Novel protein having methane or butane oxidation activity

Abstract

To provide a protein having activity for producing butanol by oxidizing butane, a microorganism expressing the protein, a composition for producing methanol or butanol using the same, and a method for producing methanol or butanol.SOLUTION: The present invention provides a protein in which ferritin monomers, fused with a butane oxidase active domain having butane oxidation activity, are self-assembled. The butane oxidase active domain may be selected from Particulate Butane monooxygenase subunit B_domain 1 (bmoB1) and Particulate Butane monooxygenase subunit B_domain 2 (bmoB2).SELECTED DRAWING: Figure 5

Description

Technical Field

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

Background Art

[0002] Methane monooxygenase (MMO) derived from methanotrophs is a very useful biocatalyst that can catalyze the oxidation reaction of various hydrocarbons (C1-C8) including methane gas under mild conditions of normal temperature and pressure to produce high-value-added products, and the development of bioprocesses utilizing this has attracted worldwide attention.

[0003] In addition, as enzymes of a similar series, ammonia monooxygenase (AMO) derived from Nitrosomonas europaea, butane monooxygenase (BMO) derived from Nocardioides sp. strain CF8, etc. are useful biocatalysts that can catalyze oxidation reactions for a wide range of hydrocarbons (AMO: C1-C10 linear / halogenated hydrocarbons, mono / polycyclic aromatic hydrocarbons, BMO: C2-C10 linear / halogenated hydrocarbons, some aromatic hydrocarbons) through a mechanism similar to that of methane monooxygenase. However, basic research such as the elucidation of the 3D structure, active domain, reaction mechanism, and substrate specificity is very insufficient to date.

[0004] At present, the production of methanol and butanol from methane and butane gas through chemical processes has many problems in terms of technology, environment, and economy, such as complex processes, environmental pollution caused by by-products (such as carbon dioxide and syngas), low reaction conversion rates, and high energy consumption due to high-temperature and high-pressure reaction conditions. In particular, due to problems such as a decrease in economic efficiency caused by high transportation and storage costs of methane gas, and the induction of a serious greenhouse effect during leakage, the production of methanol using small-scale bioplants that can be easily linked to local gas fields is very advantageous in terms of technology and economy.

[0005] Through the development efforts of bioprocesses, attempts have been made to improve the metabolic engineering strains of methane-oxidizing bacteria and various hydrocarbon-degrading bacteria for the production of high-added-value products other than methanol. However, there are problems such as the limitations of the utilization of genetic engineering tools and the difficult culturing properties of the strains. Heterologous expression using industrial strains for the large-scale production of methane monooxygenase and the like is difficult to express water-soluble proteins and requires precise interactions of enzyme complexes, so there are no successful cases of industrial application due to the high technical difficulty.

Summary of the Invention

Problems 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 containing the protein or the microorganism.

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

Means for Solving the Problems

[0010] 1. A protein in which ferritin monomers fused with an ammonia monooxygenase activity domain having methane oxidation activity or a butane monooxygenase activity domain having butane oxidation activity self-assemble.

[0011] 2. The protein according to item 1, wherein the ammonia monooxygenase activity 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 amoB1 consists of the amino acid sequence of SEQ ID NO: 1 and 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 self-assemble.

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

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

[0016] 7. The 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 above, wherein the ferritin monomer in which bmoB1 and bmoB2 are fused self-assembles.

[0018] 9. The protein according to item 6 above, wherein the ferritin monomer fused with bmoB1 and the ferritin monomer fused with bmoB2 self-assemble.

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

[0020] 11. The protein according to item 10 above, wherein each domain is fused to any one selected from the group consisting of inside the α-helix of the ferritin monomer, between adjacent α-helices, N-terminus, C-terminus, AB loop, BC loop, CD loop, DE loop, between the N-terminus and A helix, and between the E helix and 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 above, wherein the microorganism has been introduced with a vector containing a gene encoding a ferritin monomer and a gene encoding a methane monooxygenase active domain selected from amoB1 (Ammonia monooxygenase beta subunit_domain 1) and amoB2 (Ammonia monooxygenase beta subunit_domain 2), or a butane monooxygenase active domain selected from bmoB1 (Particulate Butane monooxygenase subunit B_domain 1) and bmoB2 (Particulate Butane monooxygenase subunit B_domain 2).

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

[0024] 15. A composition for methanol production, comprising the protein according to any one of Items 1 to 11, wherein the protein is a fusion of an ammonia oxidase activity 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 the step of reacting the composition according to Item 15 with methane gas.

[0028] 19. A composition for butanol production, comprising the protein according to any one of Items 1 to 11, wherein the protein is a fusion of a butane oxidase activity 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.

Advantages of the Invention

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

[0033] The protein of the present invention contains a large number of domains having methane or butane oxidation activity, and its activity is high.

[0034] The composition and method of the present invention can produce methanol or butanol in high yields.

Brief Description of the Drawings

[0035]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0036] Hereinafter, the present invention will be described in detail.

[0037] The present invention relates to a protein in which ferritin monomers fused with an ammonia monooxygenase activity domain having methane oxidation activity or a butane monooxygenase activity domain having butane oxidation activity self-assemble.

[0038] The ammonia oxidase activity domain can be used without limitation as long as it has the activity of oxidizing methane. For example, amoB1 (Ammonia monooxygenase beta subunit_domain 1) and amoB2 (Ammonia monooxygenase beta subunit_domain 2) can be used. Specifically, those containing the amino acids of SEQ ID NO: 1 can be used as amoB1, and those containing the amino acid sequence of SEQ ID NO: 2 can be used as amoB2.

[0039] The butane oxidase activity domain can be used without limitation as long as it has the activity of oxidizing butane. For example, bmoB1 (Particulate Butane monooxygenase subunit B_domain 1) and bmoB2 (Particulate Butane monooxygenase subunit B_domain 2) can be used. Specifically, those containing the amino acids of SEQ ID NO: 3 can be used as bmoB1, and those containing the amino acid sequence of SEQ ID NO: 4 can be used as bmoB2.

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

[0041] That is, in the protein of the present invention, two ammonia oxidase activity domains or butane oxidase activity 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 further be fused with an electron transfer domain containing an FAD (flavin adenine dinucleotide) binding domain.

[0043] Methane can be oxidized by the reaction of the following Mathematical Formula 1 to form methanol. However, the protein of the present invention is a self-assembled ferritin monomer in which a methane oxidation active domain and an electron transfer domain containing an FAD (flavin adenine dinucleotide) binding domain are fused, and can perform a methane oxidation reaction using NADH as a reducing agent. In particular, it can utilize the in vivo NADH during the reaction in vivo. Therefore, the use of another reducing agent is unnecessary.

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

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

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

[0047] The electron transfer domain contains an FAD binding domain, which may consist only of the FAD binding domain, may further contain an additional part other than the FAD binding domain in MMOR, may further contain at least a part of the 2Fe-2S domain other than the FAD binding domain, or may contain the FAD binding domain and the 2Fe-2S domain. For example, as the electron transfer domain, one containing the amino acid sequence of SEQ ID NO: 5 can be used.

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

[0049] In the ferritin monomer, if the protein can self-assemble to perform its function, the binding position 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. From the perspective of being exposed outside the protein to easily exert its function, it may preferably be fused to the C-terminus.

[0050] In the protein of the present invention, a linker can further be included between the ferritin monomer and each domain.

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

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

[0053] The protein of the present invention has a high expression rate to the soluble form in microorganisms and a high production yield during biosynthesis.

[0054] In addition, the present invention relates to a microorganism expressing the above 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 activity domain selected from amoB1 (Ammonia monooxygenase beta subunit_domain 1) and amoB2 (Ammonia monooxygenase beta subunit_domain 2), or a butane oxidase activity domain selected from bmoB1 (Particulate Butane monooxygenase subunit B_domain 1) and bmoB2 (Particulate Butane monooxygenase subunit B_domain 2) is introduced and which expresses the protein.

[0056] In the protein of the present invention, each domain may be all fused within one ferritin monomer, may be fused to each ferritin monomer respectively, or may be a mixture thereof. For this reason, the gene encoding the ammonia oxidase activity domain or the butane oxidase activity domain may be contained in one vector, or may be contained in two vectors respectively.

[0057] As the vector, an expression vector known in the art can be used, and examples include, but are not limited to, the BLUESCRIPT vector (Stratagene), the T7 expression vector (Invitrogen), the pET vector (Novagen), and the like.

[0058] The vector can further contain additional components known in the art, such as a promoter for protein expression, a tag for separation / purification, a transformation marker, and the like.

[0059] As the microorganism, as long as it can introduce the vector and express the protein, the type thereof is not limited, and for example, Escherichia coli can be used.

[0060] Since the microorganism of the present invention expresses the protein, it can be used for 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 for generating methanol.

[0061] The present invention also relates to a composition for producing methanol or a composition for producing butanol containing the aforementioned protein or the aforementioned microorganism.

[0062] The protein formed by self-assembly of the ferritin monomer fused with the ammonia oxidase activity domain having the aforementioned methane oxidation activity has methane oxidation activity, the protein formed by self-assembly of the ferritin monomer fused with the butane oxidase activity domain has butane oxidation activity, and the aforementioned microorganism expresses the protein. Therefore, the composition of the present invention can oxidize methane or butane by containing this and produce methanol or butanol.

[0063] The production of methanol or butanol can be carried out by treating the composition with methane gas or butane gas.

[0064] The composition of the present invention can further contain a reducing agent used for 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 including the step of reacting the aforementioned composition with methane gas or butane gas.

[0066] Methanol or butanol can be produced by reacting the composition according to the present invention with methane gas or butane gas to oxidize methane or butane, and this can be carried out by injecting methane gas or butane gas into the aforementioned composition to promote the enzyme reaction.

[0067] The production conditions of methanol or butanol are not particularly limited, and for example, they can be carried out at a temperature, pH, etc. at which the aforementioned protein or microorganism exhibits appropriate activity.

[0068] Examples 1. Production of expression vectors for protein biosynthesis According to the vector schematic diagrams shown in Table 1 below, chimeric AMO (AMO(amoB1)+sMMO(MMOR F ))), AMO-mimics (AMO-m1~AMO-m2), and BMO-mimics (BMO-m1~BMO-m2) were produced. All the plasmid expression vectors produced were purified by agarose gel and then the sequences were confirmed by complete DNA sequencing.

[0069] The PCR products thus produced were sequentially inserted into the pT7-7 and pET28a expression vectors to construct expression vectors capable of expressing each protein.

[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, pT7-BMO-m2-B2 (Figure 1).

[0071] [Table 1]

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

[0073] [Table 2]

[0074] 2. Biosynthesis and purification of recombinant proteins Escherichia coli strain BL21(DE3)[F -ompThsdS B (rB - mB - )], pGroBL21(DE3)[F - ompThsdS B (rB - mB - )] were each transformed with the expression vector prepared as described above. For cAMO, BMO-m2 excluding AMO-mimics and BMO-m1, two expression vectors were simultaneously transformed into Escherichia coli strain BL21, and transformants resistant to ampicillin and kanamycin were selected. The transformed Escherichia coli was cultured in a flask (250 mL Erlenmeyer flasks, 37 °C, 150 rpm) containing 50 mL of LB (Luria-Bertani) medium (containing 100 mg / L -1 ampicillin and 100 mg / L -1 kanamycin, 0.4 mM CuSO4).

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

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

[0077] Culture medium turbidity (O.D. 600 ) 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 Escherichia coli was centrifuged at 5,000 rpm for 5 minutes to collect the cell precipitate, which was then suspended in 5 mL of lysis solution (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and lysed using an ultrasonic disruptor (Branson Ultrasonics Corp., Danbury, CT, USA). After lysis, it was centrifuged at 13,000 rpm for 10 minutes to separate the supernatant and the insoluble aggregates.

[0078] First, the separated supernatant was subjected to Ni 2+ -NTA affinity chromatography using the binding between histidine and nickel fused to the recombinant protein. After concentrating the recombinant protein and performing buffer exchange, the purified recombinant protein was obtained. The details of each step are as follows.

[0079] 1) Ni 2+ -NTA affinity chromatography To purify the recombinant protein, the Escherichia coli cultured in the same manner as described above was collected, and its cell pellet was resuspended in 5 mL of lysis solution (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), and the cells were lysed using an ultrasonic disruptor. After the lysed cell solution was centrifuged at 13,000 rpm for 10 minutes to separate only the supernatant, each recombinant protein was separated using a Ni 2+ -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+ The 2 mL of recombinant protein eluted through Ni-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 top of the column. Then, buffer exchange was performed with Tris buffer (20 mM Tris-HCl, 250 mM NaCl, pH 8.0).

[0081] 3. Analysis of the expression rate and cytoplasmic solubility of the recombinant proteins containing the produced cAMO, AMO−, and BMO-mimics After the above process, the expression rate and cytoplasmic solubility of the purified recombinant proteins were analyzed by SDS-PAGE. For the supernatant (soluble fraction, sol) and insoluble aggregates (insoluble fraction, insol) obtained by centrifuging the disrupted cell lysate of the recombinant proteins, and the purified recombinant proteins, SDS-PAGE was performed using a 12% Tris-glycine precast gel (Tris-glycine precast gel, Invitrogen, California, U.S.A.). Then, the gel was stained with Coomassie Brilliant Blue staining solution, and the expression rate and cytoplasmic solubility of each recombinant protein were analyzed using a densitometer (GS-800 Calibrated Densitometer, Bio-Rad, California, U.S.A.) for the stained protein bands (Figure 2).

[0082] 4. Analysis of the structure of the recombinant proteins containing the produced cAMO, AMO−, and BMO-mimics After the above process, imaging was performed using a transmission electron microscope (TEM) to analyze the structure of the purified recombinant protein. To obtain a stained image of the protein, an electron microscope grid containing a naturally dried sample was incubated with a 2% (w / v) aqueous uranyl acetate solution at room temperature for 1 hour. When the protein image was observed using a Tecnai 20 (FEI, Hillsboro, Oreon, U.S.A.) electron microscope operating at 200 kV, it was confirmed that spherical nanoparticles were formed. Furthermore, by dynamic light scattering (DLS) analysis, it was confirmed that cAMO forms spherical nanoparticles with sizes of 27.9 ± 4.7 nm, AMO-m1 forms spherical nanoparticles with sizes of 29.8 ± 1.3 nm, AMO-m2 forms spherical nanoparticles with sizes of 26.5 ± 1.1 nm, BMO-m1 forms spherical nanoparticles with sizes of 17.6 ± 4.9 nm, and BMO-m2 forms spherical nanoparticles with sizes of 15.2 ± 4.0 nm (Figure 3).

[0083] To analyze the structure of the produced cAMO recombinant protein, X-ray absorption spectroscopy (XAS) and electron paramagnetic resonance (EPR) spectral analysis were performed. For X-ray absorption near-edge structure (XANES), extended X-ray absorption fine structure (EXAFS), and EPR analysis of the protein, the sample solvent-exchanged with Tris buffer was pre-frozen at -80 °C for 3 hours, and the pre-frozen sample was freeze-dried at -110 °C using a freeze dryer (FDU-2100, DRC-1000, EYELA). The XAS analysis was measured by the XAFS beamline (BL11S2) of the Aichi Synchrontron Radiation Center (Aichi). From the analysis results of cAMO EXAFS, the distance information between the copper ions present in the active site and the surrounding ligands was confirmed. In the case of the sample where the methane oxidation reaction proceeded, it was confirmed that the ligand distance was different compared to the sample where the reaction did not proceed, and an additional peak (~2.2 Å) was observed. By XANES analysis, it was confirmed that monovalent and divalent copper ions (Cu(I), Cu(II)) were mixed. Furthermore, by EPR analysis, it was confirmed that divalent copper ions were present in the valence-scrambled state (Figure 4a-c).

[0084] 5. Demonstration of methane and butane gas oxidation activities of recombinant proteins containing the produced cAMO, AMO-, and BMO-mimics To verify the methane and butane gas oxidation activities of the purified recombinant protein, 1 mL of the 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 (catalogue no. 5182-0837, Agilent). For the methane and butane oxidation reactions by the recombinant protein, 19 mL of the headspace air was removed with a syringe, and 15 mL of methane or butane gas and 4 mL of air were injected. Then, the vial was immediately subjected to an enzyme reaction in an incubator at 30 °C for up to 24 hours. And the amount of methanol or butanol, which is the oxidation product generated by the enzyme reaction, was measured by gas chromatography (7890B GC, Agilent), and the cumulative production amount was calculated (Figure 5).

[0085] of cAMO 13 To verify the C-methane gas oxidation activity, an enzyme reaction was carried out in the same manner as the aforementioned methane oxidation reaction, while replacing methane gas with 13 C-methane gas. For nuclear magnetic resonance (NMR) analysis, after heating the five reacted vials at 80 °C for 15 minutes, 19 ml of the headspace gas was directly injected into 600 μl of well-cooled ethanol using a syringe. Then, 60 μl of ethanol-d6 was added, transferred to an NMR tube (NORS55007, Sigma Aldrich), and 13 C-methanol, which is the oxidation product generated (produced) by the cAMO enzyme reaction, was confirmed by NMR analysis (Figure 6).

Claims

1. A protein in which ferritin monomers fused with a butane oxidase active domain having butane oxidation activity are self-assembled.

2. The protein according to claim 1, wherein the butane oxidase activity domain is selected from among bmoB1 (Particulate Butane Monooxygenase Subunit B_domain 1) and bmoB2 (Particulate Butane Monooxygenase Subunit B_domain 2).

3. The protein of claim 2, wherein said bmoB1 consists of the amino acid sequence of SEQ ID NO: 3 and said bmoB2 consists of the amino acid sequence of SEQ ID NO:

4.

4. The protein of claim 2, wherein ferritin monomers fused to bmoB1 and bmoB2 are self-assembled.

5. The protein of claim 2, wherein a ferritin monomer fused with bmoB1 and a ferritin monomer fused with bmoB2 are self-assembled.

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

7. The protein according to claim 1, wherein the butane oxidase activity domain is fused to any one selected from the group consisting of an 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, between the N-terminus and the A helix, and between the E helix and the C-terminus.

8. A microorganism expressing the protein according to any one of claims 1 to 7.

9. The microorganism described in claim 8, into which a vector containing a gene encoding a ferritin monomer and a gene encoding a butane oxidase activity domain selected from bmoB1 (Particulate Butane Monooxygenase Subunit B_domain 1) and bmoB2 (Particulate Butane Monooxygenase Subunit B_domain 2) has been introduced.

10. The microorganism of claim 8 , wherein the microorganism is Escherichia coli.

11. A composition for producing butanol, comprising the protein according to any one of claims 1 to 7.

12. The composition of claim 11 , wherein the composition further comprises a reducing agent.

13. The composition of claim 12, wherein the reducing agent is duroquinol.

14. 12. A method for producing butanol comprising reacting the composition of claim 11 with butane gas.

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