhance the reducing power through NADH recycling.

[0063] The microorganism of the present invention may express FDH after being introduced into a vector containing a gene encoding FDH. The gene encoding FDH may be included together with the vector containing the gene encoding the ferritin monomer, or it may be included in a separate vector.

[0064] Furthermore, the present invention relates to a methanol production composition containing the aforementioned protein or the aforementioned microorganism.

[0065] The aforementioned protein possesses methane oxidation and electron transfer activity, and the aforementioned microorganism expresses the aforementioned protein. Therefore, the composition of the present invention, when containing this protein, can oxidize methane to produce methanol.

[0066] The production of methane can be carried out by treating the composition with methane gas. When the protein is used, an additional reducing agent must be treated, but when the microorganism is used, the use of a reducing agent is unnecessary. The additional reducing agent may further include NADH.

[0067] Furthermore, the present invention relates to a method for producing methanol, which includes the step of reacting the aforementioned composition with methane gas.

[0068] Methanol can be produced by reacting the composition according to the present invention with methane gas to oxidize methane, which can be done by injecting methane gas into the aforementioned composition and advancing the enzymatic reaction.

[0069] The conditions for methanol production are not particularly limited and can be, for example, at the temperature and pH at which the aforementioned proteins or microorganisms exhibit appropriate activity.

[0070] Examples 1. Production of expression vectors for protein biosynthesis According to the vector schematic diagram shown in Table 1 below, PCR is performed to obtain chimeric MMO(pMMO(pmoB1)+sMMO(MMOR F )), chimeric AMO(AMO(amoB1)+sMMO(MMOR F We prepared sMMO-mimics (sMMO-m1~sMMO-m5), cMMO (Full reductase, FR) (pMMO (pmoB1) + sMMO (MMOR)), cAMO (FR) (AMO (amoB1) + sMMO (MMOR)), and sMMO-m2 (FR). All prepared plasmid expression vectors were purified on agarose gel and then sequenced by complete DNA sequencing.

[0071] The PCR products produced in this manner were sequentially inserted into pT7-7 and pET28a expression vectors to construct expression vectors capable of expressing each respective protein.

[0072] The expression vectors for each protein were pT7-cMMO-B1, pET28a-cMMO-B2, pT7-cAMO-B1, pET28a-cAMO-B2, pT7-sMMO-m1-B1, pET28a-sMMO-m1-B2, pT7-sMMO-m2-B1, pET28a-sMMO-m2-B2, pT7-sMMO-m3-B1, pET28a-sMMO-m3-B2, pT7-sMMO-m4-B1, pET28a-sMMO-m4-B2, pT7-sMMO-m5-B1, pET28a-sMMO-m5-B2, pT7-cMMO(FR)-B1, pET28a-cMMO(FR)-B2, pT7-cAMO(FR)-B1, pET28a-cAMO(FR)-B2, pT7-sMMO-m2(FR)-B1, pET28a-sMMO-m2(FR)-B2 (Figure 1a-c).

[0073] [Table 1]

[0074] The amino acid sequences of each protein used are as shown in Table 2 below.

[0075] [Table 2] JPEG0007868862000003.jpg80123

[0076] 2. Biosynthesis and purification of recombinant proteins E. coli strain BL21(DE3)[F - ompThsdS B (rB - mB - )] was transformed with the expression vectors prepared above, respectively. For cMMO, cAMO, sMMO-m1~m5, cMMO(FR), cAMO(FR), sMMO-m2(FR), E. coli strain BL21 was transformed with two expression vectors simultaneously, and transformants resistant to ampicillin and kanamycin were selected. The transformed E. coli was cultured in 50 mL of LB (Luria-Bertani) medium (100 mg L -1Ampicillin and 100mg L -1 The cells were cultured in flasks (250 mL Erlenmeyer flasks, 37°C, 150 rpm) containing kanamycin, 0.4 mM CuSO4, or FeSO4.

[0077] Culture medium turbidity (OD) 600 When the ) reached approximately 0.6, IPTG (Isopropyl-β-D-thiogalactopyranosid) (1 mM) was added to induce gene expression. After culturing at 20°C for 14 hours, the cultured E. coli were centrifuged at 5,000 rpm for 5 minutes to collect the cell precipitate, which was then suspended in 5 mL of lysate (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and disrupted using an ultrasonic disruptor (Branson Ultrasonics Corp., Danbury, CT, USA). After disruption, the cells were centrifuged at 13,000 rpm for 10 minutes to separate the supernatant from the insoluble aggregates. The separated supernatant was first subjected to Ni... 2+ - After NTA affinity chromatography, the recombinant protein was concentrated and then subjected to buffer exchange to obtain purified recombinant protein. Details of each step are as follows.

[0078] 1) Ni 2+ -NTA Affinity Chromatography To purify recombinant proteins, E. coli cultured in the same manner as described above was collected, and the cell pellet was resuspended in 5 mL of disruption solution (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). The cells were then disrupted using an ultrasonic disruptor. The disrupted cell saturate was centrifuged at 13,000 rpm for 10 minutes, and only the supernatant was separated. Each recombinant protein was then filtered using Ni 2+-Separation was performed using NTA columns (Quiagen, Hilden, Germany) (washing buffer: 50 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole, pH 8.0 / elution buffer: 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0).

[0079] 2) Concentration and buffer exchange Ni 2+ -Two mL of recombinant protein eluted via NTA affinity chromatography was placed in an ultracentrifugal filter (Amicon Ultra 100K, Millipore, Billerica, MA) and centrifuged at 5,000 rpm until 1 mL of solution remained on the column. Subsequently, the buffer was changed with Tris buffer (20 mM Tris-HCl, 250 mM NaCl, pH 8.0).

[0080] 3. Analysis of the expression rate and cytoplasmic solubility of recombinant proteins, including manufactured cMMOs, cAMOs, and sMMO-mimics. After the above process, the expression rate and cytoplasmic solubility of the purified recombinant proteins were analyzed by SDS-PAGE. SDS-PAGE was performed on the supernatant (soluble fraction, sol) and insoluble aggregates (insol) obtained by centrifugation of the lysed cell saturation of recombinant proteins, as well as on the purified recombinant proteins, using a 12% Tris-glycine precast gel (Invitrogen, California, USA). The gel was then stained with Coomassie blue solution, and the expression rate and cytoplasmic solubility of each recombinant protein were analyzed for the stained protein bands using a densitometer (GS-800 Calibrated Densitometer, Bio-Rad, California, USA) (Figure 2).

[0081] 4. Structural analysis of recombinant proteins, including manufactured cMMOs, cAMOs, and sMMO-mimics. After the above process, the structure of the purified recombinant protein was analyzed using a transmission electron microscope (TEM). To obtain stained images of the protein, the electron microscope grid containing the air-dried sample was incubated with a 2% (w / v) aqueous uranyl acetate solution at room temperature for 1 hour. When the protein images were observed using a Tecnai 20 (FEI, Hillsboro, Oreon, USA) electron microscope operating at 200 kV, the formation of spherical self-assemblies was confirmed. Furthermore, DLS (dynamic light scattering) analysis confirmed that cMMO forms self-assemblies with sizes of 30.3±1.9 nm, cAMO with 27.9±4.7 nm, sMMO-m1 with 23.7±6.7 nm, sMMO-m2 with 19.6±5.5 nm, sMMO-m3 with 20.5±5.4 nm, sMMO-m4 with 19.4±7.1 nm, and sMMO-m5 with 17.8±3.1 nm (Figure 3a-b).

[0082] X-ray absorption spectroscopy (XAS) and electron paramagnetic resonance (EPR) spectroscopy were performed to analyze the structures of recombinant proteins, including the manufactured cMMO, cAMO, and sMMO-m3. For X-ray absorption near-edge structure (XANES), extended X-ray absorption fine structure (EXAFS), and EPR analysis of the proteins, samples were solvent-exchanged with Tris buffer, pre-frozen at -80°C for 3 hours, and then freeze-dried at -110°C using a freeze-dryer (FDU-2100, DRC-1000, EYELA). XAS analysis was performed using the XAFS beamline (BL11S2) at the Aichi Synchrontron Radiation Center (Aichi), and EPR was measured using an EPR spectrometer (JES-FA200). cMMO EXAFS analysis confirmed the distance information between copper ions present in the active site and surrounding ligands, and XANES analysis confirmed the presence of a mixture of monovalent and divalent copper ions (Cu(I) and Cu(II)). Furthermore, EPR analysis confirmed the presence of divalent copper ions in a valence-scrambled state (Figure 4a-c).

[0083] cAMO EXAFS analysis was used to confirm the distance information between copper ions present at the active site and the surrounding ligand. In samples where the methane oxidation reaction proceeded, the ligand distance differed compared to samples where the reaction did not proceed, and an additional peak (~2.2 Å) was observed. XANES analysis confirmed the presence of both univalent and divalent copper ions (Cu(I) and Cu(II)). Furthermore, EPR analysis confirmed the presence of divalent copper ions in a valence-scrambled state (Figure 5a-c).

[0084] sMMO-m3 EXAFS analysis confirmed the distance information between iron ions present in the active site and surrounding ligands. XANES and EPR analysis revealed the presence of both divalent and trivalent iron ions (Fe(II) and Fe(III)), and confirmed that the Fe(III) / Fe(II) ratio increased in samples where the methane oxidation reaction proceeded compared to samples where the reaction did not proceed. Furthermore, EPR analysis confirmed the presence of trivalent iron ions (Figure 6a-d).

[0085] 5. Demonstration of methane gas oxidation activity of recombinant proteins including manufactured cMMOs, cAMOs, and sMMO-mimics. To verify the methane oxidation activity of purified recombinant protein, 1 mL of recombinant protein solution containing the reducing agent NADH (0.2-0.3 mM) was injected into a 20 mL septa-sealed vial (catalogue no. 5182-0837, Agilent). For the methane oxidation reaction by the recombinant protein, the air in the 19 mL headspace was removed by syringe, and 15 mL of methane gas and 4 mL of air were injected. The vial was then immediately incubated at 30°C for up to 24 hours for the enzymatic reaction. The amount of methanol, the oxidation product generated by the enzymatic reaction, was measured by gas chromatography (7890B GC, Agilent), and the cumulative production was calculated (Figure 7).

[0086] cMMO, cAMO, sMMO-m3 13 To verify the C-methane gas oxidation activity, the enzymatic reaction was carried out in the same manner as the methane oxidation reaction described above, while methane gas was used. 13The reaction was carried out under substitution with 14C-methane gas. For nuclear magnetic resonance (NMR) analysis, the five vials containing the completed reaction were heated at 80°C for 15 minutes, and then 19 ml of headspace gas was directly injected into 600 μl of well-cooled ethanol using a syringe. Subsequently, 60 μl of ethanol-d6 was added, and the mixture was transferred to an NMR tube (NORS55007, Sigma Aldrich) to analyze the oxidation products generated by the enzymatic reactions of cMMO, cAMO, and sMMO-m3. 13 14C-methanol was confirmed by NMR analysis (Figure 8).

[0087] 6. Demonstration of methane gas oxidation activity of recombinant protein in which a methane oxidation activity domain is fused with an electron transport domain containing [2Fe-2S] and FAD binding domains. Recombinant cMMO(FR), cAMO(FR), and sMMO-m2(FR) proteins, in which the electron transport domain of cMMO, cAMO, and sMMO-m2 was replaced with an electron transport domain containing a [2Fe-2S] and FAD-binding domain, were prepared by the method described above, and the methanol production after 16 hours of methane gas oxidation reaction was confirmed by gas chromatography. (cMMO(FR): ND, cMMO: 629.28 (±21.23) mol methanol / mol enzyme, cAMO(FR): 427.03 (±50.2) mol methanol / mol enzyme, cAMO: 1355.16 (±213.06) mol methanol / mol enzyme, sMMO-m2(FR): 570.60 (±50.96) mol methanol / mol enzyme, sMMO-m2: 886.62 (±46.86) mol methanol / mol enzyme). A comparison of methanol production showed that recombinant proteins fused with electron transport domains (MMOR(FR)) containing both [2Fe-2S] and FAD-binding domains performed better than recombinant proteins fused with electron transport domains (MMOR(FR)) containing only the FAD-binding domain. F We confirmed that recombinant proteins fused with ) have higher methane oxidation activity (Figure 9).

[0088] 7. Demonstration of methane gas oxidation activity of E. coli lysate expressing recombinant protein. To verify the methane gas oxidation activity using E. coli expressing cMMO, cAMO, and sMMO-m3 proteins, we used the strain BL21(DE3)[ΔyrfEΔyjaD::pncB](Eng.Life Sci., 7, 343-353, 2007), into which the pncB gene containing the genetic information for the enzyme (nicotinic acid phosphoribosyltransferase), which synthesizes the precursor of intracellular NAD(H), was introduced to overexpress the enzyme. + To reduce it to NADH and enhance the reducing power through NADH recycling, we constructed an expression vector using the pETDuet-1 vector that can co-express cMMO, cAMO, and sMMO-m3 proteins using FDH (formate dehydrogenase) (1) Journal of solid-phase biochemistry, 5, 19-33, 1980, 2) Cell, 179(6), 1255-1263, 2019) derived from Pseudomonas sp. (strain 101).

[0089] The bacterial strain (BL21(DE3)[ΔyrfEΔyjaD::pncB]) was transformed with expression vectors for cMMO, cAMO, and sMMO-m3 proteins, respectively, and protein expression was performed using the culture method described above. Furthermore, during the process of adding 1 mM IPTG, 50 mM sodium formate was added to maintain the reducing power within the strain, and the cells were cultured at 20°C for 14 hours. The cultured E. coli were centrifuged at 4,500 rpm for 10 minutes to collect the cell precipitate, and then resuspended in a lysation solution purged with methane gas. The cells were then lysed using an ultrasonic lysate. The lysated cell lysates were measured for E. coli turbidity (OD) before lysation. 600The mixture was suspended to a ratio of 30 and dispensed into GC vials in 3 mL increments. For the methane oxidation reaction by the recombinant protein-expressing E. coli lysate, the air in the 19 mL vial headspace was removed with a syringe, and 10 mL of methane gas and 9 mL of air were injected. The vials were then immediately incubated at 30°C for up to 52 hours to perform the lysate reaction. After the reaction was complete, the vials were heated at 80°C for 20 minutes to vaporize the methanol produced. The amount of methanol, the oxidation product generated by the lysate, was measured by gas chromatography, and the cumulative production was calculated (maximum methanol production of sMMO-m3-expressed lysate: 1280 MeOH(mol) / enzyme(mol), maximum methanol production of cAMO-expressed lysate: 1460 MeOH(mol) / enzyme(mol)) (Figure 10).

[0090] [Table 3]

Claims

1. It is a protein in which ferritin monomers, each fused with a methane oxidation active domain and an electron transport domain, self-assemble. The aforementioned protein is A protein formed by the self-assembly of ferritin monomers fused with the methane oxidation active domain and ferritin monomers fused with the electron transport domain; or, A protein in which ferritin monomers, formed by the fusion of the methane oxidation active domain and the electron transport domain, self-assemble; or, A ferritin monomer formed by the fusion of the methane oxidation active domain and the electron transport domain, and a protein formed by the self-assembly of ferritin monomers formed by the fusion of either the methane oxidation active domain or the electron transport domain. And, The methane oxidation active domain is MMOH (Soluble methane monooxygenase hydroxylase) or amoB1 (Ammonia monooxygenase beta subunit domain 1), and the electron transport domain consists of the amino acid sequence of SEQ ID NO: 5 or 6, or The methane oxidation active domain is pmoB1 (Particulate methane monooxygenase alpha subunit domain 1), and the electron transport domain consists of the amino acid sequence of Sequence ID No.

5. A protein in which the MMOH comprises the amino acid sequence of SEQ ID NO: 2 or 3, the amoB1 comprises the amino acid sequence of SEQ ID NO: 4, and the pmoB1 comprises the amino acid sequence of SEQ ID NO:

1.

2. The protein according to claim 1, wherein the electron transport domain includes a FAD (flavin adenine dinucleotide) binding domain.

3. The protein according to claim 1, wherein a ferritin monomer fused with a methane oxidation active domain and a ferritin monomer fused with an electron transport domain containing an FAD binding domain are self-assembled.

4. The protein according to claim 1, wherein the ferritin monomer is further fused with MMOB (Methane monooxygenase regulatory protein B).

5. The protein according to claim 4, wherein the MMOB consists of the amino acid sequence of SEQ ID NO: 7 or 8.

6. The protein according to claim 4, wherein the methane oxidation active domain is MMOH (Soluble methane monooxygenase hydroxylase), and the MMOB is fused to a ferritin monomer to which MMOH is fused.

7. The protein according to claim 4, wherein the methane oxidation active domain is MMOH (Soluble methane monooxygenase hydroxylase), and a ferritin monomer fused with the MMOH and the electron transport domain and a ferritin monomer fused with MMOB are self-assembled.

8. The protein according to claim 7, wherein the MMOB consists of the amino acid sequence of SEQ ID NO: 7 or 8.

9. The protein according to claim 1, wherein the ferritin monomer is a human ferritin heavy chain monomer.

10. The protein according to claim 1, wherein each of the domains is fused to one selected from the group consisting of the interior of an α-helix of a ferritin monomer, between adjacent α-helices, the N-terminus, the C-terminus, the AB loop, the BC loop, the CD loop, the DE loop, the space between the N-terminus and the A-helix, and the space between the E-helix and the C-terminus.

11. A microorganism expressing the protein described in any one of claims 1 to 10.

12. The microorganism according to claim 11, wherein the microorganism is introduced into a vector comprising a gene encoding a ferritin monomer, a gene encoding a methane oxidation active domain, and a gene encoding an electron transport domain including a FAD (flavin adenine dinucleotide) binding domain.

13. The microorganism according to claim 12, wherein the gene encoding the methane oxidation active domain and the gene encoding the electron transport domain including the FAD binding domain are contained in one vector or in two separate vectors.

14. The microorganism according to claim 11, wherein the microorganism is Escherichia coli.

15. The microorganism according to claim 11, wherein the microorganism further expresses FDH (formate dehydrogenase).

16. A methanol production composition comprising the protein described in any one of claims 1 to 10.

17. A method for producing methanol, comprising the step of reacting the composition according to claim 16 with methane gas.

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