Novel protein having methane or butane oxidation activity

Abstract

To provide a protein having excellent methane or butane oxidation ability, a microorganism expressing the protein, a composition for producing methanol or butanol containing the protein or the microorganism, and a method for producing methanol or butanol using the protein or the microorganism.SOLUTION: To provide a protein in which ferritin monomers fused with an ammonia oxidase activity domain having methane oxidation activity are self-assembled. The ammonia oxidase active domains are proteins selected from amoB1 and amoB2, each consisting of a unique amino acid sequence.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to a novel protein having methane or butane oxidation activity. [Background technology]

[0002] Methane monooxygenase (MMO) derived from methane-oxidizing bacteria (methanotrophs) is a highly useful biocatalyst that can catalyze the oxidation of various hydrocarbons (C1-C8), including methane gas, under mild conditions of room temperature and atmospheric pressure to produce high-value-added products. There has been worldwide interest in developing bioprocesses that utilize this enzyme.

[0003] In addition, similar enzymes, such as ammonia monoxygenase (AMO) from Nitrosomonas europaea and butane monooxygenase (BMO) from Nocardioides sp. strain CF8, are useful biocatalysts that can catalyze the oxidation of a wide range of hydrocarbons (AMO: C1-C10 linear / halogenated hydrocarbons, mono- and polycyclic aromatic hydrocarbons; BMO: C2-C10 linear / halogenated hydrocarbons, some aromatic hydrocarbons) using a mechanism similar to that of methane oxidase. However, basic research into their 3D structures, active domains, reaction mechanisms, and substrate specificities is currently insufficient.

[0004] Currently, the production of methanol and butanol through chemical processes from methane and butane gases is complicated, posing numerous technical, environmental, and economic challenges, including environmental pollution from by-products (carbon dioxide, syngas, etc.), low reaction conversion rates, and high energy consumption due to high-temperature, high-pressure reaction conditions. Methane gas, in particular, faces challenges such as expensive transportation and storage costs, which reduces its economic viability, and the severe greenhouse effect it can cause if it leaks. Therefore, producing methanol using small-scale biomass plants that can be easily connected to local gas fields offers significant technical and economic advantages.

[0005] Efforts to develop bioprocesses have been made to improve metabolically engineered strains of methane-oxidizing bacteria and various hydrocarbon-degrading bacteria to produce other high-additive products besides methanol. However, there are limitations to the use of genetic engineering tools and problems due to the difficulty of culturing strains. Heterologous expression using industrial strains to mass-produce methane oxidizing enzymes, for example, is technically difficult, as it is difficult to express water-soluble proteins and precise interactions between enzyme complexes are required, so there are no successful examples of industrial application. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a protein having excellent methane or butane oxidation ability.

[0007] An object of the present invention is to provide a microorganism that expresses the protein.

[0008] An object of the present invention is to provide a composition for producing methanol or butanol, which contains the protein or microorganism.

[0009] An object of the present invention is to provide a method for producing methanol or butanol using the protein or microorganism. [Means for solving the problem]

[0010] 1. A protein in which ferritin monomers are self-assembled to which an ammonia oxidase active domain having methane oxidation activity or a butane oxidase active domain having butane oxidation activity has been fused.

[0011] 2. The protein according to item 1, wherein the ammonia oxidase active domain is selected from amoB1 (Ammonia monooxygenase beta subunit domain 1) and amoB2 (Ammonia monooxygenase beta subunit domain 2).

[0012] 3. The protein according to item 2, wherein the amoB1 consists of the amino acid sequence of SEQ ID NO: 1, and the amoB2 consists of the amino acid sequence of SEQ ID NO: 2.

[0013] 4. The protein according to item 2, wherein ferritin monomers fused with amoB1 and amoB2 are self-assembled.

[0014] 5. The protein according to item 2, wherein amoB1-fused ferritin monomers and amoB2-fused ferritin monomers are self-assembled.

[0015] 6. A protein according to item 1, wherein the butane oxidase active domain is selected from bmoB1 (Particulate Butane monooxygenase subunit B_domain 1) and bmoB2 (Particulate Butane monooxygenase subunit B_domain 2).

[0016] 7. A protein according to item 6, wherein bmoB1 consists of the amino acid sequence of SEQ ID NO: 3, and bmoB2 consists of the amino acid sequence of SEQ ID NO: 4.

[0017] 8. The protein according to item 6, wherein ferritin monomers fused with bmoB1 and bmoB2 are self-assembled.

[0018] 9. The protein according to item 6, wherein a ferritin monomer fused with bmoB1 and a ferritin monomer fused with bmoB2 are self-assembled.

[0019] 10. The protein according to item 1, wherein the ferritin monomer is a human ferritin heavy chain monomer.

[0020] 11. A protein according to item 10, wherein each of the domains is fused to any one selected from the group consisting of within 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, between the N-terminus and the A helix, and between the E helix and the C-terminus.

[0021] 12. A microorganism expressing the protein according to any one of items 1 to 11.

[0022] 13. The microorganism according to item 12, into which a vector containing a gene encoding a ferritin monomer and a gene encoding a methane oxidase active domain selected from amoB1 (Ammonia monooxygenase beta subunit domain 1) and amoB2 (Ammonia monooxygenase beta subunit domain 2), or a butane oxidase active domain selected from bmoB1 (Particulate Butane monooxygenase subunit B domain 1) and bmoB2 (Particulate Butane monooxygenase subunit B domain 2) has been introduced.

[0023] 14. The microorganism according to item 12, wherein the microorganism is Escherichia coli.

[0024] 15. A composition for producing methanol, comprising the protein according to any one of items 1 to 11, wherein the protein is fused with an ammonia oxidase active domain.

[0025] 16. The composition according to item 15, further comprising a reducing agent.

[0026] 17. The composition according to item 15, wherein the reducing agent is duroquinol.

[0027] 18. A method for producing methanol, comprising a step of reacting the composition according to item 15 with methane gas.

[0028] 19. A composition for producing butanol, comprising the protein according to any one of items 1 to 11, wherein the protein is fused with a butane oxidase active domain.

[0029] 20. The composition according to item 19, further comprising a reducing agent.

[0030] 21. The composition according to item 19, wherein the reducing agent is duroquinol.

[0031] 22. A method for producing butanol, comprising the step of reacting the composition according to item 19 with butane gas. [Effects of the Invention]

[0032] The protein of the present invention has methane or butane oxidation activity.

[0033] The protein of the present invention contains many domains having methane or butane oxidation activity, and has high activity.

[0034] The compositions and methods of the present invention are capable of producing methanol or butanol in high yields. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 is a schematic diagram of each vector used in the examples. [Figure 2] FIG. 2 shows the results of analysis of the expression rate and cytoplasmic solubility of recombinant proteins, including cAMO, AMO-, and BMO-mimics, produced in the examples. [Figure 3] FIG. 3 confirms that recombinant proteins containing cAMO, AMO-, and BMO-mimics produced in the examples form self-assemblies. [Figure 4] Figures 4a-c show the results of X-ray absorption near-edge structure (XANES), extended X-ray absorption fine structure (EXAFS), and electron paramagnetic resonance (EPR) spectroscopy of the cAMO recombinant protein. [Figure 5] FIG. 5 shows the methane and butane gas oxidation activity of recombinant proteins containing cAMO, AMO-, and BMO-mimics prepared in the examples. [Figure 6] FIG. 6 shows the 13C-methane gas oxidation activity of the cAMO produced in the examples. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention will be described in detail below.

[0037] The present invention relates to a protein formed by self-assembly of ferritin monomers fused with an ammonia oxidase active domain having methane oxidation activity or a butane oxidase active domain having butane oxidation activity.

[0038] Any ammonia oxidase active domain can be used without limitation as long as it has the activity of oxidizing methane, and examples of such domains include amoB1 (Ammonia monooxygenase beta subunit domain 1) and amoB2 (Ammonia monooxygenase beta subunit domain 2). Specifically, amoB1 containing the amino acid sequence of SEQ ID NO: 1 can be used, and amoB2 containing the amino acid sequence of SEQ ID NO: 2 can be used.

[0039] The butane oxidase active domain can be any domain that has butane oxidation activity, and examples of such domains include bmoB1 (particulate butane monooxygenase subunit B_domain 1) and bmoB2 (particulate butane monooxygenase subunit B_domain 2). Specifically, bmoB1 can be one that contains the amino acid sequence of SEQ ID NO: 3, and bmoB2 can be one that contains the amino acid sequence of SEQ ID NO: 4.

[0040] In the protein of the present invention, each domain may be fused to one ferritin monomer, or may be fused to each ferritin monomer, or may be a mixture of these.

[0041] In other words, in the protein of the present invention, two ammonia oxidase active domains or butane oxidase active domains may be fused within one ferritin monomer, or one domain may be fused to each ferritin monomer.

[0042] The protein of the present invention may be further fused with an electron transfer domain containing a FAD (flavin adenine dinucleotide) binding domain.

[0043] Methane can be oxidized to form methanol according to the reaction of Equation 1 below, and the protein of the present invention is a self-assembled ferritin monomer that is fused with a methane oxidation active domain and an electron transfer domain containing a flavin adenine dinucleotide (FAD) binding domain, and can carry out the methane oxidation reaction using NADH as a reducing agent. In particular, it can utilize NADH in the body during the reaction in vivo, eliminating the need for a separate reducing agent.

[0044] [Mathematical formula 1] CH4+O2+NAD(P)H+H + → CH3OH+NAD(P) + +H2O

[0045] The electron transfer domain contains a flavin adenine dinucleotide (FAD) binding domain.

[0046] The FAD-binding domain may be derived from soluble MMO (methane monooxygenase) (sMMO), and specifically, may be the FAD-binding domain of MMOR, which is one of its components.

[0047] The electron transfer domain includes a FAD-binding domain, and may consist of only the FAD-binding domain, or may further include an additional portion in addition to the FAD-binding domain in the MMOR, or may further include at least a portion of a 2Fe-2S domain in addition to the FAD-binding domain, or may include a FAD-binding domain and a 2Fe-2S domain. For example, the electron transfer domain may include the amino acid sequence of SEQ ID NO: 5.

[0048] In the protein of the present invention, ferritin monomers can be derived from ferritins derived from various organisms, and in the case of vertebrates, heavy or light chain monomers can be used. For example, human ferritin heavy chain can be used.

[0049] In the ferritin monomer, the binding position of each domain is not limited as long as it can perform its function as a self-assembled protein, and it may be fused to any one selected from the group consisting of, for example, 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. Preferably, it may be fused to the C-terminus, as it can be expressed externally from the protein and easily exert its function.

[0050] The protein of the present invention may further include a linker between the ferritin monomer and each domain.

[0051] As the linker, any linker known in the art can be used without limitation, for example, S1 (G3SG3TG3SG3), S2 (GKLGGG), etc.

[0052] The protein of the present invention can be obtained, for example, by transforming an organism with a vector comprising a gene encoding a ferritin monomer and a gene encoding an ammonia oxidase active domain selected from amoB1 (Ammonia monooxygenase beta subunit domain 1) and amoB2 (Ammonia monooxygenase beta subunit domain 2), or a butane oxidase active domain selected from bmoB1 (Particulate Butane monooxygenase subunit B domain 1) and bmoB2 (Particulate Butane monooxygenase subunit B domain 2), but is not limited to this.

[0053] The protein of the present invention is highly expressed into a soluble form in microorganisms, and the production yield during biosynthesis is high.

[0054] The present invention also relates to a microorganism that expresses the protein.

[0055] The microorganism of the present invention may be one into which a vector containing a gene encoding a ferritin monomer and a gene encoding an ammonia oxidase active domain selected from amoB1 (Ammonia monooxygenase beta subunit domain 1) and amoB2 (Ammonia monooxygenase beta subunit domain 2) or a butane oxidase active domain selected from bmoB1 (Particulate Butane monooxygenase subunit B domain 1) and bmoB2 (Particulate Butane monooxygenase subunit B domain 2) has been introduced, and which expresses the protein.

[0056] In the protein of the present invention, each domain may be fused to one ferritin monomer, fused to each ferritin monomer, or a mixture of these. Therefore, the genes encoding the ammonia oxidase active domain or the butane oxidase active domain may be contained in one vector or in two vectors.

[0057] The vector may be an expression vector known in the art, including, but not limited to, a BLUESCRIPT vector (Stratagene), a T7 expression vector (Invitrogen), a pET vector (Novagen), and the like.

[0058] The vector may further include additional components known in the art, such as promoters for protein expression, tags for separation / purification, transformation markers, and the like.

[0059] The microorganism is not limited to any particular type, as long as it can express the protein when the vector is introduced into it. For example, Escherichia coli can be used.

[0060] Since the microorganism of the present invention expresses the protein, it can be used to produce methanol by oxidizing methane, and when using the microorganism of the present invention, it is not necessary to add a separate reducing agent to produce methanol.

[0061] The present invention also relates to a composition for producing methanol or a composition for producing butanol, which contains the above-mentioned protein or the above-mentioned microorganism.

[0062] The self-assembled protein of ferritin monomers fused with the ammonia oxidase active domain having methane oxidation activity has methane oxidation activity, and the self-assembled protein of ferritin monomers fused with the butane oxidase active domain has butane oxidation activity, and the microorganism expresses the proteins. Therefore, the composition of the present invention, when it contains the composition, can oxidize methane or butane to produce methanol or butanol.

[0063] Methanol or butanol can be produced by treating the composition with methane gas or butane gas.

[0064] The compositions of the present invention may further comprise a reducing agent used in the oxidation of methane or butane. The reducing agent may be, for example, duroquinol.

[0065] The present invention also relates to a method for producing methanol or butanol, which comprises reacting the aforementioned composition with methane gas or butane gas.

[0066] The composition according to the present invention can be reacted with methane gas or butane gas to oxidize the methane or butane to produce methanol or butanol, which can be done by injecting methane gas or butane gas into the composition to allow the enzymatic reaction to proceed.

[0067] The conditions for producing methanol or butanol are not particularly limited, and can be, for example, at a temperature, pH, etc. that allow the above-mentioned protein or microorganism to exhibit appropriate activity.

[0068] Example 1. Preparation of Expression Vectors for Protein Biosynthesis According to the vector diagram shown in Table 1 below, chimeric AMO (AMO(amoB1) + sMMO (MMOR)) was synthesized by PCR. F )), AMO-mimics (AMO-m1 to AMO-m2), and BMO-mimics (BMO-m1 to BMO-m2) were produced. All plasmid expression vectors were purified on agarose gel and then their sequences were confirmed by complete DNA sequencing.

[0069] The PCR products thus prepared were inserted into pT7-7 and pET28a expression vectors, respectively, to construct expression vectors capable of expressing the respective proteins.

[0070] The expression vectors for each protein were pT7-cAMO-B1, pET28a-cAMO-B2, pET28a-AMO-m1-B1, pT7-AMO-m1-B2, pT7-AMO-m2, pT7-BMO-m1, pET28a-BMO-m2-B1, and pT7-BMO-m2-B2 (Figure 1).

[0071] [Table 1]

[0072] The sequences of each protein (domain) used are shown in Table 2 below.

[0073] [Table 2]

[0074] 2. Recombinant Protein Biosynthesis and Purification E. coli strain BL21(DE3) [F -ompThsdS B (rB - mB - )], pGroBL21(DE3)[F - ompThsdS B (rB - mB - )] were transformed with the expression vectors prepared above. For cAMO and BMO-m2, except for AMO-mimics and BMO-m1, the two expression vectors were simultaneously transformed into Escherichia coli strain BL21, and transformants resistant to ampicillin and kanamycin were selected. The transformed E. coli were cultured in 50 mL of LB (Luria-Bertani) medium (100 mg L). -1 Ampicillin 100mg L -1 The cells were cultured in 250 mL Erlenmeyer flasks containing 0.4 mM kanamycin and 0.4 mM CuSO4 (37°C, 150 rpm).

[0075] AMO-m1 was prepared by simultaneously transforming two expression vectors into pGro7 / BL21, and transformants resistant to ampicillin, kanamycin, and chloramphenicol were selected. The transformed E. coli was cultured in 50 mL of Luria-Bertani (LB) medium (100 mg L). -1 Ampicillin 100mg L -1 Kanamycin, 20 mg L -1 Chloramphenicol, 0.5g L -1 The cultures were grown in flasks (250 mL Erlenmeyer flasks, 37°C, 150 rpm) containing 100 mL of PBS containing arabinose and 0.4 mM CuSO4.

[0076] For AMO-m2 and BMO-m1, the expression vector was transformed into BL21, and ampicillin-resistant transformants were selected. The transformed E. coli was then cultured in 50 mL of Luria-Bertani (LB) medium (100 mg L). -1 The cells were cultured in 250 mL Erlenmeyer flasks containing 0.4 mM CuSO4 (containing ampicillin and 0.4 mM CuSO4) at 37°C and 150 rpm.

[0077] Medium turbidity (OD 600 When the β-actin (β) ratio reached approximately 0.6, 1 mM IPTG (isopropyl-β-D-thiogalactopyranosid) was added to induce gene expression. After 14 hours of incubation at 20°C, the cultured E. coli was centrifuged at 5,000 rpm for 5 minutes to collect the bacterial pellet. The pellet was then suspended in 5 mL of disruption solution (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and disrupted using an ultrasonic disrupter (Branson Ultrasonics Corp., Danbury, CT, USA). The cells were then centrifuged at 13,000 rpm for 10 minutes to separate the supernatant and insoluble aggregates.

[0078] The separated supernatant was first purified by Ni ion exchange using histidine-nickel binding expressed in a recombinant protein. 2+ After performing NTA affinity chromatography, the recombinant protein was concentrated and buffer exchanged to obtain the purified recombinant protein. The details of each step are as follows.

[0079] 1) Ni 2+ -NTA affinity chromatography To purify the recombinant proteins, E. coli cultured as described above was harvested, and the cell pellet was resuspended in 5 mL of disruption solution (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), and the cells were disrupted using an ultrasonic disrupter. The disrupted cell solution was centrifuged at 13,000 rpm for 10 minutes, and the supernatant was separated. Each recombinant protein was then purified using Ni 2+ The samples were separated using an NTA column (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).

[0080] 2) Concentration and buffer exchange Ni2+ Two milliliters of the eluted recombinant protein from NTA affinity chromatography was placed in an ultracentrifugal filter (Amicon Ultra 100K, Millipore, Billerica, MA) and centrifuged at 5,000 rpm until 1 milliliter of solution remained on top of the column. The column was then buffer-exchanged with Tris buffer (20 mM Tris-HCl, 250 mM NaCl, pH 8.0).

[0081] 3. Analysis of Expression Rate and Cytoplasmic Solubility of Recombinant Proteins, Including Produced cAMO-, AMO-, and BMO-mimics After this process, the expression rate and cytoplasmic solubility of the purified recombinant proteins were analyzed by SDS-PAGE. The supernatant (soluble fraction, sol) and insoluble aggregates (insol) obtained by centrifugation of the recombinant protein disruption solution were subjected to SDS-PAGE using 12% Tris-glycine precast gels (Invitrogen, California, USA). The gels were then stained with Coomassie Blue staining solution, and the stained protein bands were analyzed for their expression rate and cytoplasmic solubility using a densitometer (GS-800 Calibrated Densitometer, Bio-Rad, California, USA) (Figure 2).

[0082] 4. Analysis of the Structure of Recombinant Proteins Containing Produced cAMO-, AMO-, and BMO-mimics After the above process, the purified recombinant proteins were imaged using a transmission electron microscope (TEM) to analyze their structure. To obtain stained images of the proteins, electron microscope grids containing air-dried samples were incubated with a 2% (w / v) aqueous uranyl acetate solution at room temperature for 1 hour. Protein images were observed using a Tecnai 20 (FEI, Hillsboro, Oreon, USA) electron microscope operating at 200 kV, confirming the formation of spherical nanoparticles. Furthermore, dynamic light scattering (DLS) analysis confirmed the formation of spherical nanoparticles with sizes of 27.9 ± 4.7 nm for cAMO, 29.8 ± 1.3 nm for AMO-m1, 26.5 ± 1.1 nm for AMO-m2, 17.6 ± 4.9 nm for BMO-m1, and 15.2 ± 4.0 nm for BMO-m2 (Figure 3).

[0083] To analyze the structure of the produced cAMO recombinant protein, we performed X-ray absorption spectroscopy (XAS) and electron paramagnetic resonance (EPR) spectroscopy. For X-ray absorption near-edge structure (XANES), extended X-ray absorption fine structure (EXAFS), and EPR analyses of the protein, samples were solvent-exchanged with Tris buffer and pre-frozen at -80°C for 3 hours. The pre-frozen samples were then lyophilized 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). cAMO EXAFS analysis confirmed the distance information between the copper ion in the active site and the surrounding ligands. In the sample where the methane oxidation reaction had progressed, the ligand distance was different compared to the sample where the reaction had not progressed, and an additional peak (~2.2 Å) was observed. XANES analysis confirmed the presence of a mixture of monovalent and divalent copper ions (Cu(I) and Cu(II)). Furthermore, EPR analysis confirmed the existence of divalent copper ions in a valence-scrambled state (Figure 4a-c).

[0084] 5. Demonstration of methane and butane gas oxidation activity of recombinant proteins containing the produced cAMO, AMO-, and BMO-mimics To verify the methane and butane gas oxidation activity of the purified recombinant protein, 1 mL of recombinant protein solution containing the reducing agent NADH (0.2 mM) or duroquinol (0.35 mM) was injected into a 20 mL septa-sealed vial (catalog no. 5182-0837, Agilent). For the methane and butane oxidation reaction, 19 mL of headspace air was removed using a syringe, and 15 mL of methane or butane gas and 4 mL of air were injected. The vial was then immediately placed in an incubator at 30 °C for up to 24 hours. The amount of methanol or butanol generated by the enzymatic reaction was then measured by gas chromatography (7890B GC, Agilent), and the cumulative production was calculated (Figure 5).

[0085] cAMO 13 To verify the C-methane gas oxidation activity, an enzymatic reaction was carried out in the same manner as the methane oxidation reaction described above, while methane gas was also used. 13 The reaction mixture was purged with C-methane gas. For nuclear magnetic resonance (NMR) analysis, the five vials containing the reaction mixture were heated at 80°C for 15 minutes, and then 19 ml of the headspace gas was directly injected into 600 μL of thoroughly cooled ethanol using a syringe. 60 μL of ethanol-d6 was then added, and the mixture was transferred to an NMR tube (NORS55007, Sigma-Aldrich). The cAMO (oxidation product) generated by the enzymatic reaction was analyzed. 13 C-methanol was confirmed by NMR analysis (Figure 6).

Claims

1. Ferritin monomers fused with an ammonia oxidase active domain with methane oxidation activity self-assemble, The ammonia oxidase active domain is a protein selected from amoB1 (Ammonia monooxygenase beta subunit domain 1) and amoB2 (Ammonia monooxygenase beta subunit domain 2), wherein amoB1 has the amino acid sequence of SEQ ID NO: 1, and amoB2 has the amino acid sequence of SEQ ID NO:

2.

2. The protein of claim 1, wherein amoB1 and amoB2 fused ferritin monomers are self-assembled.

3. The protein of claim 1, wherein amoB1-fused ferritin monomers and amoB2-fused ferritin monomers are self-assembled.

4. The protein of claim 1 , wherein the ferritin monomer is a human ferritin heavy chain monomer.

5. A microorganism expressing the protein according to any one of claims 1 to 4.

6. The microorganism described in claim 5, into which a vector containing a gene encoding a ferritin monomer and a gene encoding an ammonia oxidase active domain selected from amoB1 (Ammonia monooxygenase beta subunit domain 1) and amoB2 (Ammonia monooxygenase beta subunit domain 2) has been introduced.

7. The microorganism of claim 6 , wherein the microorganism is Escherichia coli.

8. A composition for methanol production, comprising the protein according to any one of claims 1 to 4.

9. The composition of claim 8 further comprising a reducing agent.

10. 10. The composition of claim 9, wherein the reducing agent is duroquinol.

11. 10. A method for producing methanol, comprising reacting the composition of claim 8 with methane gas.

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