Electron mediator, method for producing electron mediator, and electron transfer method

Abstract

The present invention relates to an electron transporter, a method for producing an electron transporter, and an electron transport method. The electron transporter (30) comprises a modified cyanobacterium (31), wherein the modified cyanobacterium (31) is at least one of: (i) a protein involved in the binding of an outer membrane (5) to a cell wall (4) in the cyanobacterium is inhibited or lost in function, and (ii) a channel protein (18) is expressed that improves the protein permeability of the outer membrane (5), and the modified cyanobacterium performs at least one of supplying electrons to the outside and taking in electrons from the outside.

Description

Technical Field

[0001] The present disclosure relates to an electron transporter comprising modified cyanobacteria, a method for producing the electron transporter, and an electron transport method. Background Art

[0002] The technology development utilizing the phenomenon of electron transfer between microbial cells and extracellular environment has received attention in recent years. For example, the bacteria of Shewanella (Shewanella) genus and Geobacter (Geobacter) genus of known Gram-negative bacteria will release the electrons produced when organic matter is alienated in the cell to the extracellular via the biomolecules with electron transfer function such as cytochrome (non-patent literature 1). It is expected that such phenomenon will be imported into, for example, sewage treatment technology, and the bioelectrochemical system (for example, power generation device) (patent literature 1) that can realize resource recovery from organic wastewater. In addition, the electrons produced in the cell by photosynthesis can be used as microbial solar cells (non-patent literature 3). In addition, for example, it has been reported that the electron transfer efficiency between cyanobacteria and the electrode is improved by modification (non-patent literature 5 and non-patent literature 6) or mediator compounds (non-patent literature 7) of the electrode surface. It should be noted that, known cyanobacteria, in addition to extracellular electron transfer, also produce various useful substances (for example, alcohols, alkanes and fatty acids, etc.) (patent literature 2 and non-patent literature 4) by photosynthesis.

[0003] On the other hand, the development of technology for improving the efficiency of material production by transferring electrons from outside the cells to microbial cells to activate the metabolism of the microbial cells is also being promoted (for example, Non-Patent Document 2).

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Publication No. 2018-142408

[0007] Patent Document 2: Japanese Patent No. 6341676

[0008] Non-patent literature:

[0009] Non-patent document 1: JA Gralnick and DK Newman, “Extracellular respiration”, Molecular Microbiology, Wiley-Blackwell, 2007, Vol. 65, pp. 1-11

[0010] Non-Patent Document 2: Junling Guo et al., “Light-driven fine chemical production in yeast biohybrids”, Science, American Association for the Advancement of Science, 2018, Vol. 362, Issue 6416, pp. 813-816

[0011] Non-Patent Document 3: Alistair J. McCormick et al., “Photosynthetic biofilms in pure culture harness solar energy in a mediatorless bio-photovoltaic cell (BPV) system”, Energy & Environmental Science, Royal Society of Chemistry, 2011, Vol. 4, pp. 4699-4709

[0012] Non-Patent Document 4: Ducat DC et al., “Engineering cyanobacteria to generate high-value products”, Trends in Biotechnology, Elsevier BV, 2011, Vol. 29, pp. 95-103

[0013] Non-Patent Document 5: Hasan K et al., “Photo-electrochemical communication between cyanobacteria (Leptolyngbia sp.) and osmium redox polymer modified electrodes”, Physical Chemistry Chemical Physics, Royal Society of Chemistry, 2014, Vol. 16, pp. 24676-24680

[0014] Non-patent document 6: Jenny Z. Zhang et al., "Photoelectrochemistry ofPhotosystem II in Vitro vs in Vivo", Journal of American Chemical Society, American Chemical Society, 2018, Vol.140, pp.6-9

[0015] Non-patent document 7: Nishio K et al., "Electrochemical detection of circadianre dox rhythm in cyanobacterial cells via extracellular electron transfer", Plant and Cell Physiology, Japanese Society of Plant Physiologists, 2015, Vol.56, pp.1053-1058 Summary of the invention

[0016] Problems to be solved by the invention

[0017] However, in the above-mentioned prior art, the extracellular electron transfer efficiency of cyanobacteria is low, and it is desired to improve the extracellular electron transfer efficiency.

[0018] Therefore, the present disclosure provides an electron transporter having improved efficiency of electron transport to the outside by including a modified cyanobacterium having improved efficiency of extracellular electron transport, a method for producing the electron transporter, and an electron transport method.

[0019] Means for solving problems

[0020] An electron transporter according to one embodiment of the present disclosure comprises a modified cyanobacterium, wherein the modified cyanobacterium is at least one of: (i) a protein involved in the binding of an outer membrane to a cell wall in the cyanobacterium is inhibited or lost in function, and (ii) a channel protein is expressed that improves the protein permeability of the outer membrane, and the modified cyanobacterium performs at least one of supplying electrons to the outside and taking in electrons from the outside.

[0021] Effects of the Invention

[0022] According to the electron transporter and the method for producing the electron transporter disclosed in the present invention, an electron transporter having improved efficiency in electron transport to the outside can be provided. In addition, according to the electron transport method disclosed in the present invention, the efficiency of at least one of supplying electrons from the electron transporter to the outside and receiving electrons from the electron transporter from the outside is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a diagram schematically showing the cell surface layer of cyanobacteria.

[0024] Figure 2 This is a schematic diagram showing an example of the electron carrier according to this embodiment.

[0025] Figure 3 This is a transmission electron microscope observation image of an ultrathin section of the modified cyanobacteria of Example 1.

[0026] Figure 4 yes Figure 3 A magnified image of the dashed area A.

[0027] Figure 5 This is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Example 2.

[0028] Figure 6 yes Figure 5 A magnified image of the dashed area B.

[0029] Figure 7 This is a transmission electron microscope image of an ultrathin section of the modified cyanobacterium of Comparative Example 1.

[0030] Figure 8 yes Figure 7 Magnified view of the dashed area C.

[0031] Fig. 9 This is a graph showing the protein amounts in the culture solutions of the modified cyanobacteria of Example 1, Example 2, Example 3, and Comparative Example 1 (n=3, error bars=SD).

[0032] Fig.10 This is an exploded perspective view schematically showing an example of the configuration of an electrochemical measuring device.

[0033] Fig.11 yes Fig.10 A schematic cross-sectional view taken along section line XI-XI.

[0034] Fig.12 This is a graph showing the results of measuring the current flowing when the culture solution of the modified cyanobacterium of Comparative Example 1 was irradiated with light.

[0035] Fig.13 This is a graph showing the results of measuring the current flowing when the culture solution of the modified cyanobacterium of Example 2 was irradiated with light.

[0036] Fig.14 This is a graph showing the results of measuring the current flowing when the culture solution of the modified cyanobacterium of Example 3 was irradiated with light. DETAILED DESCRIPTION

[0037] (Insights that form the basis of this disclosure)

[0038] In recent years, the development of technology that utilizes the phenomenon of electron transfer between microbial cells and the extracellular environment has attracted attention. For example, it is known that the bacteria of the genus Shewanella and the genus Geobacter of Gram-negative bacteria will release the electrons generated when organic matter is alienated in the cell to the outside of the cell via biological molecules with electron transfer functions such as cytochrome (non-patent document 1). In this way, if the electrons released from the microorganism are received by the external electrode, it can be used as a microbial fuel cell using organic matter as fuel, and it is expected that, for example, it can be used as a power generation device by introducing a sewage treatment process containing organic matter (patent document 1).

[0039] On the other hand, the development of technology for improving the production efficiency of substances by transferring electrons from outside the cells to microbial cells and activating the metabolism of the cells is also in progress. For example, in Non-Patent Document 2, it is reported that by attaching nanoparticles of indium phosphide to yeast cells and allowing the electrons generated by the photoelectric conversion reaction of the nanoparticles to enter the cells, the reducing power used for metabolism in the cells can be supplemented, thereby improving the production efficiency of shikimic acid from glucose raw materials.

[0040] As mentioned above, the use of microorganisms does not rely on chemical fuels and can achieve energy production and material production with low environmental load. Among them, since photosynthetic microorganisms such as cyanobacteria and algae use light as an energy source and can use carbon dioxide (CO2) in the air as a carbon raw material, they are particularly expected to be the next generation of carbon-neutral material production systems.

[0041] Cyanobacteria (cyanobacteria, also referred to as blue bacteria or blue algae) is a group in true bacteria, which produces oxygen by photosynthesis by decomposing water, and fixes CO2 in the air by the energy obtained. It should be noted that cyanobacteria, depending on the type, can also fix nitrogen (N2) in the air. In addition, as the characteristic of cyanobacteria, known growth is fast, light utilization efficiency is high, and owing to compared with other algae species, genetic manipulation is easy, therefore in photosynthetic microorganisms, active research and development has also been carried out about the utilization of cyanobacteria.

[0042] For example, when the property of cyanobacteria to transfer electrons outside the cell is utilized and the electrons generated inside the cyanobacterial cells are received by external electrodes during water electrolysis by photosynthesis, a simple structure including water, cyanobacteria and a pair of electrodes can be used as a microbial solar cell (Non-Patent Document 3).

[0043] In addition, as the example of the material production that has used cyanobacteria, report has alcohols, alkanes and lipid acid etc. (patent documentation 2 and non-patent documentation 4).Although cyanobacteria has high photosynthesis ability, for example, can't obtain sufficient luminous energy during weak light such as at night or evening, rainy day or cloudy day.Therefore, if electronics can be sent into cell from the extracellular of cyanobacteria, then can supplement the insufficient intracellular reducing power during night or weak light from outside, therefore expect to contribute to the raising of material production efficiency.

[0044] It should be noted that, as means for improving the electron transfer efficiency between cyanobacteria and an external electrode, modification of the electrode surface (Non-Patent Documents 5 and 6) and mediator compounds (Non-Patent Document 7) have been reported.

[0045] As described above, the development of technology utilizing extracellular electron transfer of cyanobacteria is expected, but the efficiency of electron transfer is still low, and the development of technology that can further improve the efficiency of extracellular electron transfer is desired.

[0046] The inventors have carried out in-depth research in order to solve the above-mentioned problem, and found that, by making the outer membrane of cyanobacteria partially break away from or improve the material permeability of outer membrane from cell wall, the electron transfer efficiency between the cell and the external electrode of modification cyanobacteria improves.More specifically, find that by the breakaway of the outer membrane of cyanobacteria or the raising of material permeability, can modify cyanobacteria and secrete the intracellular electron transfer body to extracellular and the extracellular electron transfer body is taken into at least one in the cell.Thus, the extracellular electron transfer efficiency of modification cyanobacteria improves, and therefore comprises the electron transfer body of modification cyanobacteria and can carry out efficiently with outside electron transfer.

[0047] Therefore, the present disclosure provides an electron mediator having improved electron transfer efficiency with the outside and a method for producing the electron mediator. In addition, the present disclosure provides an electron transfer method, which improves the efficiency of at least one of supplying electrons from the electron mediator to the outside and accepting electrons from the outside of the electron mediator.

[0048] (Overview of the present disclosure)

[0049] An outline of one embodiment of the present disclosure is as follows.

[0050] An electron transporter according to one embodiment of the present disclosure comprises a modified cyanobacterium, wherein the modified cyanobacterium is at least one of: (i) a protein involved in the binding of an outer membrane to a cell wall in the cyanobacterium is inhibited or lost in function, and (ii) a channel protein is expressed that improves the protein permeability of the outer membrane, and the modified cyanobacterium performs at least one of supplying electrons to the outside and taking in electrons from the outside.

[0051] By above-mentioned (i), in modified cyanobacteria, the combination (such as binding amount and binding force) of cell wall and outer membrane is partially reduced, so outer membrane becomes easy to partially detach from cell wall. Therefore, the electron generated in the cell or the substance or molecule with electron becomes easy to leak outside the outer membrane, i.e., extracellular. In addition, by above-mentioned (ii), in modified cyanobacteria, the protein permeability of outer membrane improves, so the substance permeability of outer membrane improves. Thus, modified cyanobacteria can secrete the electron generated in the cell or the substance or molecule with electron to the extracellular and the extracellular electron or the substance or molecule with electron is taken into at least one in the cell. Therefore, in modified cyanobacteria, the extracellular electron transfer efficiency improves. Therefore, the electron transfer efficiency of an electron carrier of a mode of the present disclosure and the outside improves.

[0052] It should be noted that the external refers to a substance or molecule that exists as an entity different from the electron mediator, for example, a redox substance involved in the transfer of electrons between substances, or a molecule having a redox reaction group.

[0053] For example, in the electron transporter according to one embodiment of the present disclosure, the modified cyanobacterium may generate electrons by receiving light, and release the generated electrons to the outside of the outer membrane.

[0054] Thus, when the modified cyanobacteria receives light, electrons or substances or molecules having electrons are released outside the cell. Therefore, the electron carrier of one embodiment of the present disclosure can generate electrons internally when irradiated with light and supply electrons or substances or molecules having electrons to the outside.

[0055] For example, in the electron mediator of one embodiment of the present disclosure, the modified cyanobacterium may take in electrons existing outside the outer membrane into the inside of the cell wall and utilize the electrons inside the cell wall.

[0056] Thus, the modified cyanobacteria will be present in the electrons outside the cell or substances or molecules with electrons into the cell (cytoplasm), for example, in the process of flowing in the photosynthetic electron transfer system to generate energy (ATP: adenosine triphosphate). Moreover, the modified cyanobacteria use this energy to produce organic matter based on carbon dioxide. Therefore, the electron carrier of one mode of the present disclosure can take in electrons from the outside to the inside and generate energy, producing organic matter such as protein.

[0057] For example, in one embodiment of the electron transporter of the present disclosure, in the above (i), the protein involved in the binding of the outer membrane to the cell wall may be at least one of an SLH (Surface Layer Homology) domain-retaining outer membrane protein and a cell wall-pyruvate modifying enzyme.

[0058] Thus, in modified cyanobacteria, for example, (a) the SLH domain-retaining outer membrane protein bound to the cell wall and the enzyme (i.e., cell wall-pyruvate modification enzyme) that catalyzes the reaction of pyruvate modification of the binding sugar chain on the cell wall surface, at least one of the functions is suppressed or lost, or (b) the expression of at least one of the SLH domain-retaining outer membrane protein and the cell wall-pyruvate modification enzyme is suppressed. Therefore, the binding (i.e., binding amount and binding force) of the SLH domain-retaining outer membrane protein in the outer membrane with the covalent bond type sugar chain on the cell wall surface is reduced. As a result, the outer membrane becomes easy to detach from the cell wall in the part where the outer membrane is weakened in combination with the cell wall. Thus, modified cyanobacteria can secrete the electrons or substances or molecules with electrons in the cell to the outside and the electrons or substances or molecules with electrons in the cell are taken into at least one of the cells, so the extracellular electron transfer efficiency is improved. Therefore, the electron transfer efficiency of an electron carrier of one mode of the present disclosure is improved with the outside.

[0059] For example, in one embodiment of the electron transporter disclosed herein, the SLH domain-retaining outer membrane protein may be Slr1841 composed of the amino acid sequence shown in sequence number 1, NIES970_09470 composed of the amino acid sequence shown in sequence number 2, Anacy_3458 composed of the amino acid sequence shown in sequence number 3, or a protein having more than 50% of the amino acid sequence identical to any of these SLH domain-retaining outer membrane proteins.

[0060] Thus, in the modified cyanobacteria, for example, (a) the function of any one of the SLH domain-maintained outer membrane proteins shown in the above sequence numbers 1 to 3 or more than 50% of the amino acid sequence of the protein identical to any one of these SLH domain-maintained outer membrane proteins is inhibited or lost, or (b) the expression of any one of the SLH domain-maintained outer membrane proteins shown in the above sequence numbers 1 to 3 or more than 50% of the amino acid sequence of the protein identical to any one of these SLH domain-maintained outer membrane proteins is inhibited. Therefore, in the modified cyanobacteria, (a) the function of the SLH domain-maintained outer membrane protein in the outer membrane or the protein having the same function as the SLH domain-maintained outer membrane protein is inhibited or lost, or (b) the expression level of the SLH domain-maintained outer membrane protein in the outer membrane or the protein having the same function as the SLH domain-maintained outer membrane protein is reduced. As a result, in the modified cyanobacteria, the outer membrane becomes easy to partially detach from the cell wall due to the reduced binding amount and binding force of the outer membrane binding domain (e.g., SLH domain) to the cell wall. Therefore, the electron transfer efficiency between the electron mediator of one embodiment of the present disclosure and the outside is improved.

[0061] For example, in one embodiment of the electron transporter disclosed herein, the cell wall-pyruvate modification enzyme may be Slr0688 composed of the amino acid sequence shown in sequence number 4, Synpcc7942_1529 composed of the amino acid sequence shown in sequence number 5, Anacy_1623 composed of the amino acid sequence shown in sequence number 6, or a protein having more than 50% of the amino acid sequence identical to that of any of these cell wall-pyruvate modification enzymes.

[0062] Thus, in the modified cyanobacteria, for example, (a) the function of any one of the cell wall-pyruvate modification enzymes shown in sequence numbers 4 to 6 or more than 50% of the amino acid sequence is inhibited or lost, or (b) the expression of any one of the cell wall-pyruvate modification enzymes shown in sequence numbers 4 to 6 or more than 50% of the amino acid sequence is inhibited. Therefore, in the modified cyanobacteria, (a) the function of the cell wall-pyruvate modification enzyme or a protein having an equivalent function to the enzyme is inhibited or lost, or (b) the expression level of the cell wall-pyruvate modification enzyme or a protein having an equivalent function to the enzyme is reduced. Thus, the covalently bonded sugar chains on the cell wall surface become less susceptible to modification by pyruvic acid, so the binding amount and binding force of the sugar chains of the cell wall to the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane are reduced. As a result, in the modified cyanobacteria, the covalently bonded sugar chains on the cell wall surface become less susceptible to modification by pyruvic acid, so the binding force between the cell wall and the outer membrane is weakened, and the outer membrane becomes easier to partially detach from the cell wall. Therefore, the electron transfer efficiency between the electron transporter of one embodiment of the present disclosure and the outside is improved.

[0063] For example, in the electron transporter of one embodiment of the present disclosure, in (i), the gene expressing the protein involved in the binding of the outer membrane to the cell wall may be deleted or inactivated.

[0064] Thus, in the modified cyanobacteria of above-mentioned (i), because the expression of the protein participating in the combination of cell wall and outer membrane is suppressed, or because the function of this protein is suppressed or lost, so the combination of cell wall and outer membrane (so-called binding amount and bonding force) is partially reduced.As a result, at the part where the combination of outer membrane and cell wall weakens, outer membrane becomes easy to break away from cell wall.Thus, in the modified cyanobacteria of above-mentioned (i), it is possible to secrete intracellular electrons or substances or molecules with electrons to extracellular and extracellular electrons or substances or molecules with electrons into at least one in the cell, so the extracellular electron transfer efficiency improves.Therefore, the electron transfer efficiency of an electron carrier of a mode of the present disclosure and outside improves.

[0065] For example, in the modified cyanobacterium of one embodiment of the present disclosure, the gene expressing the protein involved in the binding of the outer membrane to the cell wall may be at least one of a gene encoding an SLH domain-retaining outer membrane protein and a gene encoding a cell wall-pyruvate modification enzyme.

[0066] Thus, in modified cyanobacteria, at least one gene deletion or inactivation in the gene encoding the SLH domain-maintaining outer membrane protein and the gene encoding the cell wall-pyruvic acid modifying enzyme. Therefore, in modified cyanobacteria, for example, the expression of at least one of (a) SLH domain-maintaining outer membrane protein and the cell wall-pyruvic acid modifying enzyme is suppressed, or the function of at least one of (b) SLH domain-maintaining outer membrane protein and the cell wall-pyruvic acid modifying enzyme is suppressed or lost. Therefore, the combination (i.e., binding amount and binding force) of the SLH domain of the SLH domain-maintaining outer membrane protein in the outer membrane with the covalent bond type sugar chain of the cell wall surface is reduced. Thus, in modified cyanobacteria, at the part where the outer membrane is weakened in combination with the cell wall, the outer membrane becomes easy to detach from the cell wall, so the electrons in the cell or the substances or molecules with electrons become easy to leak out to the extracellular. Therefore, in the electron mediator of one embodiment of the present disclosure, electrons or substances or molecules having electrons in cells of the modified cyanobacterium are easily leaked to the outside of the cells, and thus the efficiency of electron transfer with the outside is improved.

[0067] In one embodiment of the electron transporter disclosed herein, the gene encoding the SLH domain-retaining outer membrane protein may be slr1841 composed of the base sequence shown in sequence number 7, nies970_09470 composed of the base sequence shown in sequence number 8, anacy_3458 composed of the base sequence shown in sequence number 9, or a gene having more than 50% of the base sequence identical to any of these genes.

[0068] Thus, in the modified cyanobacteria, the gene encoding the SLH domain-maintaining outer membrane protein shown in any of the above sequence numbers 7 to 9 or a gene identical to more than 50% of the base sequence of any of these genes is deleted or inactivated. Therefore, in the modified cyanobacteria, (a) the expression of any of the above SLH domain-maintaining outer membrane proteins or proteins with equivalent functions to any of these proteins is inhibited, or (b) the function of any of the above SLH domain-maintaining outer membrane proteins or proteins with equivalent functions to any of these proteins is inhibited or lost. Therefore, in the modified cyanobacteria, the binding amount and binding force of the binding domain (such as the SLH domain) used by the outer membrane to bind to the cell wall are reduced. As a result, the outer membrane becomes easy to partially detach from the cell wall, so that the electrons or substances or molecules with electrons in the cell become easy to leak out of the cell. Thus, the extracellular electron transfer efficiency of the modified cyanobacteria is improved, and therefore, the electron transfer efficiency of the electron carrier of one embodiment of the present invention and the outside is improved.

[0069] For example, in one embodiment of the electron transporter disclosed herein, the gene encoding the cell wall-pyruvate modification enzyme can be slr0688 composed of the base sequence shown in sequence number 10, synpcc7942_1529 composed of the base sequence shown in sequence number 11, anacy_1623 composed of the base sequence shown in sequence number 12, or a gene having more than 50% of the base sequence identical to any of these genes.

[0070] Thus, in the modified cyanobacteria, the gene encoding the cell wall-pyruvate modification enzyme shown in any of the above sequence numbers 10 to 12 or the gene identical to 50% or more of the base sequence of the gene encoding the enzyme of any of these is deleted or inactivated. Therefore, in the modified cyanobacteria, (a) the expression of any of the above cell wall-pyruvate modification enzymes or proteins with equivalent functions to any of these enzymes is inhibited, or (b) the function of any of the above cell wall-pyruvate modification enzymes or proteins with equivalent functions to any of these enzymes is inhibited or lost. Thus, the covalently bonded sugar chains on the cell wall surface become less susceptible to modification by pyruvic acid, so the binding amount and binding force of the sugar chains of the cell wall and the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane are reduced. As a result, in the modified cyanobacteria, the amount of covalently bonded sugar chains on the cell wall surface modified by pyruvic acid is reduced, so the binding force between the cell wall and the outer membrane is weakened, and the outer membrane becomes easy to partially detach from the cell wall. Thus, in the modified cyanobacteria, electrons or substances or molecules having electrons in the cell become easy to leak out of the cell, so the efficiency of extracellular electron transfer is improved. Therefore, in the electron carrier of one embodiment of the present disclosure, the efficiency of electron transfer with the outside is improved.

[0071] For example, in one embodiment of the electron transporter disclosed herein, in the above (ii), the channel protein that improves the protein permeability of the outer membrane can be CppS composed of the amino acid sequence shown in sequence number 13, CppF composed of the amino acid sequence shown in sequence number 14, or a protein having an amino acid sequence that is more than 50% identical to any of these channel proteins.

[0072] Thus, in the modified cyanobacteria of above-mentioned (ii), the channel protein that the protein permeability of outer membrane is improved is expressed, i.e., CppS (sequence number 13) or CppF (sequence number 14) or the protein with equivalent function with any one of these channel proteins.Therefore, in the modified cyanobacteria of above-mentioned (ii), because the protein permeability of outer membrane is improved, the material permeability of outer membrane is improved.As a result, the modified cyanobacteria of above-mentioned (ii) can secrete the electrons in the cell or the substance or molecule with the electrons to the extracellular and the electrons in the extracellular or the substance or molecule with the electrons are taken into at least one in the cell, so the extracellular electron transfer efficiency is improved.Therefore, the electron transfer efficiency of an electron carrier of a mode of the present disclosure and the outside is improved.

[0073] For example, in the electron transporter according to one embodiment of the present disclosure, in the above (ii), a gene encoding a channel protein that improves the protein permeability of the outer membrane may be introduced.

[0074] Thus, in the modified cyanobacteria of above-mentioned (ii), the channel protein that the protein permeability of outer membrane is improved is expressed.Therefore, in the modified cyanobacteria of above-mentioned (ii), because the protein permeability of outer membrane improves, the material permeability of outer membrane improves.As a result, the modified cyanobacteria of above-mentioned (ii) can secrete intracellular electrons or the substance or molecule with electrons to extracellular and the extracellular electrons or the substance or molecule with electrons are taken into at least one in the cell, so the extracellular electron transfer efficiency improves.Therefore, the electron transfer efficiency of an electron carrier of the present disclosure and outside improves.

[0075] For example, in the electron transporter of one embodiment of the present disclosure, the gene encoding the channel protein that improves the protein permeability of the outer membrane may be a gene derived from chloroplasts. For example, the gene encoding the channel protein that improves the protein permeability of the outer membrane may be cppS composed of the base sequence shown in sequence number 15, cppF composed of the base acid sequence shown in sequence number 16, or a gene having a base sequence that is 50% or more identical to any of these genes.

[0076] Thus, in the modified cyanobacteria of above-mentioned (ii), a gene encoding any channel protein shown in above-mentioned sequence number 15 and sequence number 16 or a gene identical with more than 50% of the base sequence of any gene in these is imported. Therefore, in the modified cyanobacteria of above-mentioned (ii), a protein having a function of improving the protein permeability in the outer membrane or a protein having equivalent functions with the protein is expressed. Thus, in the modified cyanobacteria of above-mentioned (ii), the protein permeability of the outer membrane is improved, so the material permeability of the outer membrane is improved. As a result, the modified cyanobacteria of above-mentioned (ii) can secrete the electrons in the cell or the substance or molecule with the electrons outside the cell and the electrons in the cell or the substance or molecule with the electrons outside the cell are taken in at least one, so the extracellular electron transfer efficiency is improved. Therefore, the electron transfer efficiency of an electron carrier of a mode of the present disclosure and the outside is improved.

[0077] In addition, a method for producing an electron transporter according to one embodiment of the present disclosure comprises the step of producing the following modified cyanobacteria, wherein the modified cyanobacteria is at least one of the following: (i) the function of a protein involved in the binding of the outer membrane to the cell wall in the cyanobacteria is inhibited or lost, and (ii) a channel protein is expressed that improves the protein permeability of the outer membrane.

[0078] Thus, the modified cyanobacteria manufactured is at least one of the following: (i) because the combination of cell wall and outer membrane partially weakens, so outer membrane is easily partially separated from the cell wall, and (ii) because the protein permeability of outer membrane improves, so the material permeability of outer membrane improves. Therefore, the modified cyanobacteria can secrete intracellular electrons or substances or molecules with electrons to the extracellular and extracellular electrons or substances or molecules with electrons into at least one of the intracellular. As a result, the extracellular electron transfer efficiency of the modified cyanobacteria manufactured improves. Therefore, according to the manufacture method of the electron transfer body of a mode disclosed herein, it is possible to provide an electron transfer body improved with external electron transfer efficiency.

[0079] In addition, an electron transfer method according to one embodiment of the present disclosure uses any of the above-mentioned electron mediators.

[0080] Thus, the electron transfer body comprises the modified cyanobacteria that the extracellular electron transfer efficiency has improved, therefore can efficiently supply electrons to outside (for example external electrode etc.), and accept electrons from the outside efficiently.Therefore, if use electron transfer body, then the modified cyanobacteria contained in the electron transfer body discharges the intracellular electron transfer material to extracellular, for example can efficiently supply electrons to the external electrode thus and produce electric current.In addition, for example the modified cyanobacteria contained in the electron transfer body can accept electrons from the outside to replace light energy, thereby carries out photosynthesis or respiration.Thus, the modified cyanobacteria contained in the electron transfer body also can produce useful substances such as albumen in the cell and secrete into the extracellular.

[0081] Hereinafter, the embodiments will be described in detail with reference to the drawings.

[0082] It should be noted that the embodiments described below are all general or specific examples. The numerical values, materials, steps, and the order of steps shown in the following embodiments are examples and are not intended to limit the present disclosure. In addition, the constituent elements in the following embodiments that are not recorded in the independent claims representing the highest concept are described as arbitrary constituent elements.

[0083] In addition, each figure is not necessarily a figure for strict illustration. In each figure, the same reference numeral is attached|substantially the same structure, and the overlapping description may be abbreviated or simplified.

[0084] In addition, the numerical ranges hereinbelow do not mean only the strict meaning but also include substantially equivalent ranges, for example, ranges for measuring the amount (eg, number or concentration) of a protein or its range.

[0085] In addition, in this specification, both bacterial body and cell refer to one individual of cyanobacteria.

[0086] (Implementation Method)

[0087] [1. Definition]

[0088] In this specification, the homology of base sequence and amino acid sequence is calculated by BLAST (Basic Local Alignment Search Tool, a search tool based on a local alignment algorithm) algorithm. Specifically, it is calculated by pair-wise analysis using the BLAST program available in the webpage of NCBI (National Center for Biotechnology Information, U.S. National Center for Biotechnology Information) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Information about genes of cyanobacteria and plants and proteins encoded by these genes is disclosed, for example, in the above-mentioned NCBI database and Cyanobase (http: / / genome.microbedb.jp / cyanobase / ). The amino acid sequence of the target protein and the base sequence of the genes encoding these proteins can be obtained from these databases.

[0089] Cyanobacteria, also known as blue algae or blue bacteria, are a group of prokaryotes that capture light energy through chlorophyll, and use the energy obtained to cause charge separation of the reaction center chlorophyll, thereby performing water electrolysis to produce oxygen and simultaneously perform photosynthesis. Cyanobacteria are rich in diversity. For example, in terms of cell shape, there are single-celled species such as Synechocystis sp. PCC 6803 and filamentous species such as Anabaena sp. PCC 7120 in which multiple cells are connected. In terms of growth environment, there are also thermophilic species such as Thermosynechococcus elongatus, marine species such as Synechococcus elongatus, and freshwater species such as Synechocystis. In addition, there are many species such as Microcystis aeruginosa that have gas vesicles and produce toxins, and thylakoid cyanobacteria such as Gloeobacter violaceus that do not have thylakoids but have proteins called light-collecting antennas, namely phycobilisomes, in their cell membranes that have unique characteristics.

[0090] In cyanobacteria, electrons are generated in the cell when water is decomposed by photosynthesis and organic compounds such as sugars synthesized by photosynthesis are dissimilated as their own nutrient sources. The electrons generated by the decomposition of water flow through the photosynthetic electron transport chain on the thylakoid membrane, a membrane structure existing in the cytoplasm, and in this process, a proton driving force is generated, which is used as an energy source for the organism, and is ultimately used to reduce NADP. +The reaction that produces NADPH.

[0091] On the other hand, electrons generated by organic compounds through the dissimilation of organic compounds flow in the respiratory chain existing on the cytoplasmic membrane and the thylakoid membrane, generating the above-mentioned proton driving force in the process, and are ultimately used for the reaction of reducing oxygen (O2) to produce water (H2O). Among them, it is shown that the cytoplasmic membrane and the thylakoid membrane are connected in cyanobacteria (Non-patent Document 8: van de Meene et al., 2006, Arch. Microbiol., 184 (5): 259-270).

[0092] Figure 1 FIG. 2 is a diagram schematically showing the cell surface layer of cyanobacteria. Figure 1 As shown, the cell surface layer of cyanobacteria is composed of a cell membrane (also called an inner membrane 1), a peptidoglycan 2, and an outer membrane 5, which is a lipid membrane forming the outermost layer of the cell, in order from the inside. Sugar chains 3 composed of glucosamine and mannosamine, etc. are covalently bonded to the peptidoglycan 2, and pyruvic acid is bonded to these covalently bonded sugar chains 3 (Non-patent Document 9: Jurgens and Weckesser, 1986, J. Bacteriol., 168: 568-573). In this specification, the interior including the peptidoglycan 2 and the covalently bonded sugar chains 3 is referred to as a cell wall 4. In addition, in the gap between the cell membrane (i.e., the inner membrane 1) and the outer membrane 5, there are various enzymes called periplasm, which participate in the decomposition of proteins or the formation of three-dimensional structures, the decomposition of lipids or nucleic acids, or the intake of nutrients outside the cell.

[0093] The enzyme that catalyzes the pyruvate modification reaction of the covalently bonded sugar chain 3 in the peptidoglycan 2 (hereinafter referred to as the cell wall-pyruvate modification enzyme 9) was identified in Bacillus anthracis, which is a Gram-positive bacterium, and was named CsaB (Non-patent document 10: Mesnage et al., 2000, EMBO J., 19: 4473-4484). Among the cyanobacteria whose genome base sequences have been published, many species retain genes encoding homologous proteins having an amino acid sequence homology of 30% or more to that of CsaB. Examples include slr0688 retained by Synechocystis sp. PCC 6803 and synpcc7502_03092 retained by Synechococcus sp. 7502.

[0094] In addition, the SLH domain-retaining outer membrane protein 6 (slr1841 in the figure) retaining the SLH (Surface layer homologous) domain 7 is bound to the cell wall of cyanobacteria (Non-patent document 11: Kowata et al., 2017, J. Bacteriol., 199: e00371-17). It is known that in the binding of this outer membrane protein to the cell wall, the peptidoglycan-binding sugar chain is modified by pyruvic acid (Non-patent document 12: Kojima et al., 2016, Biosci. Biotech. Biochem., 10: 1954-1959).

[0095] Channel proteins refer to membrane proteins that form a path (i.e., a channel) for selectively permeating a specified substance from the inside to the outside, or from the outside to the inside of a lipid membrane (e.g., outer membrane 5). In the outer membrane of common heterotrophic Gram-negative bacteria such as Escherichia coli and Salmonella, there are a large number of channel proteins called Porin that selectively permeate nutrients of lower molecular weight such as sugars and amino acids from the outside to the inside of the outer membrane and are taken into the cell (Non-patent document 13: Nikaido, 2003, Microbiol. Mol. Biol. Rev., 67 (4): 593-656). On the other hand, Porin does not exist in the outer membrane 5 of cyanobacteria. Instead, ion channel proteins (e.g., SLH domain-retaining outer membrane protein 6) that selectively permeate only inorganic ions exist in large quantities in the outer membrane 5. This ion channel protein accounts for about 80% of the total protein in the outer membrane 5 (Non-patent document 11). Therefore, in cyanobacteria, unless the properties of the outer membrane 5 are significantly changed using techniques such as gene introduction, it is difficult for high molecular weight substances such as proteins to pass through the outer membrane 5 and diffuse outside the cell (ie, outside the outer membrane 5).

[0096] The chloroplasts of plants originated from cyanobacteria that were symbiotic in the cells of primitive eukaryotic cells about 1.5 to 2 billion years ago, and changed into chloroplasts through subsequent evolution (Non-patent document 14: Ponce-Toledo et al., 2017, Curr. Biol., 27(3): 386-391). The chloroplasts maintained by gray algae, which are unicellular algae considered to be the most primitive plants, have peptidoglycan and retain a surface structure very similar to that of cyanobacteria. On the other hand, peptidoglycan does not exist in the chloroplasts of seed plants that are more evolved than unicellular algae. In addition, most of the outer membrane proteins of cyanobacteria were lost from the outer membrane of the chloroplasts in the early stages of the birth of chloroplasts during the process of evolution. Therefore, the structure of the outer membrane proteins of the chloroplasts of gray algae described above is very different from that of the outer membrane proteins of cyanobacteria. For example, the outer membrane 5 of cyanobacteria contains a large number of ion channel proteins that allow inorganic substances to pass through, such as Slr1841 (SLH domain-retaining outer membrane protein 6). This ion channel protein accounts for about 80% of the total protein in the outer membrane 5. On the other hand, in the outer membrane of the chloroplast of gray algae, the outer membrane contains a large amount of channel proteins (hereinafter also referred to as organic channel protein 18) that allow organic substances named CppS and CppF to pass through. This organic channel protein 18 accounts for more than 80% of the total protein in the outer membrane of the chloroplast of gray algae (Non-patent document 15: Kojima et al., 2016, J.Biol.Chem., 291: 20198-20209). CppS and CppF are channel proteins that have a channel function that selectively allows higher molecular weight organic substances (such as biological molecules such as proteins) to pass through, and are believed to function as a substance transport pathway that connects the interior of the chloroplast in the plant cell with the cytoplasm of the plant cell. CppS and CppF are widely distributed in gray algae. On the other hand, in bacteria, similar proteins to CppS and CppF exist only in bacteria belonging to the Planctomycetes phylum. It should be noted that cyanobacteria do not have CppS, CppF, and proteins similar to these (see Non-Patent Document 15).

[0097] [2. Electron transporter]

[0098] Next, the electron carrier of this embodiment will be described. Figure 2 It is a schematic diagram showing an example of the electron carrier 30 according to the present embodiment.

[0099] The electron mediator 30 has the function of supplying electrons to the outside and taking in electrons from the outside. The outside refers to a substance or molecule that exists as an entity different from the electron mediator 30, such as a redox substance related to the transfer of electrons between substances, or a molecule having a redox reaction group.

[0100] Here, donating electrons means not only donating electrons but also donating all substances or molecules having electrons. Also, taking in electrons means not only taking in electrons but also taking in all substances or molecules having electrons.

[0101] The electron transporter 30 of this embodiment includes at least one modified cyanobacterium 31 in which (i) the function of a protein involved in the binding of the outer membrane 5 to the cell wall 4 in the cyanobacterium (the parent bacterium described later) is suppressed or lost, and (ii) a channel protein (so-called organic channel protein 18) is expressed that improves the protein permeability of the outer membrane 5. In addition, the modified cyanobacterium 31 performs at least one of supplying electrons to the outside of the electron transporter 30 and taking in electrons from the outside.

[0102] For example, the electron mediator 30 may be the modified cyanobacterium 31 of (i), the modified cyanobacterium 31 of (ii), or the modified cyanobacterium 31 of both (i) and (ii).

[0103] By above-mentioned (i), in the modified cyanobacteria 31, the combination (such as binding amount and binding force) of the cell wall 4 and the outer membrane 5 is partially reduced, so the outer membrane 5 becomes easy to partially detach from the cell wall 4. Therefore, the electrons generated in the cell of the modified cyanobacteria 31 or the substance or molecule with electrons becomes easy to be exposed outside the outer membrane 5, i.e., outside the cell. In addition, by above-mentioned (ii), in the modified cyanobacteria 31, because the protein permeability of the outer membrane 5 is improved, the substance permeability of the outer membrane 5 is improved. Thus, the modified cyanobacteria 31 can secrete the electrons generated in the cell or the substance or molecule with electrons outside the cell and the electrons or the substance or molecule with electrons outside the cell are taken into at least one of the cells. As a result, the extracellular electron transfer efficiency of the modified cyanobacteria 31 is improved. Therefore, the electron transfer efficiency between the electron carrier 30 of the present embodiment and the outside is improved.

[0104] Furthermore, the electron transporter 30 may include at least one of an electron transport substance 33 , an electron mediator 35 , and a conductive substance 37 in addition to at least one of the modified cyanobacteria 31 described above (i) and (ii).

[0105] For example, Figure 2 As shown, the electron mediator 30 may include the modified cyanobacteria 31, the electron transfer substance 33, the electron mediator 35, and the conductive substance 37. Thus, in the electron mediator 30, the extracellular electron transfer efficiency of the modified cyanobacteria 31 can be further improved.

[0106] Here, the electron transfer substance 33 is a substance that is responsible for the electron transfer reaction, and is a so-called redox substance including an oxidized form that accepts electrons and a reduced form that provides electrons. The electron transfer substance 33 is not particularly limited as long as it is a redox substance related to the electron transfer system in the cell, and may be, for example, a peptide, protein, flavin, quinone, heme iron, non-heme iron such as an iron-sulfur cluster, or copper ions.

[0107] In addition, the electron mediator 35 is a substance that assists or promotes the electron transfer function of the electron transfer substance 33, and is a so-called redox active species. The electron mediator 35 can be, for example, quinones, ferrocene, ferrocyanide, cytochrome, viologen, phenazines, phenoxazines, phenothiazines, ferredoxins and their derivatives, etc., and can also be appropriately selected according to the type of the electron transfer substance 33.

[0108] The conductive material 37 is a material having the property that electrons can easily move within the material, and can be selected from one or more materials selected from carbon-based materials, conductive polymers, semiconductors, and metals. Here, the carbon-based material refers to a material with carbon as a constituent component. For example, the carbon-based material can be carbon powder such as graphite, activated carbon, and carbon black, carbon fiber such as graphite felt, carbon wool, and carbon fabric, carbon nanotubes, carbon plates, carbon paper, or carbon disks.

[0109] In addition, conductive polymer is a general term for polymer compounds having conductivity. As conductive polymer, for example, it can be a single monomer or a polymer of two or more monomers having aniline, aminophenol, diaminophenol, pyrrole, thiophene, p-phenylene, fluorene, furan, acetylene or their derivatives as structural units. More specifically, as conductive polymer, for example, it can be polyaniline, polyaminophenol, polydiaminophenol, polypyrrole, polythiophene, polyfuran, polyacetylene, etc.

[0110] In addition, the conductive material 37 can be a metal or a metal oxide. From the perspective of further improving the current yield, it can be tungsten, tungsten oxide, copper, silver, platinum, gold, niobium, iron, cobalt, titanium, molybdenum, molybdenum oxide, tin, tin oxide, nickel, nickel oxide, or alloys containing them, or their oxides.

[0111] In addition, these structures can also be appropriately selected according to the design of the electron mediator 30 .

[0112] [3. Modification of cyanobacteria]

[0113] Next, the modified cyanobacteria 31 will be described. In the present embodiment, the modified cyanobacteria 31 is included in the electron mediator 30.

[0114] The modified cyanobacteria 31, for example, generates electrons by receiving light, and releases the generated electrons to the outside of the outer membrane 5. Thus, when the modified cyanobacteria 31 receives light, it releases electrons or substances or molecules having electrons to the outside of the cell (i.e., outside the outer membrane 5). Therefore, the electron transporter 30 can generate electrons inside when receiving light, and supply electrons or substances or molecules having electrons to the outside.

[0115] In addition, the modified cyanobacteria 31, for example, takes in electrons existing on the outside of the outer membrane 5 into the inside of the cell wall 4 (i.e., in the cytoplasm), and uses the electrons on the inside of the cell wall 4. Thus, the modified cyanobacteria 31 takes in electrons existing outside the cell or substances or molecules having electrons into the cell (in the cytoplasm), for example, to generate energy (ATP: adenosine triphosphate) in the process of flowing in the photosynthetic electron transfer system. Moreover, the modified cyanobacteria 31 uses this energy to produce organic matter based on carbon dioxide. Therefore, the electron carrier 30 can take in electrons from the outside to the inside to generate energy and produce organic matter such as protein.

[0116] In addition, the modified cyanobacterium 31 is at least one of the following: (i) the function of a protein involved in the binding of the outer membrane 5 to the cell wall 4 in the cyanobacterium (hereinafter also referred to as a binding-related protein) is inhibited or lost, and (ii) a channel protein (so-called organic channel protein 18) is expressed that increases the protein permeability of the outer membrane 5.

[0117] First, the modified cyanobacterium 31 described in (i) above will be described.

[0118] In the above (i), the protein involved in the binding of the outer membrane 5 to the cell wall 4 can be, for example, at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9. In the present embodiment, the function of at least one of the proteins, for example, the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9 of the modified cyanobacterium 31 is inhibited or lost. For example, in the modified cyanobacterium 31, (a) the function of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9 can be inhibited or lost, and (b) at least one of the expression of the SLH domain-retaining outer membrane protein 6 that binds to the cell wall 4 and the expression of the enzyme that catalyzes the pyruvate modification reaction of the bound sugar chain on the surface of the cell wall 4 (i.e., the cell wall-pyruvate modification enzyme 9) can be inhibited.

[0119] Thus, in the modified cyanobacteria 31, for example, (a) the SLH domain-retaining outer membrane protein 6 bound to the cell wall and the enzyme (i.e., the cell wall-pyruvate modification enzyme 9) that catalyzes the reaction of the binding sugar chain on the cell wall surface to be modified by pyruvate are inhibited or lost, or (b) the expression of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9 is inhibited. Therefore, the binding (i.e., the binding amount and binding force) of the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane 5 to the covalently bonded sugar chain 3 on the surface of the cell wall 4 is reduced. As a result, the outer membrane 5 becomes easy to detach from the cell wall 4 in the part where the binding of the outer membrane 5 to the cell wall 4 is weakened. Thus, the modified cyanobacteria 31 can secrete the electron transfer substance 33 inside the cell to the outside of the cell, and at least one of the electron transfer substances 33 outside the cell is taken into the cell, so the extracellular electron transfer efficiency is improved. Therefore, the electron transfer efficiency of the electron carrier 30 of the present embodiment with the outside is improved.

[0120] The type of the parent microorganism of the modified cyanobacteria 31 in the present embodiment, (i) the cyanobacteria before the function of the protein involved in the binding of the outer membrane 5 to the cell wall 4 is inhibited or lost, or (ii) the cyanobacteria before the channel protein (i.e., the organic channel protein 18) that improves the protein permeability of the outer membrane 5 is expressed (hereinafter referred to as the parent bacteria) is not particularly limited, and can be any type of cyanobacteria. For example, the parent cyanobacteria can be the genus Synechocystis, the genus Synechococcus, the genus Anabaena, or the genus Thermosynechococcus, wherein it can be Synechocystis sp. PCC6803, Synechococcus sp. PCC 7942, or Thermosynechococcus elongatus BP-1.

[0121] The amino acid sequences of (i) proteins involved in the binding of the outer membrane 5 to the cell wall 4 and (ii) channel proteins (i.e., organic channel protein 18) that increase the protein permeability of the outer membrane 5 in these parent cyanobacteria, the base sequences of the genes encoding these binding-related proteins, and the locations of the genes on the chromosomal DNA or plasmid can be confirmed through the above-mentioned NCBI database and Cyanobase.

[0122] It should be noted that the protein of the combination of the outer membrane and cell wall that participates in the modification of the cyanobacteria 31 in the present embodiment, which is suppressed in function or lost, can be any protein of the parent cyanobacteria, and is not restricted by the presence position (for example on the chromosomal DNA or on the plasmid) of the gene encoding them.

[0123] For example, when the parent cyanobacteria is of the genus Synechocystis, the SLH domain-maintaining outer membrane protein 6 may be Slr1841, Slr1908, or Slr0042, etc., when the parent cyanobacteria is of the genus Synechococcus, it may be NIES970_09470, etc., when the parent cyanobacteria is of the genus Anabaena, it may be Anacy_5815 or Anacy_3458, etc., when the parent cyanobacteria is of the genus Microcystis, it may be A0A0F6U6F8_MICAE, etc., when the parent cyanobacteria is of the genus Cyanothece, it may be In the case where the parent cyanobacteria is a genus of Leptolyngbya, it can be A0A1Q8ZE23_9CYAN, in the case where the parent cyanobacteria is a genus of Calothrix, A0A1Z4R6U0_9CYAN can be cited, in the case where the parent cyanobacteria is a genus of Nostoc, it can be A0A1C0VG86_9NOSO, in the case where the parent cyanobacteria is a genus of Crocosphaera, it can be B1WRN6_CROS5, in the case where the parent cyanobacteria is a genus of Pleurocapsa, it can be K9TAE4_9CYAN, etc.

[0124] More specifically, the SLH domain-maintaining outer membrane protein 6 may be, for example, Slr1841 (sequence number 1) of Synechocystis sp. PCC6803, NIES970_09470 (sequence number 2) of Synechococcus sp. NIES-970, Anacy_3458 (sequence number 3) of Anabaena cylindrica PCC 7122, etc. In addition, it may be a protein having an amino acid sequence of 50% or more identical to these SLH domain-maintaining outer membrane proteins 6.

[0125] Thus, in the modified cyanobacteria, for example, (a) the function of any one of the SLH domain-maintained outer membrane proteins 6 shown in the above sequence numbers 1 to 3 or a protein whose amino acid sequence is 50% or more identical to any one of these SLH domain-maintained outer membrane proteins 6 is inhibited or lost, or (b) the expression of any one of the SLH domain-maintained outer membrane proteins 6 shown in the above sequence numbers 1 to 3 or a protein whose amino acid sequence is 50% or more identical to any one of these SLH domain-maintained outer membrane proteins 6 is inhibited. Therefore, in the modified cyanobacteria 31, (a) the function of the SLH domain-maintained outer membrane protein 6 in the outer membrane 5 or a protein having the same function as the SLH domain-maintained outer membrane protein 6 is inhibited or lost, or (b) the expression level of the SLH domain-maintained outer membrane protein 6 in the outer membrane 5 or a protein having the same function as the SLH domain-maintained outer membrane protein 6 is reduced. As a result, in the modified cyanobacterium 31 , the amount and strength of the binding domain (eg, SLH domain 7 ) of the outer membrane 5 to the cell wall 4 are reduced, and thus the outer membrane 5 becomes more likely to partially detach from the cell wall 4 .

[0126] It is generally believed that if more than 30% of the amino acid sequence of a protein is identical, the homology of the three-dimensional structure of the protein is high, and therefore the possibility of having the same function as the protein is high. Therefore, as an SLH domain-retaining outer membrane protein 6 whose function is inhibited or lost, for example, it can be a protein or polypeptide composed of an amino acid sequence having an amino acid sequence of more than 40%, preferably more than 50%, more preferably more than 60%, further preferably more than 70%, further preferably more than 80%, and further preferably more than 90% homology with the amino acid sequence of any one of the SLH domain-retaining outer membrane proteins 6 shown in the above sequence numbers 1 to 3, and having the function of binding to the covalently bonded sugar chain 3 of the cell wall 4.

[0127] In addition, for example, cell wall-pyruvate modification enzyme 9 may be Slr0688 when the parent cyanobacterium is of the genus Synechocystis, may be Syn7502_03092 or Synpcc7942_1529 when the parent cyanobacterium is of the genus Synechococcus, may be ANA_C20348 or Anacy_1623 when the parent cyanobacterium is of the genus Anabaena, may be CsaB (NCBI accession ID: TRU80220) when the parent cyanobacterium is of the genus Microcystis, may be CsaB (NCBI accession ID: WP_107667006.1) when the parent cyanobacterium is of the genus Cyanothece, may be CsaB (NCBI accession ID: WP_107667006.1) when the parent cyanobacterium is When it is the genus Spirulina, it can be CsaB (NCBI accession ID: WP_026079530.1) and the like; when the parent cyanobacteria is the genus Calothrix, it can be CsaB (NCBI accession ID: WP_096658142.1) and the like; when the parent cyanobacteria is the genus Nostoc, it can be CsaB (NCBI accession ID: WP_099068528.1) and the like; when the parent cyanobacteria is the genus Crocosphaera, it can be CsaB (NCBI accession ID: WP_012361697.1) and the like; when the parent cyanobacteria is the genus Pleurocapsa, it can be CsaB (NCBI accession ID: WP_036798735) and the like.

[0128] More specifically, the cell wall-pyruvate modification enzyme 9 may be, for example, Slr0688 (SEQ ID NO: 4) of Synechocystis PCC 6803, Synpcc7942_1529 (SEQ ID NO: 5) of Synechococcus PCC 7942, or Anacy_1623 (SEQ ID NO: 6) of Anabaena cylindrica PCC 7122. Alternatively, the protein may be one having an amino acid sequence 50% or more identical to that of the cell wall-pyruvate modification enzyme 9.

[0129] Thus, in the modified cyanobacteria 31, for example, (a) the function of any one of the cell wall-pyruvate modification enzymes 9 shown in the above sequence numbers 4 to 6 or a protein having an amino acid sequence of 50% or more identical to any one of these cell wall-pyruvate modification enzymes 9 is inhibited or lost, or (b) the expression of any one of the cell wall-pyruvate modification enzymes 9 shown in the above sequence numbers 4 to 6 or a protein having an amino acid sequence of 50% or more identical to any one of these cell wall-pyruvate modification enzymes 9 is inhibited. Therefore, in the modified cyanobacteria 31, (a) the function of the cell wall-pyruvate modification enzyme 9 or a protein having an equivalent function to the enzyme is inhibited or lost, or (b) the expression level of the cell wall-pyruvate modification enzyme 9 or a protein having an equivalent function to the enzyme is reduced. Thus, the covalently bonded sugar chain 3 on the surface of the cell wall 4 becomes less susceptible to modification by pyruvate, and thus the binding amount and binding strength of the sugar chain 3 of the cell wall 4 to the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 are reduced. As a result, in the modified cyanobacteria 31, the covalently bonded sugar chains 3 on the surface of the cell wall 4 become less likely to be modified by pyruvic acid, so the binding force between the cell wall 4 and the outer membrane 5 is weakened, and the outer membrane 5 becomes easier to partially detach from the cell wall 4. Therefore, the electron transfer efficiency between the electron transporter 30 of the present embodiment and the outside is improved.

[0130] In addition, as described above, it is considered that if the amino acid sequence of a protein is 30% or more identical, it is highly likely that the protein has the same function as the protein. Therefore, the cell wall-pyruvate modification enzyme 9 whose function is inhibited or lost may be, for example, a protein or polypeptide composed of an amino acid sequence having 40% or more, preferably 50% or more, more preferably 60% or more, further preferably 70% or more, further preferably 80% or more, and further preferably 90% or more homology to the amino acid sequence of any one of the cell wall-pyruvate modification enzymes 9 shown in the above sequence numbers 4 to 6, and having the function of catalyzing the reaction of modifying the covalently bonded sugar chain 3 of the peptidoglycan 2 of the cell wall 4 with pyruvate.

[0131] In addition, in the present specification, inhibiting or losing the function of the SLH domain-retaining outer membrane protein 6 means inhibiting or losing the ability of the protein to bind to the cell wall 4, inhibiting or losing the transport of the protein to the outer membrane 5, or inhibiting or losing the ability of the protein to embed into the outer membrane 5 and perform its function.

[0132] In addition, suppressing or losing the function of the cell wall-pyruvate modification enzyme 9 means suppressing or losing the function of the protein to modify the covalently bonded sugar chain 3 of the cell wall 4 with pyruvate.

[0133] The means for inhibiting or losing the functions of these proteins are not particularly limited as long as they are means commonly used for inhibiting or losing the functions of proteins. Such means may be, for example, deletion or inactivation of the gene encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9, inhibition of transcription of these genes, inhibition of translation of the transcription products of these genes, or administration of inhibitors that specifically inhibit these proteins.

[0134] In this embodiment, in the above (i), in the modified cyanobacteria 31, the gene for expressing the protein involved in the binding between the outer membrane 5 and the cell wall 4 may be deleted or inactivated.

[0135] Thus, in the modified cyanobacteria 31 of above-mentioned (i), because the expression of the protein participating in the combination of cell wall 4 and outer membrane 5 is suppressed, or the function of this protein is suppressed or lost, so the combination (so-called binding amount and binding force) of cell wall 4 and outer membrane 5 is partially reduced. As a result, at the part where the combination of outer membrane 5 and cell wall 4 weakens, outer membrane 5 becomes easy to break away from cell wall 4. Thus, in the modified cyanobacteria 31 of above-mentioned (i), it is possible to secrete the electrons in the cell or the substance or molecule with the electrons outside the cell and to take in the electrons in the cell or the substance or molecule with the electrons outside the cell, so the extracellular electron transfer efficiency is improved. Therefore, the electron transfer efficiency of the electron carrier 30 of the present embodiment and the outside is improved.

[0136] The gene that expresses the protein involved in the binding of the outer membrane 5 to the cell wall 4 can be at least one of the genes encoding the SLH domain-maintaining outer membrane protein 6 and the genes encoding the cell wall-pyruvate modification enzyme 9. In the modified cyanobacteria 31, at least one of the genes encoding the SLH domain-maintaining outer membrane protein 6 and the genes encoding the cell wall-pyruvate modification enzyme 9 is deleted or inactivated. Therefore, in the modified cyanobacteria 31, for example, (a) the expression of at least one of the SLH domain-maintaining outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9 is inhibited, or (b) the function of at least one of the SLH domain-maintaining outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9 is inhibited or lost. Therefore, the binding (i.e., the binding amount and binding strength) of the SLH domain 7 of the SLH domain-maintaining outer membrane protein 6 in the outer membrane 5 to the covalently bonded sugar chain 3 on the surface of the cell wall 4 is reduced. Thus, in the modified cyanobacteria 31, the outer membrane 5 becomes easy to detach from the cell wall 4 at the portion where the binding between the outer membrane 5 and the cell wall 4 becomes weak, so that the electrons in the cell or the substance or molecule having electrons becomes easy to leak out of the cell. Therefore, in the electron transporter 30 of the present embodiment, the electrons in the cell or the substance or molecule having electrons becomes easy to leak out of the cell in the modified cyanobacteria 31, so the efficiency of electron transfer with the outside is improved.

[0137] In this embodiment, in order to inhibit or lose the function of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9 in cyanobacteria, for example, the transcription of at least one of the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modification enzyme 9 can be inhibited.

[0138] For example, the gene encoding the SLH domain-maintaining outer membrane protein 6 may be slr1841, slr1908 or slr0042 when the parent cyanobacteria is of the genus Synechocystis, may be nies970_09470 when the parent cyanobacteria is of the genus Synechococcus, may be anacy_5815 or anacy_3458 when the parent cyanobacteria is of the genus Anabaena, may be A0A0F6U6F8_MICAE when the parent cyanobacteria is of the genus Microcystis, may be A0A0F6U6F8_MICAE when the parent cyanobacteria is of the genus Cyanothece , can be A0A3B8XX12_9CYAN etc., when the parent cyanobacteria is a genus of Leptolyngbya, can be A0A1Q8ZE23_9CYAN etc., when the parent cyanobacteria is a genus of Calothrix, can be A0A1Z4R6U0_9CYAN etc., when the parent cyanobacteria is a genus of Nostoc, can be A0A1C0VG86_9NOSO etc., when the parent cyanobacteria is a genus of Crocosphaera, can be B1WRN6_CROS5 etc., when the parent cyanobacteria is a genus of Pleurocapsa, can be K9TAE4_9CYAN etc. The base sequences of these genes can be obtained from the above-mentioned NCBI database or Cyanobase.

[0139] More specifically, the gene encoding the SLH domain-retaining outer membrane protein 6 can be slr1841 (sequence number 7) of Synechocystis PCC 6803, nies970_09470 (sequence number 8) of Synechococcus NIES-970, anacy_3458 (sequence number 9) of Anabaena cylindrica PCC 7122, or a gene whose base sequence is more than 50% identical to these genes.

[0140] Thus, in the modified cyanobacteria 31, the gene encoding any one of the SLH domain-maintaining outer membrane proteins 6 shown in the above sequence numbers 7 to 9 or a gene identical to 50% or more of the base sequence of any one of these genes is deleted or inactivated. Therefore, in the modified cyanobacteria 31, (a) the expression of any one of the SLH domain-maintaining outer membrane proteins 6 or a protein having the same function as any one of these proteins is inhibited, or (b) the function of any one of the SLH domain-maintaining outer membrane proteins 6 or a protein having the same function as any one of these proteins is inhibited or lost. Therefore, in the modified cyanobacteria 31, the binding amount and binding force of the binding domain (such as SLH domain 7) of the outer membrane 5 for binding to the cell wall 4 are reduced. As a result, the outer membrane 5 becomes easy to partially detach from the cell wall 4, so that the electron transfer substance 33 in the cell becomes easy to leak out of the cell. Thus, the extracellular electron transfer efficiency of the modified cyanobacteria 31 is improved, so the electron transfer efficiency of the electron carrier 30 of the present embodiment with the outside is improved.

[0141] As described above, if more than 30% of the amino acid sequence of a protein is identical, there is a high possibility that it has the same function as the protein. Therefore, it is believed that if more than 30% of the base sequence of the gene encoding the protein is identical, there is a high possibility that a protein with the same function as the protein is expressed. Therefore, as a gene encoding an SLH domain-retaining outer membrane protein 6 whose function is inhibited or lost, for example, it can be composed of a base sequence having a homology of more than 40%, preferably more than 50%, more preferably more than 60%, further preferably more than 70%, further preferably more than 80%, and further preferably more than 90% with the base sequence of any one of the genes encoding the SLH domain-retaining outer membrane protein 6 shown in the above-mentioned sequence numbers 7 to 9, and encoding a protein or polypeptide having the function of binding to the covalently bonded sugar chain 3 of the cell wall 4.

[0142] In addition, for example, the gene encoding cell wall-pyruvate modification enzyme 9 may be slr0688 when the parent cyanobacterium is of the genus Synechocystis, syn7502_03092 or synpcc7942_1529 when the parent cyanobacterium is of the genus Synechococcus, ana_C20348 or anacy_1623 when the parent cyanobacterium is of the genus Anabaena, csaB (NCBI accession ID: TRU80220) when the parent cyanobacterium is of the genus Microcystis, csaB (NCBI accession ID: WP_107667006.1) when the parent cyanobacterium is of the genus Cyanothece, and csaC (NCBI accession ID: WP_107667006.2) when the parent cyanobacterium is of the genus Cyanothece. When the bacterium is of the genus Spirulina, it may be csaB (NCBI accession ID: WP_026079530.1), etc., when the parent cyanobacteria is of the genus Calothrix, it may be csaB (NCBI accession ID: WP_096658142.1), etc., when the parent cyanobacteria is of the genus Nostoc, it may be csaB (NCBI accession ID: WP_099068528.1), etc., when the parent cyanobacteria is of the genus Crocosphaera, it may be csaB (NCBI accession ID: WP_012361697.1), etc., when the parent cyanobacteria is of the genus Pleurocapsa, it may be csaB (NCBI accession ID: WP_036798735), etc. The base sequences of these genes can be obtained from the above-mentioned NCBI database or Cyanobase.

[0143] More specifically, the gene encoding cell wall-pyruvate modification enzyme 9 may be slr0688 (SEQ ID NO: 10) of Synechocystis sp. PCC6803, synpcc 7942_1529 (SEQ ID NO: 11) of Synechococcus sp. PCC 7942, or anacy_1623 (SEQ ID NO: 12) of Anabaena cylindrica PCC 7122. Alternatively, the gene may be a gene having 50% or more of the same base sequence as these genes.

[0144] Thus, in the modified cyanobacteria 31, the gene encoding any one of the cell wall-pyruvate modification enzymes 9 shown in the above sequence numbers 10 to 12 or a gene that is identical to 50% or more of the base sequence of the gene encoding any of these enzymes is deleted or inactivated. Therefore, in the modified cyanobacteria 31, (a) the expression of any one of the cell wall-pyruvate modification enzymes 9 or a protein having an equivalent function to any one of these enzymes is inhibited, or (b) the function of any one of the cell wall-pyruvate modification enzymes 9 or a protein having an equivalent function to any one of these enzymes is inhibited or lost. Thus, the covalently bonded sugar chain 3 on the surface of the cell wall 4 becomes less susceptible to modification by pyruvate, and thus the binding amount and binding force of the sugar chain 3 of the cell wall 4 to the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 are reduced. As a result, in the modified cyanobacteria 31, the amount of the covalently bonded sugar chain 3 on the surface of the cell wall 4 modified by pyruvic acid is reduced, so the binding force between the cell wall 4 and the outer membrane 5 is weakened, and the outer membrane 5 becomes easy to partially detach from the cell wall 4. Thus, in the modified cyanobacteria 31, the electrons in the cell or the substances or molecules with electrons become easy to leak out of the cell, so the extracellular electron transfer efficiency is improved. Therefore, the electron transfer efficiency between the electron carrier 30 of the present embodiment and the outside is improved.

[0145] As described above, it is considered that if the base sequence of the gene encoding the protein is 30% or more identical, there is a high possibility that a protein having the same function as the protein is expressed. Therefore, as a gene encoding a cell wall-pyruvate modification enzyme 9 whose function is suppressed or lost, for example, it can be composed of a base sequence having a homology of 40% or more, preferably 50% or more, more preferably 60% or more, further preferably 70% or more, further preferably 80% or more, and further preferably 90% or more to any of the base sequences of the genes encoding the cell wall-pyruvate modification enzyme 9 shown in the above sequence numbers 10 to 12, and encoding a protein or polypeptide having a function of catalyzing the reaction of the covalently bonded sugar chain 3 of the peptidoglycan 2 of the cell wall 4 modified with pyruvate.

[0146] Next, the modified cyanobacterium 31 of (ii) above will be described.

[0147] The modified cyanobacterium 31 of (ii) above expresses the organic channel protein 18 which improves the protein permeability of the outer membrane 5 .

[0148] Here, expressing the organic channel protein 18 in the outer membrane 5 of the cyanobacterium means inserting a gene encoding the organic channel protein 18 into the chromosomal DNA or plasmid of the cyanobacterium, and transporting the organic channel protein 18 synthesized by transcription and translation of the gene to the outer membrane 5, so as to express a channel function that selectively allows the protein to permeate in the outer membrane 5 of the cyanobacterium. The means for inserting and expressing the gene are not particularly limited as long as they are commonly used means, and are not limited by the base sequence of the promoter for transcriptional activation, the ribosome binding sequence for translation, and the type of the signal sequence for transport to the outer membrane 5.

[0149] In the present embodiment, the organic channel protein 18 expressed in the outer membrane 5 of cyanobacteria may also be an outer membrane channel protein derived from chloroplasts. The organic channel protein 18 may be, for example, CppS (sequence number 13) or CppF (sequence number 14) of the gray alga Cyanophora paradoxa (blue strange diatom, hereinafter also referred to as C. paradoxa). In addition, the organic channel protein 18 may also be a protein whose amino acid sequence is more than 50% identical to that of CppS or CppF. It should be noted that proteins whose amino acid sequence is more than 50% identical to that of CppS or CppF are not limited to proteins derived from chloroplasts, and may, for example, be similar proteins of CppS or CppF derived from microorganisms such as bacteria.

[0150] Thus, in the modified cyanobacteria 31 of the above-mentioned (ii), CppS (sequence number 13) or CppF (sequence number 14) as the organic channel protein 18 that improves the protein permeability of the outer membrane 5, or a protein having the same function as any one of the organic channel proteins 18 thereof, is expressed. Therefore, the modified cyanobacteria 31 of the above-mentioned (ii) is due to the improvement of the protein permeability of the outer membrane 5, so the material permeability of the outer membrane 5 is improved. As a result, the modified cyanobacteria 31 of the above-mentioned (ii) can secrete the electrons in the cell or the substance or molecule with the electrons outside the cell and the electrons outside the cell or the substance or molecule with the electrons are taken into at least one of the cells, so the extracellular electron transfer efficiency is improved. Therefore, the electron transfer efficiency of the electron carrier 30 of the present embodiment and the outside is improved.

[0151] It is generally believed that if more than 30% of the amino acid sequence of a protein is identical, the homology of the three-dimensional structure of the protein is high, and therefore the possibility of having the same function as the protein is high. Therefore, as the organic channel protein 18, for example, it can be a protein or polypeptide having a function of improving the protein permeability of the outer membrane 5 and being composed of an amino acid sequence having 40% or more, preferably 50% or more, more preferably 60% or more, further preferably 70% or more, further preferably 80% or more, and further preferably 90% or more homology with the amino acid sequence of any one of the proteins shown in the above-mentioned sequence numbers 13 and 14.

[0152] In addition, in the present embodiment, in the above-mentioned (ii), the modified cyanobacteria 31 can also be introduced with a gene encoding an organic channel protein 18 that improves the protein permeability of the outer membrane 5. Thus, in the modified cyanobacteria 31 of the above-mentioned (ii), the organic channel protein 18 that improves the protein permeability of the outer membrane 5 is expressed. Therefore, in the modified cyanobacteria 31 of the above-mentioned (ii), because the protein permeability of the outer membrane 5 is improved, the material permeability of the outer membrane 5 is improved. As a result, the modified cyanobacteria 31 of the above-mentioned (ii) can secrete the electrons in the cell or the substance or molecule with the electrons outside the cell and the electrons outside the cell or the substance or molecule with the electrons are taken into at least one of the cells, so the extracellular electron transfer efficiency is improved. Therefore, the electron transfer efficiency of the electron carrier 30 of the present embodiment and the outside is improved.

[0153] The above-mentioned gene can be, for example, a gene derived from chloroplasts. The gene encoding the organic channel protein 18 derived from chloroplasts can be, for example, cppS (sequence number 15) or cppF (sequence number 16) of the gray alga Cyanophora paradoxa. In addition, the organic channel protein 18 can also be a gene having a base sequence of more than 50% and the same as any one of these genes. Thus, in the modified cyanobacteria 31 of the above (ii), a gene encoding the organic channel protein 18 shown in any one of the above sequence numbers 15 and 16 or a gene having a base sequence of more than 50% and the same as any one of these genes is introduced. Therefore, in the modified cyanobacteria 31 of the above (ii), a protein having a function of improving the protein permeability in the outer membrane 5 or a protein having equivalent functions to the protein is expressed. Thus, in the modified cyanobacteria 31 of the above (ii), the protein permeability of the outer membrane 5 is improved, so the material permeability of the outer membrane 5 is improved. As a result, the modified cyanobacteria 31 of (ii) above can secrete the electrons in the cell or the substance or molecule with the electrons outside the cell and take in the electrons outside the cell or the substance or molecule with the electrons inside the cell, so the extracellular electron transfer efficiency is improved. Therefore, the electron transfer efficiency between the electron carrier 30 of the present embodiment and the outside is improved.

[0154] In addition, the gene encoding the organic channel protein 18 is not limited to the gene derived from chloroplasts. The gene encoding the organic channel protein 18 may be, for example, a gene consisting of a base sequence having 40% or more, preferably 50% or more, more preferably 60% or more, further preferably 70% or more, further preferably 80% or more, and further preferably 90% or more homology to the base sequence of any one of the above-mentioned genes cppS (SEQ ID NO. 15) and cppF (SEQ ID NO. 16), and encoding a protein or polypeptide having a function of improving the protein permeability of the outer membrane 5.

[0155] [4. Method for producing electron carrier]

[0156] Next, the method for producing the electron transporter 30 of the present embodiment is described. The method for producing the electron transporter 30 includes the steps of producing the modified cyanobacteria 31 (hereinafter referred to as the step of producing the modified cyanobacteria 31), wherein (i) the function of the protein involved in the binding of the outer membrane 5 to the cell wall 4 in the cyanobacteria is suppressed or lost, and (ii) at least one of the organic channel proteins 18 that improve the protein permeability of the outer membrane 5 is expressed.

[0157] The following is an explanation of the steps for producing the modified cyanobacteria 31. The steps for producing the modified cyanobacteria 31 include: (i) a step of inhibiting or losing the function of a protein involved in the binding of the outer membrane 5 to the cell wall 4 in the cyanobacteria, and (ii) a step of expressing an organic channel protein 18 that increases the protein permeability of the outer membrane 5.

[0158] In the above (i), the protein involved in the binding between the outer membrane 5 and the cell wall 4 may be, for example, at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 .

[0159] It should be noted that the means for inhibiting or losing the function of a protein is not particularly limited, and may include, for example, deleting or inactivating the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9, inhibiting the transcription of these genes, inhibiting the translation of the transcription products of these genes, or administering inhibitors that specifically inhibit these proteins.

[0160] The means for deleting or inactivating the above-mentioned gene may be, for example, the introduction of mutations in one or more bases in the base sequence of the gene, the replacement of the base sequence with other base sequences or the insertion of other base sequences, or the deletion of part or all of the base sequence of the gene.

[0161] Means for inhibiting the transcription of the above-mentioned gene may be, for example, introduction of mutations into the promoter region of the gene, inactivation of the promoter by substitution to other base sequences or insertion of other base sequences, or CRISPR interference method (non-patent document 16: Yao et al., ACS Synth. Biol., 2016, 5: 207-212), etc. Specific methods for the above-mentioned introduction of mutations, or substitution or insertion of base sequences may be, for example, ultraviolet irradiation, site-specific introduction of mutations, or homologous recombination method, etc.

[0162] In addition, the means for inhibiting the translation of the transcription product of the above gene may be, for example, RNA (ribonucleic acid) interference method.

[0163] By using any of the above means, the modified cyanobacteria 31 can be produced by inhibiting or losing the function of the protein involved in the binding of the outer membrane 5 and the cell wall 4 in the cyanobacteria. As a result, the binding (i.e., the binding amount and binding force) between the cell wall 4 and the outer membrane 5 of the modified cyanobacteria 31 produced in the above step (i) is partially reduced, so that the outer membrane 5 becomes easy to partially detach from the cell wall 4. Therefore, in the modified cyanobacteria 31, the electrons or substances or molecules having electrons in the cell become easy to leak out of the cell, so the extracellular electron transfer efficiency is improved.

[0164] In the above (ii), the organic channel protein 18 that improves the protein permeability of the outer membrane 5 is, for example, a channel protein derived from chloroplasts, and specifically, it can be CppS composed of the amino acid sequence shown in SEQ ID NO: 13, or CppF composed of the amino acid sequence shown in SEQ ID NO: 14. In addition, the organic channel protein 18 can also be a protein having an amino acid sequence that is 50% or more identical to any of these channel proteins.

[0165] In the step of expressing the organic channel protein 18, first, a gene encoding the organic channel protein 18 that improves the protein permeability of the outer membrane 5 is inserted into the chromosomal DNA or plasmid of the cyanobacterium. Then, the organic channel protein 18 synthesized by transcription and translation of the gene is transported to the outer membrane 5, and the channel function is expressed in the outer membrane 5 of the cyanobacterium. Among them, the means of gene insertion and expression are not particularly limited as long as they are commonly used means, and are not limited by the base sequence of the promoter used for transcriptional activation, the ribosome binding sequence used for translation, and the type of the signal sequence used for transport to the outer membrane 5.

[0166] In the above manner, the organic channel protein 18 that improves the protein permeability of the outer membrane 5 can also be expressed to produce the modified cyanobacteria 31. As a result, the protein permeability of the outer membrane 5 of the modified cyanobacteria 31 produced in the above step (ii) is improved, so the material permeability of the outer membrane 5 is improved. As a result, the modified cyanobacteria 31 can secrete electrons or substances or molecules having electrons in the cell to the outside of the cell, and take in electrons or substances or molecules having electrons outside the cell, so the extracellular electron transfer efficiency is improved.

[0167] By the above steps, the following modified cyanobacteria 31 can be produced: (i) the function of the protein involved in the binding of the outer membrane 5 and the cell wall 4 in the cyanobacterium is inhibited or lost, and (ii) at least one of the organic channel proteins 18 that improve the protein permeability of the outer membrane 5 is expressed.

[0168] Thus, the modified cyanobacteria 31 manufactured by the above method can secrete the electrons in the cell or the substance or molecule with the electrons outside the cell and take in the electrons in the cell or the substance or molecule with the electrons outside the cell, so the extracellular electron transfer efficiency is improved. Therefore, according to the manufacturing method of the electron carrier 30 of the present embodiment, it is possible to provide an electron carrier 30 with improved electron transfer efficiency with the outside.

[0169] [5. Electron transfer method]

[0170] The electron transfer method of the present embodiment uses an electron mediator 30 including any of the modified cyanobacteria 31 described above.

[0171] The electron transporter 30 comprises a modified cyanobacterium 31 in which (i) the function of a protein involved in the binding of the outer membrane 5 to the cell wall 4 in the cyanobacteria is inhibited or lost, and (ii) at least one of the organic channel proteins 18 that express the improved protein permeability of the outer membrane 5 is expressed, and the modified cyanobacterium 31 performs at least one of (I) supplying electrons to the outside and (II) obtaining electrons from the outside.

[0172] As described above, in this embodiment, electrons are electrons or substances or molecules having electrons. In addition, donating electrons means not only donating electrons but also donating all substances or molecules having electrons. In addition, taking in electrons means not only taking in electrons but also taking in all substances or molecules having electrons.

[0173] In addition, the electron transporter 30 may be at least one of the modified cyanobacteria 31 in (i) and (ii) above, such as Figure 2 As shown, in addition to the modified cyanobacteria 31 , at least one of an electron transfer substance 33 , an electron mediator 35 , and a conductive substance 37 may be included.

[0174] First, the above-mentioned (I) supplying electrons to the outside is described. The modified cyanobacteria 31 supplying electrons to the outside means, for example, that the modified cyanobacteria 31 receives light and generates electrons or a substance or molecule having electrons. The substance or molecule having electrons may be, for example, the electron transfer substance 33.

[0175] In addition, modified cyanobacteria 31 can also release the generated electronics or the material or molecule with electronics outside the outer membrane.For example, modified cyanobacteria 31 can also release a part of the electron transfer material 33 participating in the photosynthesis electron transport chain to the extracellular (i.e. outside the outer membrane 5). The electron transfer material 33 released to the extracellular can be, for example, taken into the cell of other modified cyanobacteria 31 and participate in the generation of the organism energy source, and can also cause the movement of electrons via other multiple modified cyanobacteria 31.And, the released electron transfer material 33 can also supply electrons to the electrode by the redox reaction with the outside (for example external electrode).The movement of electrons can be confirmed by measuring the current value.For example, if the modified cyanobacteria 31 is cultivated, the electrode is configured in the cell suspension (so-called culture fluid) and the potential is applied from the outside, then the electron transfer between the cell and the electrode occurs with high efficiency, therefore produces electric current.

[0176] Next, the situation of the above-mentioned (II) taking in electrons from the outside is described. The modification of cyanobacteria 31 taking in electrons from the outside refers to that, for example, the modification of cyanobacteria 31 will take in electrons present in the outer membrane outside or substances or molecules with electrons into the inner side of cell wall 4. In addition, the modification of cyanobacteria 31 can utilize electrons or substances or molecules with electrons in the inner side (cytoplasm) of cell wall 4. As described in the prior art, usually, cyanobacteria have higher photosynthesis ability and produce various organic substances in the cell. The modification of cyanobacteria 31 also produces various organic substances in the cell (in the cell wall and in the thylakoid) in the same manner as cyanobacteria. At this time, the modification of cyanobacteria 31 can take in electrons present in the outer membrane 5 or substances or molecules with electrons (it should be noted that it can also be a part of electron transfer material 33) into the inside of cell wall 4, and accept electrons from electron transfer material 33 in the inner side (i.e., in the cytoplasm) of cell wall 4 for material production. In addition, the modification of cyanobacteria 31 can also use the electrons taken in from the outside for respiration (dissimilation of organic substances). In this way, the modified cyanobacteria 31 can utilize electrical energy instead of light energy, so even in an environment where sunlight is insufficient, the reducing power in the cell is not easy to become insufficient. Therefore, the modified cyanobacteria 31 can utilize both light energy and electrical energy, so the material production efficiency in the cell is improved.

[0177] In addition, for example, the modified cyanobacteria 31 included in the electron mediator 30 can utilize electrical energy instead of light energy to stably generate necessary energy and reducing power, dissipate substances, and produce substances in the cell.

[0178] According to the above content, the electron carrier 30 of the present embodiment comprises the modified cyanobacteria 31 with improved extracellular electron transfer efficiency, and therefore can efficiently supply electrons to the outside (such as external electrodes, etc.), and efficiently accept electrons from the outside. Therefore, if, for example, the electron carrier 30 is used, the modified cyanobacteria 31 included in the electron carrier 30 releases the electron transfer substance in the cell to the outside, and thus, for example, can efficiently supply electrons to the external electrodes and generate electric current.

[0179] Example

[0180] Hereinafter, the electron mediator, the method for producing the electron mediator, and the electron transfer method of the present disclosure will be specifically described in Examples, but the present disclosure is not limited only to the following Examples.

[0181] In the following embodiments, in cyanobacteria, (i) by making the function of the protein associated with the combination of outer membrane and cell wall be suppressed or lost, make the modified cyanobacteria (embodiment 1 and embodiment 2) that the outer membrane of cyanobacteria is partially separated from the cell wall, (ii) by making the channel protein that the outer membrane expression improves the permeability of protein, make the modified cyanobacteria (embodiment 3) that has improved the permeability of the substance of outer membrane.Evaluate whether these 3 kinds of modified cyanobacteria secrete intracellular electron transfer material 33, supply electrons to the outside and take in at least one of the electrons from the extracellular to the cell, i.e. whether the extracellular electron transfer efficiency between the cell and the external electrode is improved.Evaluate by the quantitative and identification of the protein secreted into the extracellular by these modified cyanobacteria and the mensuration of the intracellular current value produced by photosynthesis.

[0182] It should be noted that the cyanobacteria species used in this example is Synechocystis PCC6803 (hereinafter referred to as "cyanobacteria").

[0183] (Example 1)

[0184] In Example 1, a modified cyanobacterium in which the expression of the slr1841 gene (SEQ ID NO: 7) encoding an SLH domain-maintaining outer membrane protein was suppressed was produced.

[0185] (1) Construction of modified cyanobacterial strains in which the expression of slr1841 gene is inhibited

[0186] As a gene suppression method, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference method was used. In this method, the expression of slr1841 gene (sequence number 7) can be suppressed by introducing the gene encoding dCas9 protein (hereinafter referred to as dCas9 gene) and slr1841_sgRNA (single-guide Ribonucleic Acid) gene into the chromosomal DNA of cyanobacteria.

[0187] The mechanism of gene expression inhibition by the present method is as follows.

[0188] First, a Cas9 protein deficient in nuclease activity (dCas9) and an sgRNA (slr1841_sgRNA) that binds complementarily to the base sequence of the slr1841 gene (SEQ ID NO: 7) form a complex.

[0189] Next, the complex recognizes the slr1841 gene on the chromosomal DNA of the cyanobacteria and specifically binds to the slr1841 gene. Since the binding becomes a steric hindrance, the transcription of the slr1841 gene is inhibited. As a result, the expression of the slr1841 gene of the cyanobacteria is inhibited.

[0190] Hereinafter, the method for introducing the above two genes into the chromosomal DNA of cyanobacteria will be specifically described.

[0191] (1-1) Introduction of dCas9 gene

[0192] Using the chromosomal DNA of Synechocystis LY07 strain (hereinafter also referred to as LY07 strain) (refer to non-patent literature 16) as a template, the primers psbA1-Fw (sequence number 17) and psbA1-Rv (sequence number 18) described in Table 1 were used to amplify the dCas9 gene and the operator gene for controlling the expression of the dCas9 gene, and the spectinomycin resistance marker gene that serves as a marker for gene introduction by PCR (polymerase chain reaction) method. Among them, in the LY07 strain, the above three genes are inserted into the psbA1 gene on the chromosomal DNA in a connected state, so they can be amplified as one DNA fragment by PCR. Here, the obtained DNA fragment is recorded as "psbA1::dCas9 box". Using the In-FusionPCR cloning method (registered trademark), the psbA1::dCas9 box is inserted into the pUC19 plasmid to obtain the pUC19-dCas9 plasmid.

[0193] Table 1

[0194] Primer name Base sequence Serial Number psbA1-Fw 5′-CAGTGAATTCGAGCTCGGTATATAGCGTTGCAGTCCCTGG-3′ 17 psbA1-Rv 5′-GATTACGCCAAGCTTGCATGACCGCGGTCACTTCATAACC-3′ 18 slr2030-Fw 5′-CAGTGAATTCGAGCTCGGTAATAACCGTTGTCCCTTTTGTTTCATCG-3′ 19 sgRNA_slr1841-Rv 5′-TGTTAGTGAGCCCTGCTGTTAGCTCCCAGTATCTCTATCACTGAT-3′ 20 sgRNA_slr1841-Fw 5′-ACAGCAGGGCTCACTAACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAA-3′ 21 slr2031-Rv 5′-GATTACGCCAAGCTTGCATGGGGAACAAGCTGAATCTGGGCATC-3′ 22 sgRNA_slr0688-Rv 5′-TTTTAGTCTGTTTGCTGCATAGCTCCCAGTATCTCTATCACTGAT-3′ 23 sgRNA_slr0688-Fw 5′-TGCAGCAACAGACTAAAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAA-3′ 24 slr0042-Fw 5′-TAGATATAATGTGTGGTAAATTAGAGGAATTCATCGCA-3′ 25 slr0042-Rv 5′-CGCTCTTCGATCTGATTTAAAATGTGAACGTCGTCCGTAG-3′ 26 PL22-Rv 5′-TGTTAGTGAGCCCTGCTGTTAGCTCCCAGTATCTCTATCACTGAT-3′ 27 KmR 5′-ACAGCAGGGCTCACTAACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAA-3′ 28 cppS-Fw 5′-TCCCTGGTTCCGCTGGGGCTATGGCCCCGGAGTGCACCGT-3′ 29 cppS-Rv 5′-ATTTGATGCCTGGCTCTAGTTTACAGAAGCTCGATCTCGA-3′ 30 tetR-Rv 5′-TAGCAGGTACTGCAAACGCTATGCCGCTCCCGGTGTTCGC-3′ 31 tetR-Fw 5′-TTAGAACTGGTAAACGATACTTACTGATCGATAACGAGCG-3′ 32

[0195] 1 μg of the obtained pUC19-dCas9 plasmid was mixed with a cyanobacterial culture solution (cell concentration OD730 = about 0.5), and the pUC19-dCas9 plasmid was introduced into the cells of the cyanobacteria by natural transformation. Screening was performed by growing the transformed cells on a BG-11 agar medium containing 20 μg / mL of spectinomycin. In the screened cells, homologous recombination occurred between the psbA1 gene on the chromosomal DNA and the psbA1 upstream fragment region and the psbA1 downstream fragment region on the pUC19-dCas9 plasmid. Thus, a dCas9 strain of Synechocystis sp. with a dCas9 box inserted in the psbA1 gene region was obtained. Among them, the composition of the BG-11 medium used is shown in Table 2.

[0196] Table 2

[0197] Element Content (mg / L) EDTA-Na 1 Ammonium Ferric Citrate 6 <![CDATA[NaNO3]]> 1500 <![CDATA[MgSO4]]> 75 <![CDATA[K2HPO4]]> 39 <![CDATA[CaCl2]]> 28.6 <![CDATA[H3BO4]]> 2.8 <![CDATA[MnCl2]]> 1.8 <![CDATA[ZnSO4]]> 0.2 <![CDATA[CuSO4]]> 0.08 <![CDATA[Na2MoO4]]> 0.02 <![CDATA[Co(NO3)2]]> 0.005 TES-KOH (pH 7.5) 4580

[0198] (1-2) Introduction of slr1841_sgRNA gene

[0199] In the CRISPR interference method, a sequence of about 20 bases complementary to the target sequence is introduced into a region called a protospacer on the sgRNA gene, so that the sgRNA specifically binds to the target gene. The protospacer sequences used in this example are shown in Table 3.

[0200] Table 3

[0201] Protospacer target gene Base sequence Serial Number slr1841 5′-ACAGCAGGGCTCACTAACA-3′ 33 slr0688 5′-TGCAGCAAACAGACTAAAA-3′ 34

[0202] In the Synechocystis LY07 strain, the sgRNA gene (excluding the pre-spacer sequence region) and the kanamycin resistance marker gene are inserted into the slr2030-slr2031 gene on the chromosomal DNA in a linked form. Therefore, by giving the primers used when amplifying the sgRNA gene by PCR a pre-spacer sequence (sequence number 33) complementary to the slr1841 gene (sequence number 7), it is easy to obtain an sgRNA (slr1841_sgRNA) that specifically recognizes slr1841.

[0203] First, using the chromosomal DNA of the LY07 strain as a template, the two DNA fragments were amplified by PCR using the primer set of slr2030-Fw (sequence number 19) and sgRNA_slr1841-Rv (sequence number 20) and the set of sgRNA_slr1841-Fw (sequence number 21) and slr2031-Rv (sequence number 22) recorded in Table 1.

[0204] Next, the mixed solution of the above DNA fragments was used as a template and amplified by PCR using the primers slr2030-Fw (SEQ ID NO: 19) and slr2031-Rv (SEQ ID NO: 22) listed in Table 1, thereby obtaining a DNA fragment (slr2030-2031::slr1841_sgRNA) in which (i) the slr2030 gene fragment, (ii) slr1841_sgRNA, (iii) kanamycin resistance marker gene, and (iv) the slr2031 gene fragment were sequentially connected. Using the In-Fusion PCR cloning method (registered trademark), slr2030-2031::slr1841_sgRNA was inserted into the pUC19 plasmid to obtain the pUC19-slr1841_sgRNA plasmid.

[0205] The pUC19-slr1841_sgRNA plasmid was introduced into the Synechocystis dCas9 strain by the same method as described above (1-1), and the transformed cells were screened on a BG-11 agar medium containing 30 μg / mL kanamycin. Thus, a transformant Synechocystis dCas9slr1841_sgRNA strain (hereinafter also referred to as slr1841 suppression strain) in which slr2030-slr2031 gene on chromosomal DNA was inserted was obtained.

[0206] (1-3) Suppression of slr1841 gene

[0207] The promoter sequences of the dCas9 gene and the slr1841_sgRNA gene are designed to be induced to express in the presence of anhydrotetracycline (aTc). In this example, the expression of the slr1841 gene was inhibited by adding aTc at a final concentration of 1 μg / mL to the culture medium.

[0208] (Example 2)

[0209] In Example 2, a modified cyanobacterium in which the expression of the slr0688 gene encoding a cell wall-pyruvate modification enzyme is suppressed was obtained by the following steps.

[0210] (2) Construction of modified cyanobacterial strains in which the expression of slr0688 gene is inhibited

[0211] By the same steps as above (1-2), the sgRNA gene containing the protospacer sequence (sequence number 34) complementary to the slr0688 gene (sequence number 10) was introduced into the Synechocystis dCas9 strain to obtain the Synechocystis dCas9 slr0688_sgRNA strain. Among them, the same conditions as those in (1-2) were used except that the primers slr2030-Fw (sequence number 19) and sgRNA_slr0688-Rv (sequence number 23) and the primers slr0688-Fw (sequence number 24) and slr2031-Rv (sequence number 22) described in Table 1 were used; and (i) the slr2030 gene fragment, (ii) slr0688_sgRNA, (iii) the kanamycin resistance marker gene, and (iv) the slr2031 gene fragment were sequentially connected to form a DNA fragment (slr2030-2031::slr0688_sgRNA) inserted into the pUC19 plasmid using the In-Fusion PCR cloning method (registered trademark) to obtain the pUC19-slr0688_sgRNA plasmid.

[0212] Furthermore, the expression of the slr0688 gene was suppressed by the same procedure as in the above (1-3).

[0213] (Comparative Example 1)

[0214] In Comparative Example 1, the same steps as (1-1) of Example 1 were used to obtain the Synechocystis dCas9 strain.

[0215] Next, the state of the cell surface layer was observed for each of the strains obtained in Example 1, Example 2, and Comparative Example 1. The details are described below.

[0216] (3) Observation of the state of the cell surface of the strain

[0217] Ultrathin sections were prepared for the modified cyanobacterium Synechocystis dCas9 slr1841_sgRNA strain obtained in Example 1 (the so-called slr1841 inhibition strain), the modified cyanobacterium Synechocystis dCas9slr0688_sgRNA strain obtained in Example 2 (hereinafter also referred to as the slr0688 inhibition strain), and the modified cyanobacterium Synechocystis dCas9 strain obtained in Comparative Example 1 (hereinafter referred to as the control strain), and the state of the cell surface (in other words, the outer membrane structure) was observed using an electron microscope.

[0218] (3-1) Cultivation of strains

[0219] The slr1841 inhibitor strain of Example 1 was inoculated into a BG-11 medium containing 1 μg / mL aTc at an initial bacterial cell concentration of OD730 = 0.05 and then cultured under a light intensity of 100 μmol / m 2 The culture was shaken at 100 μg / s and 30°C for 5 days. The slr0688-inhibited strain of Example 2 and the control strain of Comparative Example 1 were also cultured under the same conditions as in Example 1.

[0220] (3-2) Preparation of Ultrathin Sections of Strain

[0221] The culture solution obtained in (3-1) above was centrifuged at 2,500 g for 10 minutes at room temperature to recover the cells of the slr1841 inhibitor strain of Example 1. Next, the cells were rapidly frozen with liquid propane at -175°C and fixed at -80°C for 2 days using an ethanol solution containing 2% glutaraldehyde and 1% tannic acid. The fixed cells were dehydrated with ethanol, and the dehydrated cells were immersed in propylene oxide and then sunk into a resin (Quetol-651) solution. Then, the resin was allowed to stand at 60°C for 48 hours to solidify and the cells were embedded in the resin. The cells in the resin were thinly sliced ​​into 70 nm thickness using an ultra-thin slicer (Ultracut) to prepare ultra-thin sections. The ultra-thin sections were stained with 2% uranyl acetate and 1% lead citrate solution to prepare a transmission electron microscope sample of the slr1841 inhibitor strain of Example 1. The same operation was performed on the slr0688-suppressed strain of Example 2 and the control strain of Comparative Example 1 to prepare samples for transmission electron microscopy.

[0222] (3-3) Observation using an electron microscope

[0223] The ultrathin sections obtained in (3-2) were observed using a transmission electron microscope (JEOL JEM-1400Plus) at an accelerating voltage of 100 kV. The observation results are shown in Figure 3 to Figure 8 .

[0224] First, the slr1841-suppressed strain of Example 1 will be described. Figure 3 This is a TEM (Transmission Electron Microscope) image of the slr1841-suppressed strain of Example 1. Figure 4 yes Figure 3 A magnified image of the dashed area A. Figure 4 (a) is Figure 3 The enlarged TEM image of the dashed area A is shown in Figure 1. Figure 4 (b) depicts Figure 4 (a) is an enlarged TEM image of the sample.

[0225] like Figure 3 As shown, in the slr1841-suppressed strain of Example 1, the outer membrane was partially peeled off from the cell wall (ie, the outer membrane was partially peeled off), and the outer membrane was partially bent.

[0226] In order to confirm the state of the cell surface in more detail, the dotted area A was magnified and observed. Figure 4 (a) and Figure 4 As shown in (b), a portion where the outer membrane was partially peeled off was confirmed (the dot-dash area a1 and a2 in the figure). In addition, a portion where the outer membrane was greatly bent was confirmed next to the dot-dash area a1. It is considered that this portion is a portion where the bond between the outer membrane and the cell wall is weakened, and because the culture fluid penetrates from the outer membrane into the extracellular cytoplasm, the outer membrane expands outward and bends.

[0227] Next, the slr0688-suppressed strain of Example 2 is described. Figure 5 This is a TEM image of the slr0688 inhibitor strain of Example 2. Figure 6 yes Figure 5 A magnified image of the dashed area B. Figure 6 (a) is Figure 5 The enlarged TEM image of the dashed area B is shown in Figure 1. Figure 6 (b) depicts Figure 6 (a) is an enlarged TEM image of the sample.

[0228] like Figure 5 As shown, the outer membrane was partially peeled off from the cell wall and partially curved in the slr0688-inhibited strain of Example 2. In addition, it was confirmed that the outer membrane was partially detached from the cell wall in the slr0688-inhibited strain.

[0229] In order to confirm the state of the cell surface in more detail, the dotted line area B was magnified and observed. Figure 6 (a) and Figure 6 As shown in (b), a portion where the outer membrane is greatly bent (the dotted line region b1 in the figure) and a portion where the outer membrane is partially peeled off (the dotted line regions b2 and b3 in the figure) are confirmed. In addition, a portion where the outer membrane is detached from the cell wall is also confirmed near each of the dotted line regions b1, b2 and b3.

[0230] Next, the control strain of Comparative Example 1 will be described. Figure 7 This is a TEM image of the control strain of Comparative Example 1. Figure 8 yes Figure 7 A magnified image of the dashed area C. Figure 8 (a) is Figure 7 The enlarged TEM image of the dashed area C, Figure 8 (b) depicts Figure 8(a) is an enlarged TEM image of the sample.

[0231] like Figure 7 As shown, the cell surface layer of the control strain of Comparative Example 1 was neat, and the inner membrane, cell wall, outer membrane, and S layer were stacked in order. That is, in the control strain, there was no portion where the outer membrane was separated from the cell wall, no portion where the outer membrane was peeled off (i.e., peeled off) from the cell wall, and no portion where the outer membrane was bent as in Examples 1 and 2.

[0232] (Example 3)

[0233] In Example 3, the Synechocystis cppS tetR strain (hereinafter also referred to as cppS-introduced strain) was obtained by introducing the chloroplast outer membrane channel protein CppS (SEQ ID NO: 13) retained by the gray alga Cyanophora paradoxa into the outer membrane of cyanobacteria through the following procedure.

[0234] (4) Import of the chloroplast outer membrane channel protein CppS into the outer membrane of cyanobacteria

[0235] (4-1) Construction of cppS gene expression cassette

[0236] A gene cassette was prepared by connecting the cppS gene, a promoter region (PL22) for controlling the expression of the cppS gene, an outer membrane translocation signal sequence in cyanobacteria (slr0042-signal), and a kanamycin resistance marker gene (KmR) as a marker for gene introduction according to the following procedure.

[0237] First, the chromosomal DNA of cyanobacteria was used as a template, and the primers slr0042-Fw (sequence number 25) and slr0042-Rv (sequence number 26) described in Table 1 were used to amplify by PCR to obtain the slr0042 gene. Next, the chromosomal DNA of Synechocystis strain LY07 (Non-Patent Document 16) was used as a template, and the primers slr2030-Fw (sequence number 19) and PL22-Rv (sequence number 27) and the primers KmR-Fw (sequence number 28) and slr2031-Rv (sequence number 22) described in Table 1 were used to amplify by PCR to obtain PL22 and KmR. In the LY07 strain, these are present as being inserted into the slr2030-slr2031 gene on the chromosome, so when the above four primers are used for amplification by PCR, the slr2030 gene fragment is amplified in a form connected to the 5' end side of PL22, and the slr2031 gene fragment is amplified in a form connected to the 3' end side of KmR. Next, amplification is performed by PCR using the mixed solution of the slr0042 gene, PL22 and KmR obtained in the above step as a template and the four primers (SEQ ID NOs. 19, 22, 27, and 28) listed in Table 1, thereby obtaining a gene cassette (slr2030-2031::slr0042-KmR cassette) in which the slr2030 gene fragment, PL22, slr0042 gene, KmR and slr2031 gene fragment are connected in order from the 5' end. Using the In-Fusion PCR cloning method (registered trademark), the slr2030-2031::slr0042-KmR cassette was inserted into the pUC19 plasmid to obtain the pUC19-slr0042 plasmid.

[0238] In parallel with the above steps, total cDNA was prepared from gray algae C. paradoxa NIES-547 using SMART cDNA library synthesis kit (Clontech). Using this cDNA as a template, primers cppS-Fw (sequence number 29) and cppS-Rv (sequence number 30) described in Table 1 were used to amplify by PCR to obtain the cppS gene (sequence number 13). Using In-Fusion PCR cloning (registered trademark), the cppS gene (sequence number 13) was inserted into the pUC19-slr0042 plasmid to obtain the pUC19-CppS plasmid. Wherein, through this step, the cppS gene is inserted in the form of being connected to the 3' end side of the outer membrane transfer signal sequence of the sr0042 gene, and the region other than the outer membrane transfer signal sequence of the sr0042 gene is removed in the form of an insertion replacement with the coding region of cppS.

[0239] (4-2) Construction of the promoter activity control box (tetR) of PL22

[0240] The promoter activity of PL22 is induced only in the presence of dehydrotetracycline (aTc) by the control of the TetR repressor. Therefore, it is necessary to introduce the tetR gene for controlling the activity of PL22 into the modified cyanobacteria.

[0241] First, tetR gene and spectinomycin resistance marker gene (SpcR) as a marker for gene introduction were obtained by amplification by PCR using the chromosome DNA of LY07 strain as a template and the primers psbA1-Fw (SEQ ID NO: 17) and tetR-Rv (SEQ ID NO: 31) and tetR-Fw (SEQ ID NO: 32) and psbA1-Rv (SEQ ID NO: 18) as described in Table 1. In the LY07 strain, these are present as inserted into the psbA1 gene on the chromosome, so when amplification is performed by PCR using the above primers, the upstream fragment of the psbA1 gene is connected to the 5' end of the tetR gene, and the downstream fragment of the psbA1 gene is connected to the 3' end of the SpcR. Next, a mixed solution of tetR gene and SpcR was used as a template and amplified by PCR method using primers psbA1-Fw (SEQ ID NO: 17) and psbA1-Rv (SEQ ID NO: 18) listed in Table 1, thereby obtaining a gene cassette (psbA1::tetR cassette) to which the upstream fragment of psbA1 gene, tetR, SpcR, and the downstream fragment of psbA1 gene were linked in order from the 5' end. The psbA1::tetR cassette was inserted into the pUC19 plasmid using the In-Fusion PCR cloning method (registered trademark) to obtain the pUC19-tetR plasmid.

[0242] (4-3) Introduction of cppS gene expression cassette and tetR cassette

[0243] 1 μg of the pUC19-cppS plasmid obtained by the above steps was mixed with a cyanobacterial culture solution (cell concentration OD730 = about 0.5), and the plasmid was introduced into the cells by natural transformation. The transformed cells were grown on a BG-11 agar medium containing 30 μg / mL kanamycin for screening. In the screened cells, homologous recombination occurred between the slr2030-2031 gene on the chromosome and the slr2030 gene fragment region and the slr2031 gene fragment region on the pUC19-cppS plasmid. Thus, a Synechocystis cppS strain with a cppS gene expression cassette inserted in the slr2030-2031 gene region was obtained. The composition of the BG-11 medium used is shown in Table 2.

[0244] Next, 1 μg of the pUC19-tetR plasmid was mixed with a culture solution of Synechocystis cppS (cell concentration OD730 = about 0.5), and the plasmid was introduced into the cells by natural transformation. The transformed cells were grown on a BG-11 agar medium containing 30 μg / mL of kanamycin and 20 μg / mL of spectinomycin, and the Synechocystis cppS tetR strain was obtained by screening. In the same manner as described above, in this strain, the tetR cassette was inserted into the psbA1 gene on the chromosomal DNA.

[0245] (4-4) Induction of cppS gene expression

[0246] The cppS gene of the above-mentioned Synechocystis cppS tetR strain is induced to express in the presence of anhydrotetracycline (aTc). In this example, the expression of the cppS gene was induced by culturing in BG-11 medium with the addition of aTc at a final concentration of 1 μg / mL.

[0247] (5) Protein secretion productivity test

[0248] By the following method, it was confirmed whether the protein present in the extracellular cytoplasm (the gap between the outer membrane and the inner membrane) was secreted outside the cell.

[0249] More specifically, the slr1841 suppression strain of Example 1, the slr0688 suppression strain of Example 2, the cppS introduction strain of Example 3, and the control strain of Comparative Example 1 were cultured respectively, and the amount of protein secreted outside the cell (hereinafter also referred to as the secreted protein amount) was measured. Based on the amount of protein in the culture solution, the secretion productivity of the protein of each of the above strains was evaluated. Among them, the secretion productivity of the protein refers to the ability to produce protein by secreting the protein produced in the cell to the outside of the cell. The specific method is described below.

[0250] (5-1) Cultivation of strains

[0251] The slr1841-suppressed strain of Example 1 was cultured in the same manner as in (3-1) above. The culture was performed independently three times. In addition, the strains of Example 2, Example 3, and Comparative Example 1 were also cultured under the same conditions as the strain of Example 1.

[0252] (5-2) Quantification of extracellular protein secretion

[0253] The culture solution obtained in (5-1) above was centrifuged at 2,500g for 10 minutes at room temperature to obtain a culture supernatant. The obtained culture supernatant was filtered using a membrane filter with a pore size of 0.22μm to completely remove the cells of the slr1841 inhibitor strain of Example 1. The total protein content contained in the filtered culture supernatant was quantified by the BCA (Bicinchoninic acid) method. This series of operations was performed on the three culture solutions obtained by independent culture, and the average value and standard deviation of the amount of protein secreted to the extracellular space by the slr1841 inhibitor strain of Example 1 were calculated. In addition, for the strains of Example 2, Example 3 and Comparative Example 1, the protein content of the three culture solutions obtained in (5-1) above was quantified under the same conditions, and the average value and standard deviation of the protein content in the three culture solutions were calculated.

[0254] The results are shown in Fig. 9 . Fig. 9 It is a graph showing the protein amount in the culture solution of the modified cyanobacteria of Examples 1 to 3 and Comparative Example 1 (n=3, error bar=SD).

[0255] like Fig. 9 As shown, the amount of protein (mg / L) secreted into the culture supernatant of the slr1841-suppressed strain of Example 1, the slr0688-suppressed strain of Example 2, and the cppS-introduced strain of Example 3 was increased by about 25 times compared with the control strain of Comparative Example 1.

[0256] Although the data are omitted, the absorbance of the culture medium was measured (730 nm) and the amount of secreted protein per 1 g of dry weight of the bacteria (mg protein / g dry weight of the bacteria) was calculated. The results showed that the amount of secreted protein per 1 g of dry weight of the bacteria (mg protein / g dry weight of the bacteria) of the slr1841 inhibition strain of Example 1, the slr0688 inhibition strain of Example 2, and the cppS introduction strain of Example 3 were all increased by about 36 times compared with the control strain of Comparative Example 1.

[0257] In addition, Fig. 9In the experiment, the amount of secreted protein (mg / L) of the "modified cyanobacteria in which the outer membrane of cyanobacteria is partially separated from the cell wall" of Example 1 and Example 2 was compared. Compared with the slr1841 inhibition strain of Example 1 in which the expression of the gene (slr1841) encoding the SLH domain-maintaining outer membrane protein was inhibited, the slr0688 inhibition strain of Example 2 in which the expression of the gene (slr0688) encoding the cell wall-pyruvate modification enzyme was inhibited secreted more protein in the culture supernatant. This is considered to be related to the fact that the number of covalently bonded sugar chains on the cell wall surface is greater than the number of SLH domain-maintaining outer membrane proteins (Slr1841) in the outer membrane. That is, considering that the slr0688 inhibition strain of Example 2 has a further reduced amount and binding force between the outer membrane and the cell wall compared to the slr1841 inhibition strain of Example 1, the amount of protein secreted is greater than that of the slr1841 inhibition strain of Example 1.

[0258] In addition, Fig. 9 In the experiment, the amount of secreted protein (mg / L) of the slr0688-suppressed strain of Example 2 was compared with the amount of secreted protein (mg / L) of the cppS-introduced strain of Example 3 in which the gene (cppS) encoding the organic channel protein CppS was expressed in the outer membrane of the cyanobacterium. The slr0688-suppressed strain of Example 2 expressed slightly more secreted protein than the cppS-introduced strain of Example 3 (about 20 mg / L).

[0259] Confirmed by the above result, owing to having suppressed the function of the protein related to the combination of adventitia and cell wall, the adventitia of cyanobacteria and the combination of cell wall partially weaken, and adventitia partially breaks away from the cell wall.Also confirmed, because adventitia and the combination of cell wall weaken, the protein produced in the cell of cyanobacteria becomes easy to be secreted into the extracellular.Therefore, according to the disclosed modified cyanobacteria, the secretion productivity of protein is demonstrated and significantly improves.

[0260] (5-3) Identification of secreted proteins

[0261] Next, the secretory proteins contained in the culture supernatant obtained in (5-2) above were identified by LC-MS / MS. The method is described below.

[0262] (5-3-1) Sample preparation

[0263] Add 8 times the amount of cold acetone relative to the volume of the culture supernatant, let it stand at 20°C for 2 hours, and then centrifuge at 20,000g for 15 minutes to obtain a protein precipitate. Add 100mM Tris pH8.5 and 0.5% sodium dodecanoate (SDoD) to the precipitate, and dissolve the protein using a closed ultrasonic crusher. After adjusting the protein concentration to 1μg / mL, add dithiothreitol (DTT) at a final concentration of 10mM and let it stand at 50°C for 30 minutes. Next, add iodoacetamide (IAA) at a final concentration of 30mM and let it stand at room temperature (shaded) for 30 minutes. In order to stop the reaction of IAA, add cysteine ​​at a final concentration of 60mM and let it stand at room temperature for 10 minutes. Add 400ng of trypsin and let it stand at 37°C overnight to fragment the protein into peptides. After adding 5% TFA (Trifluoroacetic Acid), centrifuge at 15,000g for 10 minutes at room temperature to obtain a supernatant. This operation removes SDoD. After desalting using a C18 spin column, the sample is dried using a centrifugal evaporator. Then, 3% acetonitrile and 0.1% formic acid are added, and the sample is dissolved using a closed ultrasonic crusher. Prepare in such a way that the peptide concentration reaches 200ng / μL.

[0264] (5-3-2) LC-MS / MS analysis

[0265] The sample obtained in (5-3-1) was analyzed under the following conditions using an LC-MS / MS device (UltiMate 3000RSLCnano LC System).

[0266] Sample injection amount: 200ng

[0267] Column: CAPCELL CORE MP 75μm×250mm

[0268] Solvent: Solvent A is 0.1% formic acid aqueous solution, solvent B is 0.1% formic acid + 80% acetonitrile

[0269] Gradient program: 4 minutes after sample injection, solvent B is 8%, 27 minutes after solvent B is 44%, 28 minutes after solvent B is 80%, and measurement is terminated after 34 minutes.

[0270] (5-3-3) Data Analysis

[0271] The obtained data were analyzed under the following conditions to identify proteins and peptides and calculate quantitative values.

[0272] Software: Scaffold DIA

[0273] Database: UniProtKB / Swiss Prot database (Synechocystis PCC 6803)

[0274] Fragmentation: HCD

[0275] Precursor Tolerance: 8ppm

[0276] Fragment Tolerance: 10ppm

[0277] Data Acquisition Type: Overlap DIA

[0278] Peptide Length: 8-70

[0279] Peptide Charge: 2-8

[0280] Max Missed Cleavages: 1

[0281] Fixed Modification: Cysteine ​​Carbamidomethylation

[0282] Peptide FDR: less than 1%

[0283] Table 4 shows the ten proteins in order of the protein with the largest relative quantitative value among the identified proteins.

[0284] Table 4

[0285] Protein name Uniprot Login ID Gene name 1 Carboxyl terminal protease P73458 prc 2 Sll0314 protein Q55648 sll0314 3 Sll0470 protein Q55847 sll0470 4 Ssr1853 protein P72639 ssr1853 5 Sll0319 protein P74789 sll0319 6 Slr0581 protein Q55408 slr0581 7 Carbon dioxide-concentration mechanism P72761 ccmK2 8 Protein CcmK homolog 2 Q55386 slr0924 9 Slr0924 protein Q59991 slr0042 10 Slr0042 protein P73817 sll1951

[0286] All 10 kinds of proteins are contained in the culture supernatants of Examples 1 to 3, respectively. In all of these proteins, the extracellular plasm (referring to the gap between the outer membrane and the inner membrane) transfer signal is maintained. According to the results, it was confirmed that in the slr1841 inhibition strain of Example 1 and the slr0688 inhibition strain of Example 2, the binding between the outer membrane and the cell wall is weakened, so the outer membrane is partially detached from the cell wall, and thus the proteins in the extracellular plasm become easy to leak out of the outer membrane (i.e., outside the bacteria). In addition, it was confirmed that in the cppS introduction strain of Example 3, the protein permeability of the outer membrane is improved by the expression of the cppS gene, and the proteins in the extracellular plasm become easy to pass through the channel protein CppS and be secreted outside the outer membrane (i.e., outside the bacteria). Therefore, the modified strains of Examples 1 to 3 show that the material permeability of the outer membrane is improved.

[0287] (6) Evaluation of the extracellular electron transfer efficiency of modified cyanobacteria

[0288] The extracellular electron transfer efficiency was evaluated for the slr0688-suppressed strain of Example 2, the cppS-introduced strain of Example 3, and the control strain of Comparative Example 1. More specifically, the culture fluids of these modified strains were irradiated with light, and the extracellular electron transfer efficiency was evaluated by detecting the intracellular current generated by water decomposition caused by photosynthesis using an external electrode. The following describes the device used and the current measurement procedure.

[0289] (6-1) Electrochemical measurement device

[0290] First, the configuration of the electrochemical measurement device 100 will be described with reference to the drawings. Fig.10 It is an exploded perspective view schematically showing an example of the configuration of the electrochemical measuring device 100 . Fig.11 yes Fig.10 A schematic cross-sectional view taken along section line XI-XI.

[0291] like Fig.10 and Fig.11 As shown, the electrochemical measurement device 100 includes a measurement unit 10 and a light irradiation unit 20. The measurement unit 10 includes: a reaction tank 12 having a container 11 for containing a culture solution 40 of cyanobacteria; a first electrode 13 disposed inside the reaction tank 12 in a manner of contacting the culture solution 40 in the container 11; a second electrode 14 disposed inside the reaction tank 12 in a manner of contacting the culture solution 40 in the container 11; a potentiostat 15 for controlling the potential of the first electrode 13; and a reference electrode 16 disposed inside the reaction tank 12 in a manner of contacting the culture solution 40 in the container 11.

[0292] The reaction tank 12 has electrical insulation properties and does not penetrate the culture solution 40. The reaction tank 12 may be made of a material that is not corroded or damaged by the culture solution 40, and may be made of, for example, plastic or ceramic.

[0293] The measuring unit 10 applies a voltage or flows a current between the first electrode 13 and the second electrode 14, and measures a current or a potential corresponding to the voltage or current. Here, the measuring unit 10 applies a voltage between the first electrode 13 and the second electrode 14, and measures a current.

[0294] The first electrode 13 is a so-called working electrode, which is an electrode that electrochemically responds sensitively to trace substances in the culture solution 40 on the electrode surface. The second electrode 14 is a so-called counter electrode, which is an electrode for setting a potential difference with the working electrode (first electrode 13) or for flowing a current.

[0295] The first electrode 13 and the second electrode 14 are made of a conductive material. As the conductive material, for example, a carbon material, a conductive polymer material, a semiconductor or a metal can be used. For example, as a carbon material, it can be carbon nanotubes, Ketjen black, glassy carbon, graphene, fullerene, carbon fiber, carbon fiber fabric, or carbon aerogel. In addition, as a conductive polymer material, for example, it can be polyaniline, polyacetylene, polypyrrole, poly(3,4-ethylenedioxythiophene), poly(p-phenylene vinylene), polythiophene, or poly(p-phenylene sulfide). In addition, as a semiconductor, for example, it can also be silicon, germanium, indium tin oxide (ITO: Indium Tin Oxide), titanium oxide, copper oxide or silver oxide. In addition, as a metal, for example, it can be gold, platinum, silver, titanium, aluminum, tungsten, copper, iron or palladium. Here, the first electrode 13 is an indium tin oxide (ITO) electrode, and the second electrode 14 is a platinum electrode. Among them, the conductive material is not particularly limited as long as it is a conductive material that will not decompose due to its own oxidation reaction.

[0296] The reference electrode 16 is an electrode that maintains a constant potential without reacting with substances in the culture solution 40, and is used to control the potential difference between the first electrode 13 and the reference electrode 16 to be constant using the potentiostat 15. Here, the reference electrode 16 is a silver / silver chloride electrode.

[0297] The potentiostat 15 applies a voltage between the first electrode 13 and the second electrode 14 , and controls the potential between the first electrode 13 and the reference electrode 16 to a predetermined value.

[0298] The light irradiation unit 20 includes a light source 21 and a housing 22 for holding the light source 21. Although the detailed structure is omitted in the figure, the light source 21 includes, for example, one or more light emitters (such as LEDs (light emitting diodes)) and a reflective surface surrounding the light emitters. The light irradiation unit 20 can be arranged at a predetermined distance from the measurement unit 10 in the positive direction of the Z axis.

[0299] In addition, Fig.11 In the embodiment, the first electrode 13, the second electrode 14, and the reference electrode 16 are each led out to the bottom of the reaction tank 12 (i.e., in the negative direction of the Z axis), but as long as they can be electrically connected to the constant potential instrument 15, they may not fly out of the reaction tank 12. In addition, the first electrode 13, the second electrode 14, and the reference electrode 16 may be led out to the side of the reaction tank 12, respectively.

[0300] (6-2) Measurement of current

[0301] For the control strain of Comparative Example 1, the slr0688-suppressed strain of Example 2, and the cppS-introduced strain of Example 3, the amount of current generated by each of the three strains was measured according to the following procedure.

[0302] The structure of the electrochemical measuring device 100 used is as follows.

[0303] First electrode 13: Indium tin oxide electrode (surface area: 3.14 cm 2 )

[0304] Second electrode 14: platinum electrode

[0305] Reference electrode 16: Silver / silver chloride electrode

[0306] Light source 21: emits about 120 μmol / m 2 / s white light source

[0307] (6-2-1) Preparation of culture medium

[0308] First, the control strain of Comparative Example 1 was inoculated into a BG-11 medium containing 1 μg / mL aTc to an initial bacterial cell concentration of OD730 = 0.1. 2 / s and 30°C for 3 days. The slr0688-suppressed strain of Example 2 and the cppS-introduced strain of Example 3 were also cultured under the same conditions.

[0309] (6-2-2) Measurement of current

[0310] After 4 mL of the culture solution 40 of the control strain of Comparative Example 1 obtained in (6-2-1) above was injected into the reaction tank 12, the potential of the first electrode 13 relative to the reference electrode 16 was controlled by the constant potential meter 15 to be +0.0 V. Then, the culture solution 40 in the container 11 was irradiated with about 120 μmol / m 2 / s white light was used to measure the current flowing between the first electrode 13 and the second electrode 14. Next, the potential of the first electrode 13 relative to the reference electrode 16 was changed in the order of +0.1V, +0.2V, +0.25V and +0.3V, and the current flowing between the first electrode 13 and the second electrode 14 was measured in the same manner while controlling the respective potentials. The results are shown in Fig.12 . Fig.12 This is a graph showing the results of measuring the current flowing when the culture solution 40 of the control strain of Comparative Example 1 was irradiated with light.

[0311] like Fig.12As shown, in the culture solution 40 of the control strain of Comparative Example 1, when the potential of the first electrode 13 relative to the reference electrode 16 is +0.0V, +0.1V, +0.2V, +0.25V and +0.3V, respectively, the measured current values ​​(maximum value after baseline correction) are approximately +0.0nA, approximately +0.0nA, approximately +0.0nA, approximately +0.0nA and approximately +1.0nA.

[0312] Next, the current values ​​of the slr0688-suppressed strain of Example 2 and the cppS-introduced strain of Example 3 were measured in the same manner as in Comparative Example 1. The results are shown in Fig.13 and Fig.14 . Fig.13 This is a graph showing the results of measuring the current flowing when the culture solution 40 of the slr0688-suppressed strain of Example 2 was irradiated with light. Fig.14 This is a graph showing the results of measuring the current flowing when the culture solution 40 of the cppS-introduced strain of Example 3 was irradiated with light.

[0313] like Fig.13 As shown, in the culture solution 40 of the slr0688 inhibitory strain of Example 2, when the potential of the first electrode 13 relative to the reference electrode 16 was +0.0V, +0.1V, +0.2V, +0.25V and +0.3V, respectively, the measured current values ​​(maximum value after baseline correction) were about +90nA, about +250nA, about +560nA, about +750nA and about +1100nA.

[0314] In addition, if Fig.14 As shown, in the culture solution 40 of the cppS-introduced strain of Example 3, when the potential of the first electrode 13 relative to the reference electrode 16 was +0.0V, +0.1V, +0.2V, +0.25V and +0.3V, respectively, the measured current values ​​(maximum value after baseline correction) were about +10nA, about +20nA, about +70nA, about +150nA and about +260nA.

[0315] like Fig.13 and Fig.14 As shown, the current value increases in a potential-dependent manner.

[0316] As can be seen from the above, when the potential of the first electrode 13 with respect to the reference electrode 16 was controlled to +0.3 V, the current values ​​of the culture solution 40 of all the three strains were measured, and therefore these current values ​​were extracted and compared in Table 5.

[0317] Table 5

[0318] Strains used in the assay Photocurrent when +0.3V is applied (μA) Comparative Example 1 Synechocystis dCas9 strain <0.001 Example 2 Synechocystis dCas9 slr0688_sgRNA strain 1.1 Example 3 Synechocystis cppS tetR strain 0.26

[0319] As shown in Table 5, the current (hereinafter referred to as photocurrent) flowing in the culture solution 40 by irradiating the culture solution 40 with light, when the potential of the first electrode 13 relative to the reference electrode 16 was controlled to +0.3 V, was increased 1000 times for the culture solution 40 of the slr0688-suppressed strain of Example 2 compared with the culture solution 40 of the control strain of Comparative Example 1. In addition, for the culture solution 40 of the cppS-introduced strain of Example 3, the photocurrent when the potential of the first electrode 13 relative to the reference electrode 16 was controlled to +0.3 V was increased to about 300 times that of the culture solution 40 of the control strain of Comparative Example 1.

[0320] The above results indicate that the modified cyanobacteria of the present embodiment improves the extracellular electron transfer efficiency by about 300 to 1000 times by partially separating the outer membrane of the cyanobacteria from the cell wall or improving the material permeability of the outer membrane of the cyanobacteria.

[0321] (7) Investigation

[0322] From the above results, it can be seen that, as in Examples 1 and 2, in the modified cyanobacteria modified in a manner that weakens the combination of the outer membrane of cyanobacteria with the cell wall and makes the protein in the cell easy to leak out, due to the rupture and peeling of the outer membrane, organic substances other than protein (such as quinones, etc.) also become easy to leak out of the cell. Therefore, it can be seen that compared with the modified cyanobacteria in which the protein permeability of the outer membrane of cyanobacteria is improved as in Example 3, the photocurrent is about 5 times higher.

[0323] Therefore, it was found that in order to further improve the efficiency of electron transfer between the modified cyanobacteria and the outside, it is more effective to peel off part of the outer membrane than to express the organic channel protein.

[0324] It should be noted that in the disclosed electron transporter, the protein production efficiency of modified cyanobacteria has also improved, and after the recovery protein, the modified cyanobacteria can also be used repeatedly, so the outer membrane can be appropriately separated and peeled off, which is very beneficial in this regard. In addition, according to the identification result of the above-mentioned protein, by modifying the gene of the protein in the extracellular plasm of the coding, the desired protein can also be secreted into the modified cyanobacteria. In the production of such useful substances, if the disclosed electron transporter is used, then by giving the electric energy except that the sunlight, useful substances can be stably and efficiently produced.

[0325] The electron carrier, the method for producing the electron carrier, and the electron transfer method of the present disclosure are described above based on the embodiments. However, the present disclosure is not limited to these embodiments. As long as it does not depart from the gist of the present disclosure, the embodiments are implemented in a manner that can be thought of by a person skilled in the art, and other methods constructed by combining some of the constituent elements in the embodiments are also included in the scope of the present disclosure.

[0326] In the above-mentioned embodiment, the example that the function of the protein of the combination of the outer membrane and the cell wall that participates in the cyanobacteria is suppressed or lost, the combination of the outer membrane and the cell wall weakens and the extracellular electron transfer efficiency is improved has been described, but the disclosure is not limited to this. For example, the outer membrane can be weakened by applying external force to the cyanobacteria and the combination of the cell wall, and the outer membrane can also be weakened. In addition, the outer membrane can also be weakened by adding enzyme or medicament in the culture solution of the cyanobacteria.

[0327] Industrial Applicability

[0328] According to the electron transporter, the method for manufacturing the electron transporter, and the electron transport method disclosed in the present invention, electrons can be efficiently supplied to the outside of the electron transporter, so that sludge or sewage can be treated and electricity can be generated at the same time. In addition, according to the present invention, the electron transporter can efficiently transfer electrons from the outside, so the electron transporter can use electrical energy when the amount of sunlight is insufficient. Therefore, according to the present invention, it is possible to efficiently manufacture useful substances in the fields of food, medicine, or chemistry, improve soil, treat wastewater, or generate electricity.

[0329] Explanation of symbols

[0330] 1 Endometrium

[0331] 2 Peptidoglycan

[0332] 3 Sugar chains

[0333] 4 Cell Wall

[0334] 5 Outer membrane

[0335] 6 SLH domain-maintaining outer membrane protein

[0336] 7 SLH domain

[0337] 8.18 Organic channel protein

[0338] 9 Cell wall-pyruvate modifying enzymes

[0339] 10 Measurement unit

[0340] 11 Accommodation

[0341] 12 Reaction tank

[0342] 13. First electrode

[0343] 14. Second electrode

[0344] 15. Potentiostat

[0345] 16 Reference electrode

[0346] 20 Light irradiation part

[0347] 21 Light Source

[0348] 22 Housing

[0349] 30 Electron transporters

[0350] 31 Modified cyanobacteria

[0351] 33 Electron transfer substances

[0352] 35 Electron Mediator

[0353] 37 Conductive material

[0354] 40 Culture medium

[0355] 100 Electrochemical measurement device Sequence Listing <110> Panasonic Intellectual Property Management Co., Ltd. <120> Electron mediator, method for producing electron mediator, and electron transfer method <130> P1030103WO01 <150> JP 2019-210255 <151> 2019-11-21 <160> 34 <170> PatentIn version 3.5 <210> 1 <211> 630 <212> PRT <213> Synechocystis PCC6803 <400> 1 Met Leu Lys Leu Ser Trp Lys Ser Leu Leu Val Ser Pro Ala Val Ile 1 5 10 15 Gly Ala Ala Leu Val Ala Gly Ala Ala Ser Ala Ala Pro Asp Asn Val 20 25 30 Thr Asn Ala Gln Val Leu Asp Gln Leu Asp Gln Tyr Thr Ala Glu Gly 35 40 45 Gln Ser Ser Ala Ile Asp Gln Val Thr Ser Val Ser Glu Leu Arg Asp 50 55 60 Val Gln Pro Thr Ala Trp Ala Tyr Glu Ala Leu Lys Ser Leu Val Glu 65 70 75 80 Arg Tyr Gly Cys Ile Val Gly Tyr Pro Asp Arg Thr Phe Arg Gly Asp 85 90 95 Arg Ala Leu Ser Arg Trp Glu Phe Ala Ala Gly Leu Asn Ala Cys Met 100 105 110 Asn Val Met Glu Arg Leu Ile Gln Glu Asn Val Ala Val Leu Arg Glu 115 120 125 Asp Ile Asp Lys Leu Lys Arg Leu Met Gln Glu Phe Glu Ala Glu Leu 130 135 140 Ala Ala Leu Gly Ala Arg Val Asp Asn Leu Glu Ala Arg Thr Ser Phe 145 150 155 160 Leu Glu Asp His Gln Phe Ser Thr Thr Thr Lys Leu Thr Gly Glu Val 165 170 175 Ile Phe Ala Pro Thr Ala Ile Phe Gly Thr Lys Lys Thr Leu Asn Asn 180 185 190 Gln Thr Asp Asn Asn Gln Ala Val Phe Gln Asn Arg Val Arg Leu Gln 195 200 205 Phe Asn Thr Ser Phe Ser Gly Glu Asp Leu Leu Val Thr Arg Leu Ala 210 215 220 Ala Gly Asn Gly Asn Arg Phe Lys Ser Phe Tyr Pro Thr Gln Asn Val 225 230 235 240 Ile Asp Pro Glu Thr Gly Asn Pro Ile Gly Ile Val Gly Asp Tyr Tyr 245 250 255 Glu Ser Pro Thr Phe Thr Gln Val His Gln Leu Ser Pro Gly Asp Asn 260 265 270 Asn Asn Val Ala Val Asp Trp Leu Ala Tyr Tyr Val Pro Leu Asp Leu 275 280 285 Gly Glu Asn Phe Arg Leu Asn Asn Tyr Ile Ala Ala Trp Gly Gly Ile 290 295 300 Trp Asp Asp Phe Val Pro Thr Leu Asn Pro Tyr Phe Asp Asp Tyr Thr 305 310 315 320 Gly Gly Lys Gly Ser Leu Ser Gln Phe Thr Ala Gln Asn Pro Ile Tyr 325 330 335 Ser Ile Gly Gly Gly Thr Gly Ile Gly Thr Ser Leu Glu Leu Gly Phe 340 345 350 Leu Ser Asn Leu Leu Gly Pro Thr Ser Leu Ser Leu Gly Tyr Leu Ala 355 360 365 Ser Thr Gly Asn Asn Pro Ser Ser Gly Gly Ser Val Thr Asn Pro Ala 370 375 380 Thr Gly Asn Asn Tyr Asp Phe Ser Ser Gly Gly Asn Gly Leu Phe Asn 385 390 395 400 Gly Gly Tyr Ser Ala Leu Ala Gln Ile Thr Thr Asn Ile Phe Asp Arg 405 410 415 Val Ser Leu Gly Phe Thr Tyr Val Asn Ala Tyr Thr Thr Pro Asp Ala 420 425 430 Ala Ile Phe Gly Lys Gly Gly Thr Gln Gly Ile Val Gly Thr Thr Ala 435 440 445 Ala Asn Leu Asn Arg Ser Glu Leu Asn Asp Asp Phe Val Asn Gly Leu 450 455 460 Gly Val Gly Ala Pro Pro Val Asp Ala Ala Thr Tyr Asn Asn Asn Val 465 470 475 480 Ser Gly Gly Ser Thr Ser Gly Asn Val Ile Asn Pro Tyr Asp Phe Gly 485 490 495 Gly Lys Gln Thr Asn Ser Tyr Gly Val Gln Met Ala Trp Asn Ile Ala 500 505 510 Asp Trp Leu Ser Phe Ser Ala Tyr Gly Ser Tyr Thr Asn Val Thr Leu 515 520 525 Ile Gly Lys Asp Asn Gly Asp Ile Trp Thr Tyr Gly Gly Gly Phe Ala 530 535 540 Phe Pro Asp Leu Gly Lys Glu Gly Asn Val Leu Gly Ile Phe Ala Gly 545 550 555 560 Val Gln Pro Tyr Val Ser Gly Phe Thr Asn Asn Thr Leu Gly Thr Thr 565 570 575 Phe Val Thr Thr Ala Asn Pro Leu Gln Val Glu Leu Phe Tyr Lys Tyr 580 585 590 Gln Leu Thr Asp Asn Leu Ser Ile Thr Pro Gly Val Ile Trp Ile Ser 595 600 605 Lys Pro Glu Gln Thr Thr Asn Ala Thr Asp Ala Phe Ile Gly Thr Val 610 615 620 Arg Gly Thr Phe Thr Phe 625 630 <210> 2 <211> 532 <212> PRT <213> Synechococcus sp. NIES-970 <400> 2 Met Ser Lys Ala Met Arg Asn Leu Leu Ser Gly Ala Thr Val Ala Leu 1 5 10 15 Gly Ala Thr Val Ala Phe Ser Gly Ala Ala Ile Ala Glu Glu Asn Gln 20 25 30 Ala Val Leu Asp Gln Ile Ser Gln Tyr Ser Thr Asp Asn Ser Val Ala 35 40 45 Gln Val Asn Ser Val Phe Gln Leu Ser Asp Val Ser Pro Ser Asp Trp 50 55 60 Ala Phe Asp Ala Leu Arg Asn Leu Val Glu Asn Tyr Asn Cys Ile Val 65 70 75 80 Gly Tyr Pro Asp Gly Thr Phe Arg Gly Thr Arg Pro Leu Ser Arg Tyr 85 90 95 Glu Phe Ala Ala Gly Leu Asn Ala Cys Leu Gln Gln Ile Glu Arg Met 100 105 110 Leu Glu Gly Gly Gly Glu Val Thr Gln Glu Asp Leu Ala Ala Leu Arg 115 120 125 Arg Leu Leu Asn Glu Phe Glu Ala Glu Leu Ala Thr Leu Gly Ala Arg 130 135 140 Val Asp Asp Leu Asp Gly Arg Val Glu Phe Leu Glu Asp Asn Gln Phe 145 150 155 160 Ser Thr Thr Thr Lys Leu Asn Gly Glu Val Ile Phe Asn Leu Gly Gly 165 170 175 Ala Phe Gly Asp Ser Arg Ala Asp Gly Pro Gly Pro Ile Asp Asp Glu 180 185 190 Phe Thr Phe Ser Asn Arg Val Arg Leu Asn Leu Asp Thr Ser Phe Thr 195 200 205 Gly Arg Asp Arg Leu Arg Thr Arg Leu Glu Ala Gly Asn Ile Thr Arg 210 215 220 Leu Asp Arg Ala Thr Gly Val Asp Ala Ala Arg Leu Gly Phe Asp Thr 225 230 235 240 Asn Asn Gly Asn Asp Val Glu Ile Gly Lys Leu Phe Tyr Arg Phe Pro 245 250 255 Val Gly Asp Asn Ile Thr Ala Tyr Val Gly Ala Val Gly Leu Gly Leu 260 265 270 Asp Asp Ile Phe Asp Val Thr Asn Pro Tyr Phe Glu Ser Ser Gly Asn 275 280 285 Gly Ala Leu Ser Arg Phe Gly Arg Phe Asn Pro Ile Tyr Arg Gly Pro 290 295 300 Asp Gly Ala Gly Ala Gly Phe Asn Phe Asp Leu Gly Glu Arg Val Thr 305 310 315 320 Phe Ser Ala Ala Tyr Leu Ala Asp Asp Gly Gly Ala Ser Ser Pro Val 325 330 335 Ser Lys Asn Gly Leu Phe Asn Gly Asn Tyr Thr Ala Gly Ala Gln Leu 340 345 350 Asp Phe Ala Pro Ser Asp Asn Ile Met Leu Ser Ala Val Tyr Thr Arg 355 360 365 Arg Tyr Gln Gln Gly Gly Asp Val Asn Leu Ser Gly Ser Thr Gly Ser 370 375 380 Asp Phe Ala Ala Asp Pro Phe Gly Phe Ala Ala Ser Ser Asn Asn Phe 385 390 395 400 Gly Leu Gln Gly Ser Trp Leu Ile Gly Glu Lys Phe Asn Leu Gly Gly 405 410 415 Trp Val Gly Tyr Thr Asn Ala Asp Leu Lys Asp Pro Val Val Thr Gly 420 425 430 Asn Ala Asp Val Trp Asn Trp Ala Val Asn Ala Ala Leu Leu Asp Val 435 440 445 Gly Gly Glu Gly Asn Thr Leu Gly Phe Ile Phe Gly Gln Pro Pro Lys 450 455 460 Leu Thr Glu Ala Asp Ile Asp Phe Gly Gly Gly Pro Ile Thr Phe Asp 465 470 475 480 Gly Ser Asp Ser Thr Tyr Leu Ala Glu Leu Gln Tyr Arg Tyr Lys Val 485 490 495 Asn Asn Asn Ile Thr Ile Thr Pro Gly Ala Tyr Val Leu Trp Asn Pro 500 505 510 Asn Gln Asn Asp Ala Asn Asp Thr Ile Val Val Gly Thr Val Arg Thr 515 520 525 Thr Phe Ser Phe 530 <210> 3 <211> 567 <212> PRT <213> PCC 7122 <400> 3 Met Ser Asn Leu Leu Trp Lys Ser Leu Val Val Ser Pro Ala Val Leu 1 5 10 15 Gly Ala Ala Leu Leu Val Ser Gly Thr Ala Ile Ala Ser Pro Ala Ala 20 25 30 Gly Asn Ser Ala Ser Asn Ala Ala Thr Glu Val Ser Ile Leu Glu Thr 35 40 45 Gln Ala Ala Thr Glu Val Val Gln Gln Pro Val Val Leu Ala Gln Val 50 55 60 Lys Gln Asp Thr Gln Val Leu Asp Gln Val Lys Arg Tyr Ser Asn Glu 65 70 75 80 Gly Gln Asn Leu Gln Ser Gln Val Thr Ser Val Ser Gln Phe Ser Asp 85 90 95 Val Gln Pro Thr Asp Trp Ala Phe Gln Ala Leu Gln Ser Leu Val Glu 100 105 110 Arg Tyr Gly Cys Ile Ala Gly Tyr Pro Asn Gly Thr Tyr Arg Gly Asn 115 120 125 Arg Ala Leu Thr Arg Tyr Glu Phe Ala Ala Gly Leu Asn Ala Cys Leu 130 135 140 Asp Arg Val Asn Glu Leu Ile Ala Thr Ala Thr Ala Asp Met Val Thr 145 150 155 160 Lys Gln Asp Leu Ala Thr Leu Gln Arg Leu Gln Glu Glu Phe Ser Ala 165 170 175 Glu Leu Ala Thr Leu Arg Gly Arg Val Asp Ala Leu Glu Ala Arg Thr 180 185 190 Ala Glu Leu Glu Ala Asn Gln Phe Ser Thr Thr Thr Lys Leu Gln Gly 195 200 205 Glu Ala Ile Phe Ala Leu Thr Asp Ala Phe Asn Gly Asp Ile Ala Gly 210 215 220 Lys Thr Val Phe Gln Asp Arg Val Arg Leu Asn Leu Gln Ser Ser Phe 225 230 235 240 Thr Gly Arg Asp Met Leu Asn Thr Arg Leu Ala Ala Gly Asn Ala Gln 245 250 255 Asn Phe Gly Asn Thr Gly Glu Ser Arg Gln Thr Phe Asp Val Gly Ser 260 265 270 Thr Gly Thr Asn Asn Val Val Leu Asp Lys Leu Thr Tyr Glu Ala Pro 275 280 285 Val Gly Pro Ala Gln Val Tyr Ile Ala Ala Thr Gly Gly Lys His Ser 290 295 300 His Tyr Ala Ala Val Asn Asn Pro Tyr Phe Phe Asp Lys Thr Asp Gly 305 310 315 320 Gly Asn Gly Ala Leu Ser Thr Phe Ser Ser Glu Asn Pro Ile Tyr Arg 325 330 335 Ile Gly Gly Gly Ser Gly Ile Ala Leu Asn Val Pro Phe Gly Gln Gly 340 345 350 Gly Gly Ile Leu Lys Pro Ser Ser Leu Thr Leu Gly Tyr Leu Ala Ser 355 360 365 Glu Ala Asn Ser Pro Ala Ala Gly Glu Gly Leu Phe Asp Gly Asn Tyr 370 375 380 Ala Ala Leu Gly Gln Leu Asn Phe Asn Val Gly Asn Arg Ile Ala Leu 385 390 395 400 Ala Ala Thr Tyr Val His Gly Tyr His Gly Ala Ser Ser Arg Leu Phe 405 410 415 Asp Ser Gly Glu Gly Asp Asp Ile Val Gly Thr Ser Leu Ala Asn Asn 420 425 430 Leu Ser Leu Gly Ser Ala Ser Ser Ser Asn Ser Tyr Gly Val Ser Ala 435 440 445 Ala Phe Arg Pro Ser Asp Lys Leu Ser Val Ser Gly Phe Val Ser Tyr 450 455 460 His Asp Ile Thr Gly Phe Gly Ala Gly Asp Asp Tyr Glu Ala Trp Ser 465 470 475 480 Tyr Gly Val Gly Val Ala Leu Pro Asp Phe Gly Lys Lys Gly Asn Val 485 490 495 Leu Gly Ile Phe Gly Gly Ala Gln Pro Tyr Ser Phe Asn Asn Asn Gly 500 505 510 Gly Ala Asn Ser Gly Asp Ile Pro Tyr Gln Val Glu Gly Phe Tyr Lys 515 520 525 Tyr Arg Val Ser Asp Asn Ile Ser Val Thr Pro Gly Val Ile Trp Leu 530 535 540 Pro Ser Ile Gly Gln Ser Ser Ser Ser Glu Asp Thr Phe Ile Gly Thr 545 550 555 560 Leu Arg Thr Thr Phe Thr Phe 565 <210> 4 <211> 300 <212> PRT <213> Synechocystis sp. PCC6803 <400> 4 Met Arg Val Ile Leu Cys Gly Tyr Tyr Gly Gln Asp Asn Ala Gly Asp 1 5 10 15 Glu Ala Leu Leu Val Cys Leu Leu Gln Met Leu Pro Ala Thr Val Glu 20 25 30 Pro Val Val Leu Ser Ala Asn Pro Gln Val Thr Thr Ala Arg Tyr Gly 35 40 45 Val Lys Ala His Tyr Asn Arg Asp Trp Gly Lys Ile Trp Gln Leu Leu 50 55 60 Gly Gln Cys Asp Gly Phe Ile Trp Gly Gly Gly Ser Leu Met Gln Asp 65 70 75 80 Val Thr Ser Val Val Ser Pro Leu Tyr Tyr Gly Gly Leu Met Ala Ile 85 90 95 Ala Gln Met Arg Arg Leu Lys Thr Ile Ala Trp Ala Gln Gly Ile Gly 100 105 110 Pro Leu Arg Arg Pro Pro Leu Arg Trp Phe Thr Gln Gln Val Leu Arg 115 120 125 Gly Cys Ser Gly Ile Ser Val Arg Asp Gln Ala Ser Leu Gln Leu Val 130 135 140 Lys Gln Trp Gly Leu Lys Ala Phe Leu Ala Ala Asp Pro Val Trp Ser 145 150 155 160 Leu Ala Ala Glu Asn Ala Ser Ser Ser Pro Ser Pro Leu Pro Met Val 165 170 175 Ala Val Asn Leu Arg Ala His Pro Leu Leu Thr Leu Glu Arg Leu Ala 180 185 190 Val Ile Thr Glu Ala Leu Arg Asp Phe Gln His Gln Thr Gln Thr His 195 200 205 Val Arg Leu Ile Pro Leu Gln Lys Ser Gln Asp Leu Ala Ile Ala Glu 210 215 220 Ala Ile Ala Val Glu Leu Pro Gly Ser His Glu Ile Leu Tyr Arg Pro 225 230 235 240 Asp Pro Arg Glu Cys Lys Gly Leu Phe Arg Gly Ala Glu Phe Thr Ile 245 250 255 Gly Met Arg Leu His Ser Leu Ile Met Ala Ala Ala Glu Gly Ser Ala 260 265 270 Cys Phe Ala Leu Ser Tyr Asp Pro Lys Val Ser Arg Leu Ile Ala Glu 275 280 285 Val Gly Leu Pro Gly Arg Glu Leu Ala Asp Leu Pro 290 295 300 <210> 5 <211> 339 <212> PRT <213> Synechocystis sp. PCC7942 <400> 5 Met Arg Ala Leu Leu Cys Gly Tyr Tyr Gly Glu Gly Asn Gly Gly Asp 1 5 10 15 Glu Ala Leu Leu Ala Ala Leu Leu Gln Leu Leu Pro Pro Thr Val Glu 20 25 30 Pro Leu Val Leu Ser Gly Asn Pro Ala Ala Thr Arg Thr Cys Tyr Gly 35 40 45 Val Glu Ala Cys Asp Arg Arg Ser Gly Ala Ala Val Trp Gln Ala Leu 50 55 60 Arg Arg Ser Asp Leu Phe Ile Phe Gly Gly Gly Ser Leu Ile Gln Asp 65 70 75 80 Val Thr Ser Trp Thr Ser Pro Leu Tyr Tyr Cys Gly Leu Met Gly Leu 85 90 95 Ala Gln Gln Leu Gly Leu Lys Thr Ile Ala Trp Ala Gln Gly Ile Gly 100 105 110 Pro Leu Gln Arg Lys Arg Thr Arg Trp Leu Ala Arg Arg Thr Phe Gln 115 120 125 His Cys Asp Arg Ile Thr Val Arg Asp Arg Gly Ser Ala Ala Leu Leu 130 135 140 Lys Glu Trp Gly Ile Ser Val Ala Ile Ala Pro Asp Pro Val Trp Ala 145 150 155 160 Leu Glu Pro Leu Pro Leu Asp Gln Pro Leu Ser Asn Gln Glu Ala Ile 165 170 175 Ala Val Cys Leu Arg Pro His Pro Glu Leu Thr Pro Glu Arg Ser Asp 180 185 190 Arg Leu Lys Ala Ala Leu Thr Thr Leu Gln Ser Gln Thr Gln Ala Pro 195 200 205 Ile Leu Leu Ile Pro Phe His His Gln Gln Asp Arg Pro Leu Ala Ala 210 215 220 Gln Trp Ala Ser Glu Ile Pro Gln Ala Glu Val Leu Asp Trp Gln His 225 230 235 240 Pro Arg Gln Leu Leu Ser Tyr Phe Gln Ser Val Arg Leu Thr Ile Ala 245 250 255 Met Arg Phe His Gly Leu Val Met Ala Ala Ala Ala Gly Asn Arg Cys 260 265 270 Phe Gly Leu Ser Tyr Asp Pro Lys Val Arg Arg Phe Leu Asp Ala Leu 275 280 285 Glu Gln Pro Gly Trp Asp Leu Pro Glu Ile Pro Asp Ser Ala Glu Ala 290 295 300 Ile Ala Ala Ala Trp Leu Glu Ser Trp Gln Ala Glu Pro Ile His Ala 305 310 315 320 Ala Ala Ile Ala Asp Leu Arg Gln Gln Val Asp Val His Arg Gln Ala 325 330 335 Leu Val Phe <210> 6 <211> 350 <212> PRT <213> PCC 7122 <400> 6 Met Val Lys Ile Arg Ala Leu Leu Ser Gly Tyr Tyr Gly Lys Gly Asn 1 5 10 15 Gly Gly Asp Glu Ala Leu Leu Ala Thr Leu Leu Gln Met Leu Pro Pro 20 25 30 Asp Val Thr Pro Val Val Leu Ser Gly Asn Pro Glu Glu Thr Lys Lys 35 40 45 Arg Tyr Gly Val Glu Ser Tyr Asn Arg Met Ala Phe Leu Gln Ile Ile 50 55 60 Lys Ala Leu Arg Ser Cys Asp Ala Phe Ile Trp Gly Gly Gly Ser Leu 65 70 75 80 Met Gln Asp Ala Thr Ser Ser Ile Ser Pro Phe Tyr Tyr Gly Gly Leu 85 90 95 Met Ala Leu Ala Gln Ala Met Gly Leu Lys Thr Val Ala Trp Gly Gln 100 105 110 Gly Ile Gly Pro Leu Leu Arg Ser Gln Thr Arg Trp Leu Ala Lys Arg 115 120 125 Asn Phe Ala Leu Cys Thr Lys Ile Ser Val Arg Asp Arg Ser Ser Ala 130 135 140 Ala Leu Leu Ser Asn Trp Gly Ile Pro His Ile Leu Ala Pro Asp Pro 145 150 155 160 Val Trp Ala Leu Glu Ser Lys Pro Val Thr Glu Leu Ala Asp Leu Pro 165 170 175 Lys Pro Arg Ile Ala Val Ile Leu Arg Asn His Pro Gln Leu Thr Glu 180 185 190 Thr Arg Leu Ala Asn Leu Ile His Ala Leu Val Asp Leu Gln Gln Ala 195 200 205 Thr Gln Ala Phe Ile Leu Leu Leu Pro Phe Gln Lys Ser Glu Asp Leu 210 215 220 Asp Ile Ala Glu Lys Ile Gln Leu Gln Leu Lys Asp Val Ser Lys Ile 225 230 235 240 Ile Cys Ala Glu Asp Pro Gln Leu Leu Lys Gly Ile Phe Arg Gly Val 245 250 255 Glu Met Ala Ile Gly Met Arg Leu His Ser Leu Ile Met Ala Ala Ser 260 265 270 Glu Gly Cys Arg Cys Phe Ala Leu Ser Tyr Asp Pro Lys Val Asn His 275 280 285 Leu Met Glu Asp Leu Glu Ile Pro Gly Trp Asp Leu Lys Asp Leu Pro 290,295,300 Thr Asp Val Asp Met Served Thr Thr Trp Ile Gln Tyr Tyr His Asn 305 310 315 320 Ser Glu Ala Leu Ser Glu Lys Ile Gln Ser Leu Val Asn Gly Ala 325 330 335 Phe Leu His Arg Asp Leu Arg Ala Ser Leu Ser Asn Lys 340 345 350 <210> 7 <211> 1352 <212> DNA <213> PCC6803 <400> 7 atgcttaaac tatcttggaa gtcctgtta gtgagccctg ctgtgatcgg tgccgccctt 60 gtggctggtg ctgctagtgc tgctcccgac aacgttacga atgctcaagt tttagaccaa 120 ttagaccaat acacctgccga aggtcagtct tctgccattg atcagtac cagcgttagc 180 gaactccgcg atgtgcacc cactgcctgg gcctatgaag cgctcaagg cttggtggaa 240 cgctacggtt gtatcgttgg ttaccctgac cgtaccttcc gtggtgatcg agccctcagc 300 cgttgggaat ttgccgctgg tcttaacgcc tgtatgaacg ttatggaacg tttgattcag 360 gaaaacgtgg ctgttctccg ggaggacatc gacaaactga aacgcctcat gcaggaattc 420 gaggcagaat tggctgcttt gggtgctcgg gttgataacc tcgaagcccg cacctctttc 480 ctcgaagacc atcagttttc taccaccacc aaattgaccg gggaagtaat atttgctccc 540 accgccattt ttggtaccaa aaagactctc aacaaccaaa ccgataacaa ccaagcggta 600 ttccagaacc gggttcgttt acagttcaac accagcttct ccggtgaaga cctgttggtc 660 actcgtctcg ccgctggtaa cggtaatcgc ttcaagagct tctatcctac tcagaatgtt 720 attgatcccg agaccggtaa ccccatcggt attgtaggtg actactatga aagccccacc 780 tttacccagg tgcaccagct ttcccccggc gacaacaaca acgtagccgt tgactggtta 840 gcttactatg ttcccttgga tctgggcgaa aacttccgtc tcaacaacta cattgctgct 900 tggggcggta tctgggatga cttcgttcct accctgaacc cctatttcga cgactacact 960 ggcggcaaag gttccttgag tcagttcacc gcccagaacc ccatctactc cattggtggc 1020 ggtactggta taggtacaag cctagagttg ggcttcctca gcaatctgtt gggccccact 1080 tccttgagct tgggttactt ggctagcacg ggcaacaacc ccagcagtgg tggttctgtt 1140 actaaccctg ccactggcaa caattatgac ttcagcagcg gtggtaacgg tttgtttaac 1200 ggtggctata gcgccttggc tcaaattacc accaacattt tcgaccgggt tagcctaggc 1260 ttcacctacg ttaatgccta caccacccct gatgccgcta tcttcggtaa aggtggcact 1320 caaggtatcg tcggtaccac tgcggctaac ct 1352 <210> 8 <211> 1353 <212> DNA <213> NIES-970 <400> 8 atgtctaaag caatgagaaa tcttctcagt ggtgcaaccg ttgctctcgg tgcaactgtt 60 gctttttctg gcgcagcgat cgccgaagaa aaccaagccg tcctcgacca gatcagtcaa 120 tacagcactg ataactccgt tgctcaggtc aacagcgttt tccagctcag cgacgtttcc 180 ccttccgatt gggctttga cgcgctccgc aacttggttg aaaactacaa ctgtatcgtt 240 ggttatcctg atggtacttt ccgtggcact cgccccctca gccgttatga gttcgccgca 300 ggtctgaatg cttgtttgca acagatcgaa cggatgctcg aaggtggcgg tgaagtaacc 360 caagaagatc tcgcagccct ccgtcgtctg ctcaacgaat ttgaagcaga actagctacc 420 ctcggcgctc gtgttgatga ccttgatggc cgtgttgagt tccttgaaga caatcagttc 480 tccaccacca ccaaactgaa tggggaagtt atcttcaacc ttggtggcgc ttttggcgat 540 agcagagctg atggtcctgg cccaatcgat gatgagttca cattcagcaa tcgtgttcgt 600 ctgaacctag acactagctt cactggtcgc gatcgcctcc gcactcgtct tgaagctggc 660 aatatcactc gcttagaccg tgcaactggt gtagatgcag cccgtctcgg ctttgacact 720 aacaacggta atgatgttga aattgggaaa ttgttctacc gcttccctgt aggtgacaac 780 attactgcct atgtcggtgc cgttggccta ggtcttgatg acatatttga tgtaaccaac 840 ccatattttg agagcagcgg taatggtgcc ctctctcgtt tcggtcgttt taaccccatc 900 taccgtggcc ccgatggtgc tggtgctggt tttaacttcg atcttggtga aagagttacc 960 ttctccgcag cttaccttgc ggatgatggt ggtgctagca gccctgtctc gaagaatggc 1020 ctgttcaatg gcaactacac tgcgggtgct cagctcgatt ttgctccctc tgacaacatc 1080 atgctgtctg ccgtttatac ccgtcgctac caacagggtg gcgatgtcaa cctgagcggt 1140 agcactggta gtgatttgc tgcagatcct tttggttttg ctgcttcttc caacaacttc 1200 ggcttacaag gtagctggtt gatcggtgaa aaattcaacc ttggtggctg ggtcggctac 1260 accaatgctg atctcaaaga tcctgttgtc accggtaacg ctgatgtttg gaactgggct 1320 gttaacgccg ctctacttga tgtcggcggc gaa 1353 <210> 9 <211> 1360 <212> DNA <213> PCC 7122 PCC 7122 <400> 9 atgtctaatc tattgtggaa atccttggtg gttagcccag ccgtgttggg agccgcctta 60 ttagtttcag gaacagcgat cgcatcgccc gccgcaggca actcagcctc aaatgctgct 120 actgaagtat caatactaga aacacaagcg gcaacagagg ttgttcaaca acctgtagta 180 ttggctcaag tcaagcaaga cactcaggtt ttagatcaag ttaaacgcta cagtaacgaa 240 ggtcaaaatc tacaatctca agtaacatca gtctctcagt tttccgatgt ccaaccaact 300 gactgggcat tccaagcttt gcagtccttg gtggaacgtt atggttgtat cgcaggttat 360 cccaatggta catatcgcgg aaaccgtgct ttaacccgtt atgaatttgc cgctggtttg 420 aatgcctgtt tagaccgggt gaatgaattg attgccacag caacagctga catggtgaca 480 aaacaggatt tagccacctt acagcgcttg caagaagaat tttctgctga attggcaact 540 ttacgcggtc gtgtcgatgc tttggaagca cgcactgctg agttagaagc taatcaattc 600 tctactacca ccaaattgca aggtgaagct atttttgctc tgaccgatgc ttttaatggt 660 gacattgccg gtaagaccgt ttttcaagat agagtgcgtt tgaacctgca aagcagcttc 720 acaggtcgag atatgttgaa tacccgtttg gctgcgggta atgcacaaaa ctttggaaac 780 actggggaaa gtagacaaac ctttgacgtt ggtagcactg ggactaacaa tgttgtgtta 840 gacaaactga cctatgaagc tcctgttggt ccagcacagg tctacatagc agctacaggt 900 ggtaaacaca gccattatgc tgctgttaac aacccttact tctttgataa aactgatggt 960 ggtaacggtg ctttaagtac ctttagttct gaaaacccca tctatcgaat tggtggtggt 1020 tctggtattg cccttaatgt accctttggt caaggtggcg gtattctcaa accaagttca 1080 ttgacattgg gttatttggc ttctgaggct aatagtcctg ctgctggtga aggtttgttc 1140 gacggtaact atgctgcttt aggtcagtta aactttaacg ttggtaatcg cattgcctta 1200 gctgctactt atgtacatgg ttatcatggt gctagtagcc gtttgtttga ctcaggagag 1260 ggtgatgata tcgtaggtac atcactagct aataatctca gtttaggtag cgcctcgtcc 1320 agcaactcct acggtgtttc tgctgccttc agacctagcg 1360 <210> 10 <211> 1032 <212> DNA <213> Synechocystis sp. PCC6803 <400> 10 atgcgtgtca ttctgtgtgg ttactacggc caagacaatg ctggggatga agccctttta 60 gtctgtttgc tgcaaatgct tcctgcgacg gtggagccag tggtgctctc cgccaatccc 120 caggtcacca cagcgagata cggtgttaaa gcccactaca accgagactg gggcaaaatt 180 tggcagcttt tgggtcaatg tgacggtttt atctgggggg gaggcagttt gatgcaggat 240 gtgaccagcg tggttagtcc cctatactac ggtggtttga tggcgatcgc ccagatgcgg 300 cgtttaaaaa ccattgcctg ggcccagggc attggccccc ttagaaggcc gccgttgcgc 360 tggtttaccc aacaggtgct ccggggttgt agcggcatca gcgtacggga tcaagcttct 420 ttgcaattag ttaaacaatg gggcttaaag gcttttttag cggcggatcc ggtgtggtct 480 ttagccgctg aaaatgcgtc ttcttccccc agccctttgc ccatggtggc ggtgaatttg 540 cgggcccatc ctttgttaac cttggaacgc ttggcggtca ttaccgaagc cctaagggat 600 tttcaacacc agacccaaac ccatgtgcgg ctcattcccc tacaaaaatc ccaggatttg 660 gccattgccg aagcgatcgc cgtggaattg ccggggagtc atgaaattct ttaccgcccc 720 gaccccaggg aatgtaaagg tttgttccgg ggggcggaat tcaccattgg catgcggctc 780 cacagtttaa tcatggccgc ggcggagggc agtgcctgct ttgcccttag ttatgaccct 840 aaagttagtc gtttaatcgc agaggtggga ctaccgggcc gggaattggc ggatttaccc 900 agggatagca acgaactcag ccaggtttgg cagggacatt tccggcaaag acaaccctct 960 accggggtag agttcttgca aaaatctgcc cttcagcatc aaactttgtt gcagaaaatt 1020 tttgttgact ag 1032 <210> 11 <211> 1020 <212> DNA <213> Synechocystis sp. PCC7942 <400> 11 atgcgggcgt tgctctgtgg ctattacgga gagggcaatg ggggagatga ggcgctgttg 60 gcagcgctgt tgcaactctt gccgccgaca gtggaaccgc tggtgctgtc ggggaatcct 120 gcggcaactc gcacttgcta cggcgtagaa gcttgcgatc gccgatcggg ggctgcggtt 180 tggcaggcac tgcgacgcag tgatctattc atctttggcg gcggcagcct gattcaagat 240 gtcacaagct ggacgagccc tctctactac tgcggtctga tggggctggc acaacagtta 300 ggtctcaaaa cgatcgcctg ggcccagggc attgggccac tgcagcgcaa acgaacccgc 360 tggttggcac gccgaacgtt tcagcactgc gatcgcatta cagtgcgcga tcgcggttct 420 gctgccttgc taaaagagtg ggggatttct gtggcgattg ccccggatcc cgtttgggct 480 ttagagccgc tgcctttaga tcagccgctg tcgaaccaag aggcgatcgc ggtttgtctg 540 cggccccacc ctgagttgac gcctgagcgc agcgatcgct tgaaagctgc cctgacaact 600 ctgcaatcgc aaacccaagc acccatcctg ctgattccct tccaccacca gcaagatcga 660 ccactggccg cgcaatgggc aagcgaaatt ccccaagcag aagttttgga ctggcagcat 720 ccgcgccagc tcctcagcta ctttcagtcg gtgcgattaa cgatcgccat gcgcttccat 780 ggattagtga tggcagcagc ggctggaaat cgttgctttg gcctcagcta tgaccccaaa 840 gtgcgccgtt tcctggatgc ccttgagcaa cccggctggg atttgccgga gattccagac 900 tctgctgagg cgatcgcggc cgcttggtta gagtcttggc aagcagaacc gattcatgcc 960 gctgcgatcg cggacctccg tcagcaagtg gatgtccatc gccaggcttt agttttttga 1020 <210> 12 <211> 1053 <212> DNA <213> Cylindrospermum sp. PCC 7122 <400> 12 ctacttatta ctcaatgatg ctctcaataa atcccgatgt aaaaaagctc catttactaa 60 agattgtatt ttctcagatg ataatgcttc gctattgtga taatattgaa tccaggtttt 120 actcattaaa tctacatcag ttggtaatc ctttaaatcc catccaggta tttctaatc 240. ttccatcaaa tggttaacct tggggtcata actcaaagca aaacaacgac aaccttcact agctgccata attaactgt gtagacgcat ccctattgcc atttctacac cacgaaatat accttttaaa agttgtggat cttctgcaca gataatttta ctaacatctt ttaattgcag ttgaattttt tctgcaatat ctaaatcttc acttttttga aaaggcaata ataaaataaa tgcttgggta gcttgttgta aatcaacta ggcatggatt aaatttgcta accgagtttc tgtaagttgg ggatgatttc ttaaataac ggcaattcta ggttttggta aatctgctaa ttctgttact ggtttagatt ctaaagccca aactggatca ggtgcaagaa tatgaggtat gccccaattt gataataaag ccgcactaga gcgatcgcgt acactgattt tagtacaaag agcaaaattt cgctttgcta accaacgagt ttgcgatcgc aataaaggcc ctattccctg 720 tccccaagct acagttttca aacccatcgc ttgagctaa gccattaatc ccccataata aaaagggcta atgctgctag tcgcatcctg cattaaactt ccaccacccc agatgaaagc atcacaggaa cgcaaagctt tgataatctg caaaaacgcc atacggttgt aactttccac 900 accataacgc tttttagttt cctcaggatt cccagaaagc accacaggcg ttacatcagg 960 tggtagcatt tgcagaagtg tcgccaacaa agcttcatca ccaccattac ctttaccgta 1020 atacccagac aataacgccc gtatcttcac cat 1053 <210> 13 <211> 335 <212> PRT <213> Cyanidioschyzon merolae NIES-547 <400> 13 Met Ala Pro Glu Cys Thr Val Val Asn Ser Gly Ile Thr Ser Val Gly 1 5 10 15 Asp Val Arg Thr Ser Ala Thr Ala Gly Val Gln Thr Asn Ser Val Tyr 20 25 30 Val Thr Ala Gln Leu Pro Gln Pro Val Val Glu Ala Gln Thr Phe Pro 35 40 45 Val Pro Ile Thr Val Asn Lys Thr Val Ser Lys Thr Tyr Ala Ala Gln 50 55 60 Asp Ile Pro Lys Asn Glu Lys Glu Ser Val Ser Thr Val Gln Ile Pro 65 70 75 80 Val Lys Glu Tyr Thr Lys Ile His Tyr Asp Tyr Glu Leu Pro Glu Asn 85 90 95 Asn Val Glu Thr Val Asp Glu Val Lys Asp Ile Ile Val Ser Asp Phe 100 105 110 Ile Ser Glu Thr Tyr Glu Phe Asp Phe Pro Glu Asp Thr Ile Lys Glu 115 120 125 Glu Lys Val Ser Ile Pro Val Tyr Glu Glu Val Val Thr Thr Lys Thr 130 135 140 Tyr Lys Val Asn Leu Pro Gln Asp Ser Glu Thr Arg Val Glu Asn Thr 145 150 155 160 Phe Thr Val Pro Ala Thr Lys Tyr Thr Thr Thr Thr Tyr Asp Tyr Cys 165 170 175 Gly Gln Ile Pro Glu Cys Lys Thr Asp Tyr Lys Glu Tyr Thr Gln Lys 180 185 190 Leu Asp Val Thr Lys Thr Glu Glu Thr Lys Tyr Lys Tyr Glu Val Pro 195 200 205 Leu Lys Asp Lys Ala Leu Cys Pro Glu Asp Leu Thr Val Glu Gly Lys 210 215 220 Ile Lys Thr Ala Lys Val Val Asn Tyr Asp Leu Pro Ala Asp Ser Val 225 230 235 240 Lys Glu Glu Thr Ser Glu Glu Thr Ile Asn Gln Thr Val Val Tyr Ala 245 250 255 Thr Asp Phe Lys Pro Leu Pro Cys Asn Ser Gly Asn Cys Pro Ala Lys 260 265 270 Gly Ala Val Pro Ser Val Tyr Asp Phe Gly Ser Asn Pro Ile Leu Asn 275 280 285 Pro Gln Leu Asp Ala Glu Thr Gln Ala Val Asp Leu Gln Ala Asp Tyr 290 295 300 Met Asp Met Ile Ile Gly Ala Ser Ser Glu Ala Val Asp Ser Asp Ile 305 310 315 320 Leu Ile Asp Ser Gln Ser Glu Glu Ala Val Glu Ile Glu Leu Leu 325 330 335 <210> 14 <211> 375 <212> PRT <213> Cyanidium sp. NIES-547 <400> 14 Met Ser Arg Pro Glu Asn Val Ala Ser Leu Pro Ile Gly Glu Gly Ala 1 5 10 15 Ala Pro Ala His Pro Ile Pro Ser Ile Pro Ala Ala Glu Cys Lys Pro 20 25 30 Ala Ala Arg Ile Glu Pro Glu Thr Ser Gln Lys Tyr Gly Thr Leu Tyr 35 40 45 Thr Thr Gln Val Pro Tyr Ile Gln Thr Thr Thr Glu Tyr Glu Glu Tyr 50 55 60 Asp Ile Pro Glu Asp Ser Val Ile Val Glu Thr Arg Pro Ile Lys Val 65 70 75 80 Pro Phe Ser Glu Thr Thr Thr Lys Thr Trp Glu Val Glu Leu Pro Pro 85 90 95 Ala Gly Gln Glu Val Ser Tyr Gly Thr Leu Ala Leu Pro Val Glu Lys 100 105 110 Val Val His Thr Ser Tyr Asp Tyr Gln Leu Pro Ala Lys Gln Ala Thr 115 120 125 Thr Glu Pro Ser Pro Ala Glu Ile Thr Ala Lys Lys Ile Thr Ser Ile 130 135 140 Asp Tyr Asp Tyr Glu Leu Pro Glu Asn Lys Ala Val Tyr Thr Thr Lys 145 150 155 160 Val Tyr Met Ile Pro Ile Glu Glu Thr Ile Val Gln Thr Lys Thr Tyr 165 170 175 Ser Ala Lys Leu Pro Ser Asp Tyr Glu Ala Glu Glu Lys Thr Asp Val 180 185 190 Glu Ile Ala Gly Arg Lys Leu Thr Thr His Thr Tyr Glu Tyr Lys Val 195 200 205 Pro Ser Cys Asp Thr Lys Val Tyr Ser Asp Leu Val Asp Val Asn Val 210 215 220 Lys Gln Ser Val Thr Lys Ser Tyr Ala Tyr Lys Leu Pro Asp Cys Glu 225 230 235 240 Lys Pro Val Glu Thr Lys Phe Ala Thr Ala Asp Gly Val Glu Thr Ile 245 250 255 Thr Leu Thr Lys Asp Tyr Leu Leu Pro Cys Glu Asn Thr Thr Thr Lys 260 265 270 Leu Asn Pro Thr Glu Ala Ser Val Lys Ala Ala Ala Ser Ser Ala Ala 275 280 285 Thr Cys Asn Asn Cys Gly Ser Ser Pro Ser Val Asn Glu Val Arg Tyr 290 295 300 Gln Tyr Asp Pro Ala Lys Val Thr Val Thr Lys Gln Leu Glu Glu Ser 305 310 315 320 Met Gln Gln Ala Glu Asp Asp Ala Ile Ala Gly Glu Leu Ala Asn Met 325 330 335 Ser Ser Asp Ala Glu Ser Met Pro Leu Ala Glu Ser Ser Asp Val Ala 340 345 350 Asn Val Glu Ile Ser Glu Glu His Cys Glu Leu Ile Ser Glu Leu Asp 355 360 365 Asn Ile Asp Ala Glu Leu Leu 370 375 <210> 15 <211> 1008 <212> DNA <213> Cyanidioschyzon merolae NIES-547 <400> 15 atggccccgg agtgcaccgt cgtgaactct ggcatcacca gcgttggtga tgtccgcacc 60 tctgccaccg ctggcgtgca gaccaactcc gtctacgtga cggcccagct gccccagccg 120 gtcgttgagg cgcagacctt cccggtcccg atcaccgtca acaagacggt gtcgaagacc 180 tatgcggccc aggacatccc gaagaacgag aaggagtccg tgtcgactgt ccagatcccg 240 gtgaaggagt acaccaagat ccactacgac tatgagctcc cggagaacaa cgtcgagacc 300 gtcgatgagg tgaaggacat catcgtgtcc gacttcatct ccgagaccta cgagttcgac 360 ttcccggagg acaccatcaa ggaggagaag gtgtcgatcc cggtgtacga ggaggtcgtg 420 accaccaaga cctacaaggt caacctgccc caggacagcg agacccgcgt cgagaacact 480 ttcaccgtgc cggcgaccaa gtacaccacc accacctacg actactgcgg tcagatcccg 540 gagtgcaaga ctgactacaa ggagtacacc cagaagctcg acgtgacgaa gaccgaggag 600 accaagtaca agtacgaggt gccgctcaag gacaaggcgc tctgcccgga ggacctcacc 660 gtcgagggca agatcaagac cgctaaggtc gtgaactacg atctgcccgc ggactccgtg 720 aaggaggaga ccagcgagga gaccattaac cagactgtcg tgtacgcgac tgacttcaag 780 cccctgccct gcaactctgg caactgcccg gcgaagggtg cggtgccgtc ggtgtacgac 840 ttcggctcga accccatcct gaacccccag ctcgacgcgg agacgcaggc ggttgacctc 900 caggcggatt acatggacat gatcatcggc gctagctctg aggcggtgga ctctgacatc 960 ctcatcgaca gccaaagcga ggaggcggtc gagatcgagc ttctgtaa 1008 <210> 16 <211> 1128 <212> DNA <213> Cyanidium sp. NIES-547 <400> 16 atgtcgcgcc ccgagaacgt tgcgagcctg ccgatcggcg agggcgctgc cccggcgcac 60 ccgattccca gcatcccggc ggctgagtgc aagcccgcgg ctcgcattga gcccgagacc 120 tcgcagaagt atggcactct ctacaccacc caggtgccgt acatccagac taccactgag 180 tacgaggagt acgacatccc ggaggactcg gtgatcgtcg agacccgccc gattaaggtc 240 ccgttctcgg agaccaccac gaagacctgg gaggtcgagc tcccgcccgc gggccaggag 300 gtttcgtacg gcactctcgc cctgccggtt gagaaggtcg tgcacacctc gtacgactac 360 cagctgccgg cgaagcaggc gaccaccgag ccgtccccgg ctgagatcac ggcgaagaag 420 atcacctcga tcgactacga ctatgagctt cctgagaaca aggcggttta caccacgaag 480 gtgtacatga tccccattga ggagaccatc gtccagacca agacctactc ggctaagctc 540 ccgtcggact acgaggcgga ggagaagact gatgttgaga tcgccggccg gaagctgacc 600 acgcacacct acgagtacaa ggtcccgagc tgcgacacca aggtttacag cgacctcgtc 660 gacgtgaacg tgaagcagtc cgtgaccaag tcgtacgcct acaagcttcc ggactgcgag 720 aagcctgttg agactaagtt cgcgaccgcg gacggtgttg agaccattac tctcaccaag 780 gactacctcc tgccctgcga gaacaccacc accaagctca acccgactga ggcgtcggtc 840 aaggcggctg cgtcgtcggc ggcgacctgc aacaactgcg gctctagccc ctcggtcaac 900 gaggtccgct accagtatga cccggccaag gtcactgtga ccaagcagct ggaggagtcg 960 atgcagcagg cggaggatga tgcgattgcg ggcgagctgg cgaacatgtc gtctgatgcg 1020 gagtcgatgc ccctggcgga gtcgagcgat gtcgcgaacg ttgagatcag cgaggagcac 1080 tgcgagctca ttagcgaact cgacaacatt gatgccgagc tgctgtaa 1128 <210> 17 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 17 cagtgaattc gagctcggta tatagcgttg cagtccctgg 40 <210> 18 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 18 gattacgcca agcttgcatg accgcggtca cttcataacc 40 <210> 19 <211> 47 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 19 cagtgaattc gagctcggta ataaccgttg tcccttttgt ttcatcg 47 <210> 20 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 20 tgttagtgag ccctgctgtt agctcccagt atctctatca ctgat 45 <210> twenty one <211> 51 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> twenty one acagcagggc tcactaacag ttttagagct agaaatagca agttaaaata a 51 <210> twenty two <211> 44 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> twenty two gattacgcca agcttgcatg gggaacaagc tgaatctggg catc 44 <210> twenty three <211> 45 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> twenty three ttttagtctg tttgctgcat agctcccagt atctctatca ctgat 45 <210> twenty four <211> 51 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> twenty four tgcagcaaac agactaaaag ttttagagct agaaatagca agttaaaata a 51 <210> 25 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 25 tagatataat gtgtggtaaa ttagaggaat tcatcgca 38 <210> 26 <211> 41 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 26 cgctcttccg atctgattta aaatgtgaac gtcgtccgta g 41 <210> 27 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 27 tgttagtgag ccctgctgtt agctcccagt atctctatca ctgat 45 <210> 28 <211> 51 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 28 acagcagggc tcactaacag ttttagagct agaaatagca agttaaaata a 51 <210> 29 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 29 tccctggttc cgctggggct atggccccgg agtgcaccgt 40 <210> 30 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 30 atttgatgcc tggctctagt ttacagaagc tcgatctcga 40 <210> 31 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 31 tagcaggtac tgcaaacgct atgccgctcc cggtgttcgc 40 <210> 32 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Synthetic primers <400> 32 ttagaactgg taaacgatac ttactgatcg ataacgagcg 40 <210> 33 <211> 19 <212> DNA <213> Synechocystis PCC6803 <400> 33 acagcagggc tcactaaca 19 <210> 34 <211> 19 <212> DNA <213> Synechocystis PCC6803 <400> 34 tgcagcaaac agactaaaa 19

Claims

1. The purpose of modifying cyanobacteria for improving the efficiency of electron transfer with the outside, wherein: (i) the function of the protein participating in the combination of outer membrane and cell wall in cyanobacteria is suppressed, the outer membrane has a peeling part that is partially peeled off from the cell wall so that the electrons in the cell leak to the extracellular, or (ii) a channel protein that improves the protein permeability of the outer membrane is expressed, The modified cyanobacteria supplies electrons to the outside, in, The protein involved in the binding of the outer membrane to the cell wall is at least one of an SLH (Surface Layer Homology) domain-maintaining outer membrane protein and a cell wall-pyruvate modifying enzyme, The SLH domain-maintaining outer membrane protein is Slr1841 consisting of the amino acid sequence shown in SEQ ID NO: 1, The cell wall-pyruvate modification enzyme is Slr0688 consisting of the amino acid sequence shown in SEQ ID NO: 4, The channel protein is CppS composed of the amino acid sequence shown in sequence number 13.

2. The use according to claim 1, wherein The modified cyanobacteria generates electrons by receiving light, and releases the generated electrons to the outside of the outer membrane.

3. The purposes of modifying cyanobacteria for improving the efficiency of electron transfer with the outside, wherein: (i) the function of the protein participating in the combination of outer membrane and cell wall in cyanobacteria is suppressed, the outer membrane has a peeling part that is partially peeled off from the cell wall so that the electrons in the cell leak to the extracellular, or (ii) a channel protein that improves the protein permeability of the outer membrane is expressed, The modified cyanobacteria supplies electrons to the outside, in, In (i), the gene expressing the protein involved in the binding of the outer membrane to the cell wall is deleted or inactivated, In the above (ii), a gene encoding a channel protein that increases the protein permeability of the outer membrane is introduced. The gene expressing the protein involved in the binding of the outer membrane to the cell wall is at least one of a gene encoding an SLH (Surface Layer Homology) domain-maintaining outer membrane protein and a gene encoding a cell wall-pyruvate modifying enzyme, The gene encoding the SLH domain-maintaining outer membrane protein is slr1841 consisting of the base sequence shown in SEQ ID NO: 7, The gene encoding the cell wall-pyruvate modification enzyme is slr0688 consisting of the base sequence shown in SEQ ID NO: 10, The gene encoding the channel protein is cppS consisting of the base sequence shown in SEQ ID NO:

15.

4. A method for improving the efficiency of electron transfer between modified cyanobacteria and outside, wherein: in the modified cyanobacteria: (i) the function of the protein participating in the combination of outer membrane and cell wall in the cyanobacteria is suppressed, the outer membrane has a peeling-off part that is partially peeled off from the cell wall so that the electrons in the cell leak to the extracellular, or (ii) a channel protein that is expressed to improve the protein permeability of the outer membrane, in, The protein involved in the binding of the outer membrane to the cell wall is at least one of an SLH (Surface Layer Homology) domain-maintaining outer membrane protein and a cell wall-pyruvate modifying enzyme, The SLH domain-maintaining outer membrane protein is Slr1841 consisting of the amino acid sequence shown in SEQ ID NO: 1, The cell wall-pyruvate modification enzyme is Slr0688 consisting of the amino acid sequence shown in SEQ ID NO: 4, The channel protein is CppS composed of the amino acid sequence shown in sequence number 13.

5. An electron transfer method using the modified cyanobacterium according to any one of claims 1 to 3.

Images