Novel proteins with 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] This 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 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 lineage, such as ammonia monoxygenase (AMO) from Nitrosomonas europaea and butane monooxygenase (BMO) from Nocardioides sp. CF8 (strain CF8), are useful biocatalysts that can catalyze oxidation reactions in a wide range of hydrocarbons (AMO: C1-C10 chain / halogenated hydrocarbons, mono / polycyclic aromatic hydrocarbons; BMO: C2-C10 chain / halogenated hydrocarbons, some aromatic hydrocarbons) through mechanisms similar to methane oxidase. However, basic research on their 3D structure, active domains, reaction mechanisms, and substrate specificity has been severely lacking to date.

[0004] Currently, the production of methanol and butanol through chemical processes of methane and butane gases presents 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. In particular, methane gas production presents significant technical and economic advantages due to its high transportation and storage costs, resulting in reduced economic viability, and the serious greenhouse effect it can cause when released. Therefore, methanol production using small-scale bioplants that can be easily integrated with local gas fields is highly advantageous.

[0005] While bioprocess development efforts have attempted to improve metabolically engineered strains of methane-oxidizing bacteria and various hydrocarbon-degrading 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 using industrial strains for mass production of methane oxidases and other enzymes. 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 Initiative] [Problems that the invention aims to solve]

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

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

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

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

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

[0011] 2. In item 1 above, 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).

[0012] 3. In item 2 above, amoB1 is a protein consisting of the amino acid sequence of SEQ ID NO: 1, and amoB2 is a protein consisting of the amino acid sequence of SEQ ID NO: 2.

[0013] 4. A protein in which ferritin monomers formed by the fusion of amoB1 and amoB2 are self-assembled, as described in item 2 above.

[0014] 5. A protein in which ferritin monomers fused with amoB1 and ferritin monomers fused with amoB2 self-assemble, as described in item 2 above.

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

[0016] 7. In item 6 above, bmoB1 is a protein consisting of the amino acid sequence of SEQ ID NO: 3, and bmoB2 is a protein consisting 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 is self-assembled.

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

[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 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, N-terminus, C-terminus, AB loop, BC loop, CD loop, 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 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 methanol production composition comprising a protein described in any of items 1 to 11 above, wherein the protein is fused with an ammonia oxidase active domain.

[0025] 16. A composition further comprising a reducing agent as described in item 15 above.

[0026] 17. A composition in which, in item 15 above, the reducing agent is duroquinol.

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

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

[0029] 20. In item 19 above, the composition further comprises a reducing agent.

[0030] 21. A composition in which, in item 19 above, the reducing agent is duroquinol.

[0031] 22. A method for producing butanol, comprising the step of reacting the composition described in 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 numerous domains having methane or butane oxidation activity, and this activity is high.

[0034] The compositions and methods of the present invention can produce methanol or butanol in high yield. [Brief explanation of the drawing]

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

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

[0037] The present invention relates to a protein formed by the 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] The ammonia oxidase activity domain can be any domain that has the activity to oxidize methane, such as amoB1 (Ammonia monooxygenase beta subunit domain 1) and amoB2 (Ammonia monooxygenase beta subunit domain 2). Specifically, amoB1 can be a domain containing the amino acids of SEQ ID NO: 1, and amoB2 can be a domain containing the amino acid sequence of SEQ ID NO: 2.

[0039] The butane oxidase activity domain can be any domain that has butane oxidase activity, and for example, bmoB1 (Particulate Butane monooxygenase subunit B_domain 1) and bmoB2 (Particulate Butane monooxygenase subunit B_domain 2) can be used. Specifically, bmoB1 can be a subdomain containing the amino acids of SEQ ID NO: 3, and bmoB2 can be a subdomain containing the amino acid sequence of SEQ ID NO: 4.

[0040] 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.

[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 further have an electron transport domain containing an FAD (flavin adenine dinucleotide) binding domain fused to it.

[0043] 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 containing an FAD (flavin adenine dinucleotide) binding 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.

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

[0045] The electron transport domain includes a 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 constituent elements.

[0047] The electron transport domain includes an FAD-binding domain, which may consist solely of the FAD-binding domain, or may further include additional parts other than 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 can include the amino acid sequence of Sequence ID No. 5.

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

[0049] 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.

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

[0051] As a linker, any known in this field can be used without restriction; 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 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), and obtaining the protein from that organism.

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

[0054] Furthermore, the present invention relates to a microorganism that expresses the aforementioned protein.

[0055] The microorganism of the present invention may be expressed by introducing 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).

[0056] 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 ammonia oxidase active domain or the butane oxidase active domain may be contained in one vector, or in two separate vectors.

[0057] 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.

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

[0059] 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.

[0060] 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.

[0061] Furthermore, the present invention relates to a methanol production composition or a butanol production composition containing the aforementioned protein or microorganism.

[0062] Proteins formed by the self-assembly of ferritin monomers fused with the aforementioned methane-oxidizing ammonia oxidase active domain possessing methane-oxidizing activity have methane-oxidizing activity, and proteins formed by the self-assembly of ferritin monomers fused with the butane oxidase active domain possess butane-oxidizing activity, and the aforementioned microorganisms express the aforementioned proteins. Therefore, by including the composition of the present invention, methane or butane can be oxidized to 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 may further contain a reducing agent used for the oxidation of methane or butane. The reducing agent may be, for example, duroquinol.

[0065] Furthermore, the present invention relates to a method for producing methanol or butanol, which includes 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 the methane or butane, which can be done by injecting methane gas or butane gas into the aforementioned composition and advancing the enzymatic reaction.

[0067] The production conditions for methanol or butanol are not particularly limited and can be, for example, at temperatures and pH levels that allow the aforementioned proteins or microorganisms to exhibit appropriate activity.

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

[0069] 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.

[0070] The expression vectors used 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. Biosynthesis and Purification of Recombinant Proteins E. 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, an 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 (OD) 600 When the saturation level reached approximately 0.6, IPTG (Isopropyl-β-D-thiogalactopyranosid) (1 mM) was added to induce gene expression. After incubation 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.

[0078] First, the separated supernatant liquid was used to analyze the binding of histidine and nickel to the recombinant protein. 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.

[0079] 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).

[0080] 2) Concentration and buffer exchange Ni2+ -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).

[0081] 3. Analysis of the expression rate and cytoplasmic solubility of recombinant proteins, including manufactured 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. 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).

[0082] 4. Structural analysis of recombinant proteins, including manufactured cAMO, AMO-, and BMO-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 200kV, it was confirmed that spherical nanoparticles were formed. Furthermore, dynamic light scattering (DLS) analysis confirmed that the cAMO formed spherical nanoparticles with sizes of 27.9±4.7 nm, AMO-m1 with 29.8±1.3 nm, AMO-m2 with 26.5±1.1 nm, BMO-m1 with 17.6±4.9 nm, and BMO-m2 with 15.2±4.0 nm (Figure 3).

[0083] To analyze the structure of the synthesized cAMO recombinant proteins, X-ray absorption spectroscopy (XAS) and electron paramagnetic resonance (EPR) spectroscopy were performed. 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). cAMO EXAFS analysis revealed the distance between copper ions in the active site and surrounding ligands. 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 4a-c).

[0084] 5. Demonstration of methane and butane gas oxidation activity of recombinant proteins, including manufactured cAMO, AMO-, and BMO-mimics. To verify the methane and butane gas oxidation activity of purified recombinant proteins, 1 mL of recombinant protein solution containing the reducing agent NADH (0.2 mM) or duloquinol (0.35 mM) was injected into a 20 mL septa-sealed vial (catalogue no. 5182-0837, Agilent). For the methane and butane oxidation reaction by the recombinant protein, the air in the 19 mL headspace was removed by syringe, and 15 mL of methane or butane gas and 4 mL of air were injected. The vial was then immediately incubated at 30°C for up to 24 hours to allow the enzymatic reaction to occur. The amount of methanol or butanol, the oxidation product generated by the enzymatic reaction, was 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, the enzymatic reaction was carried out in the same manner as the methane oxidation reaction described above, while methane gas was used. 13 The 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 cAMO enzymatic reaction. 13 14C-methanol was confirmed by NMR analysis (Figure 6).

Claims

1. Ferritin monomers fused with a butane oxidase activity domain possessing butane oxidation activity self-assemble, The butane oxidase active domains are bmoB1 (Particulate Butane monooxygenase subunit B_domain 1) and bmoB2 (Particulate Butane monooxygenase subunit B_domain 2), The bmoB1 polymer consists of the amino acid sequence of SEQ ID NO: 3, and the bmoB2 polymer consists of the amino acid sequence of SEQ ID NO:

4.

2. The polymer according to claim 1, wherein ferritin monomers formed by the fusion of bmoB1 and bmoB2 are self-assembled.

3. The polymer according to claim 1, wherein the ferritin monomers fused with bmoB1 and the ferritin monomers fused with bmoB2 are self-assembled.

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

5. The polymer according to claim 1, wherein the butane oxidase active domain is fused to any one selected from the group consisting of the interior 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, the space between the N-terminus and the A-helix, and the space between the E-helix and the C-terminus.

6. A microorganism that expresses the multimer described in any one of claims 1 to 5.

7. The microorganism according to claim 6, wherein the microorganism is introduced into a vector containing a gene encoding a ferritin monomer and genes encoding bmoB1 (Particulate Butane monooxygenase subunit B_domain 1) and bmoB2 (Particulate Butane monooxygenase subunit B_domain 2).

8. The microorganism according to claim 6, wherein the microorganism is Escherichia coli.

9. A composition for producing butanol, comprising the polymer described in any one of claims 1 to 5.

10. The composition according to claim 9, further comprising a reducing agent.

11. The composition according to claim 10, wherein the reducing agent is duloquinol.

12. A method for producing butanol, comprising the step of reacting the composition according to claim 9 with butane gas.

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