Engineered Cyanobacteria

Abstract

This electron carrier (30) comprises a modified cyanobacterium (31) that has at least one modification selected from (i) suppression or deletion of the function of a protein which participates in the binding of the outer membrane (5) to the cell wall (4) in the cyanobacterium, and (ii) expression of an organic substance channel protein (18) which improves the protein permeability of the outer membrane 5, wherein the modified cyanobacterium (31) supplies electrons to the outside and / or incorporates electrons from the outside.

Description

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

[0001] The present disclosure relates to electron carriers, including engineered cyanobacteria, methods for producing electron carriers, and methods for electron transfer.

[0002] Technological developments utilizing the phenomenon of electron transfer between microbial cells and the extracellular environment have attracted attention in recent years. For example, Gram-negative bacteria belonging to the genera Shewanelle and Geobacter are known to release electrons generated during the intracellular catabolism of organic matter via electron-transferring biomolecules such as cytochromes (Non-Patent Document 1). By incorporating this phenomenon into wastewater treatment technology, it is expected that bioelectrochemical systems (e.g., power generation devices) that can recover resources from organic wastewater can be developed (Patent Document 1). Furthermore, electrons generated intracellularly by photosynthesis can be used as microbial solar cells (Non-Patent Document 3). It has also been reported that the efficiency of electron transfer between cyanobacteria and electrodes can be improved by, for example, modifying the electrode surface (Non-Patent Documents 5 and 6) or using mediator compounds (Non-Patent Document 7). In addition to extracellular electron transfer, cyanobacteria are also known to produce various useful substances (e.g., alcohols, alkanes, and fatty acids) through photosynthesis (Patent Documents 2 and 4).

[0003] Meanwhile, technological developments are also underway to improve the efficiency of substance production by sending electrons from outside the cells into microbial cells and activating the metabolism of the microbial cells (for example, Non-Patent Document 2).

[0004] JP 2018-142408 A Japanese Patent No. 6341676 A

[0005] J. A. Gralnick and D. K. Newman, “Extracellular respiration”, Molecular Microbiology, Wiley-Blackwell, 2007, Vol.65, pp.1-11Junling 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.813-816Alistair 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.4699-4709Ducat DC et al., “Engineering cyanobacteria to generate high-value products”, Trends in Biotechnology, Elsevier BV, 2011, Vol.29, pp.95-103Hasan 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.24676-24680Jenny Z. Zhang et al., “Photoelectrochemistry of Photosystem II in Vitro vs in Vivo”, Journal of American Chemical Society, American Chemical Society, 2018, Vol.140, pp.6-9Nishio K et al., “Electrochemical detection of circadian redox rhythm in cyanobacterial cells via extracellular electron transfer”, Plant and Cell Physiology, Japanese Society of Plant Physiologists, 2015, Vol.56, pp.1053-1058.

[0006] However, in the above-mentioned conventional techniques, the efficiency of extracellular electron transport in cyanobacteria is low, and there is a demand for an improvement in the efficiency of extracellular electron transport.

[0007] Therefore, the present disclosure provides an electron carrier that includes a modified cyanobacterium with improved extracellular electron transport efficiency, thereby improving the efficiency of electron transport with the outside, a method for producing the electron carrier, and an electron transport method.

[0008] An electron carrier according to one aspect of the present disclosure includes a modified cyanobacterium in which at least one of (i) the function of a protein involved in binding between the outer membrane and the cell wall in the cyanobacterium is suppressed or lost, and (ii) a channel protein that improves the protein permeability of the outer membrane is expressed, and the modified cyanobacterium performs at least one of supplying electrons to the outside and taking in electrons from the outside.

[0009] The electron carrier and the method for producing the electron carrier of the present disclosure can provide an electron carrier with improved electron transfer efficiency with the outside. Furthermore, the electron transfer method of the present disclosure improves the efficiency of at least one of the electron donation from the electron carrier to the outside and the electron acceptance by the electron carrier from the outside.

[0010] FIG. 1 is a schematic diagram showing the cell surface of a cyanobacterium. FIG. 2 is a schematic diagram showing an example of an electron carrier according to the present embodiment. FIG. 3 is a transmission electron microscope image of an ultrathin section of the modified cyanobacterium of Example 1. FIG. 4 is an enlarged image of the dashed area A in FIG. 3. FIG. 5 is a transmission electron microscope image of an ultrathin section of the modified cyanobacterium of Example 2. FIG. 6 is an enlarged image of the dashed area B in FIG. 5. FIG. 7 is a transmission electron microscope image of an ultrathin section of the modified cyanobacterium of Comparative Example 1. FIG. 8 is an enlarged view of the dashed area C in FIG. 7. FIG. 9 is a graph showing the protein amounts (n=3, error bars=SD) in the culture solutions of the modified cyanobacteria of Examples 1, 2, and 3, and Comparative Example 1. FIG. 10 is an exploded perspective view showing an example of the configuration of an electrochemical measurement device. FIG. 11 is a schematic cross-sectional view taken along the XI-XI cross-section line in FIG. 10. Fig. 12 shows the results of measuring the current that flows when the culture solution of the modified cyanobacterium in Comparative Example 1 is irradiated with light. Fig. 13 shows the results of measuring the current that flows when the culture solution of the modified cyanobacterium in Example 2 is irradiated with light. Fig. 14 shows the results of measuring the current that flows when the culture solution of the modified cyanobacterium in Example 3 is irradiated with light.

[0011] (Findings that Form the Basis of the Present Disclosure) In recent years, technological developments that utilize the phenomenon of electron transfer between microbial cells and the extracellular environment have attracted attention. For example, it is known that gram-negative bacteria of the genera Shewanelle and Geobacter release electrons generated during the intracellular catabolism of organic matter to the outside of the cell via biomolecules with electron transfer function, such as cytochromes (Non-Patent Document 1). In this way, if the electrons released from the microorganisms are accepted by an external electrode, they can be used as a microbial fuel cell that uses organic matter as fuel. This is expected to be applied, for example, to a wastewater treatment process containing organic matter and used as a power generation device (Patent Document 1).

[0012] Meanwhile, technological development is also underway to send electrons from outside the cells to microbial cells, activate the cellular metabolism, and improve the efficiency of substance production. For example, Non-Patent Document 2 reports that by adhering indium phosphide nanoparticles to yeast cells and taking up electrons generated by the photoelectric conversion reaction of the nanoparticles into the cells, the reducing power used in intracellular metabolism can be replenished, and the efficiency of shikimic acid production from glucose as a raw material can be improved.

[0013] As described above, the use of microorganisms can realize energy production and material production that does not depend on chemical fuels and has a low environmental impact. Among them, photosynthetic microorganisms such as cyanobacteria and algae use light as an energy source to convert carbon dioxide (CO ) from the air. ２ ) can be used as a raw material, making it particularly promising as a carbon-neutral, next-generation material production system.

[0014] Cyanobacteria (also known as blue-green bacteria or blue-green algae) are a group of bacteria that use photosynthesis to split water and produce oxygen, and then use the energy they obtain to convert CO2 from the air. ２ Some species of cyanobacteria fix nitrogen (N ２ Furthermore, cyanobacteria are known to have the characteristics of rapid growth and high light utilization efficiency, and are easier to genetically manipulate than other algae species, so research and development into the use of cyanobacteria, among photosynthetic microorganisms, is actively being conducted.

[0015] For example, by utilizing the property of cyanobacteria to transfer electrons outside the cell, electrons generated within the cyanobacterial cell during water electrolysis by photosynthesis can be accepted by an external electrode, allowing the cell to be used as a microbial solar cell with a simple configuration comprising water, cyanobacteria, and a pair of electrodes (Non-Patent Document 3).

[0016] Furthermore, examples of substance production using cyanobacteria have been reported, including alcohols, alkanes, and fatty acids (Patent Document 2 and Non-Patent Document 4). Although cyanobacteria have a high photosynthetic capacity, they are unable to obtain sufficient light energy during periods of weak light, such as at night or in the evening, or on rainy or cloudy days. Therefore, if electrons could be transported from outside the cyanobacterial cell into the cell, the intracellular reducing power that is insufficient at night or during periods of weak light could be replenished from the outside, which is expected to lead to improved substance production efficiency.

[0017] As a means for improving the efficiency of electron transfer between cyanobacteria and an external electrode, modification of the electrode surface (Non-Patent Documents 5 and 6) and the use of a mediator compound (Non-Patent Document 7) have been reported.

[0018] As described above, there is hope for the development of technologies that utilize the extracellular electron transport of cyanobacteria, but the electron transport efficiency is still low, and there is a need to develop technologies that can further improve the efficiency of extracellular electron transport.

[0019] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have found that the efficiency of electron transfer between the modified cyanobacterium cell and an external electrode can be improved by partially detaching the outer membrane of the cyanobacterium from the cell wall or by improving the permeability of the outer membrane to substances. More specifically, they have found that detaching the outer membrane of the cyanobacterium or improving its permeability to substances enables the modified cyanobacterium to perform at least one of the following: secreting an intracellular electron carrier outside the cell, or taking up an extracellular electron carrier into the cell. This improves the extracellular electron transfer efficiency of the modified cyanobacterium, allowing the electron carrier containing the modified cyanobacterium to efficiently transfer electrons to the outside.

[0020] Therefore, the present disclosure provides an electron carrier with improved electron transfer efficiency with the outside, a method for producing the electron carrier, and an electron transfer method in which the efficiency of at least one of donating electrons from the electron carrier to the outside and accepting electrons from the outside is improved.

[0021] (Summary of the Present Disclosure) An overview of one aspect of the present disclosure is as follows.

[0022] An electron carrier according to one aspect of the present disclosure includes a modified cyanobacterium in which at least one of (i) the function of a protein involved in binding between the outer membrane and the cell wall in the cyanobacterium is suppressed or lost, and (ii) a channel protein that improves the protein permeability of the outer membrane is expressed, and the modified cyanobacterium performs at least one of supplying electrons to the outside and taking in electrons from the outside.

[0023] Due to the above (i), in the modified cyanobacterium, the bond between the cell wall and the outer membrane (e.g., the amount and strength of the bond) is partially reduced, making it easier for the outer membrane to partially detach from the cell wall. As a result, electrons or substances or molecules having electrons generated within the cell are more likely to leak out of the outer membrane, i.e., outside the cell. Furthermore, due to the above (ii), in the modified cyanobacterium, the protein permeability of the outer membrane is improved, thereby improving the substance permeability of the outer membrane. This allows the modified cyanobacterium to perform at least one of the following: secrete electrons or substances or molecules having electrons generated within the cell outside the cell; and take up extracellular electrons or substances or molecules having electrons into the cell. Therefore, the efficiency of extracellular electron transfer is improved in the modified cyanobacterium. Therefore, the electron carrier according to one embodiment of the present disclosure improves the efficiency of electron transfer with the outside.

[0024] The term "external" refers to a substance or molecule that exists as an individual separate from the electron carrier, such as a redox substance involved in the transfer of electrons between substances, or a molecule having a redox reactive group.

[0025] For example, in an electron carrier according to one embodiment of the present disclosure, the modified cyanobacterium may receive light to generate electrons and release the generated electrons outside the outer membrane.

[0026] This allows the modified cyanobacterium to release electrons or electron-containing substances or molecules outside the cell when exposed to light. Therefore, the electron carrier according to one embodiment of the present disclosure can generate electrons internally when irradiated with light and supply electrons or electron-containing substances or molecules to the outside.

[0027] For example, in an electron carrier according to one aspect of the present disclosure, the modified cyanobacterium may take up electrons present outside the outer membrane into the inside of the cell wall and utilize the electrons inside the cell wall.

[0028] As a result, the engineered cyanobacterium takes in electrons or electron-containing substances or molecules present outside the cell (into the cytoplasm) and generates energy (adenosine triphosphate (ATP) during, for example, the process of flowing through the photosynthetic electron transport system. The engineered cyanobacterium then uses this energy to produce organic matter from carbon dioxide. Therefore, the electron carrier according to one embodiment of the present disclosure can take in electrons from the outside to generate energy and produce organic matter such as proteins.

[0029] For example, in the electron carrier according to one embodiment of the present disclosure, in (i), the protein involved in binding between the outer membrane and the cell wall may be at least one of an SLH (Surface Layer Homology) domain-containing outer membrane protein and a cell wall-pyruvate modifying enzyme.

[0030] As a result, in the modified cyanobacterium, for example, (a) the function of at least one of an SLH domain-containing outer membrane protein that binds to the cell wall and an enzyme that catalyzes the reaction of modifying bound glycans on the surface of the cell wall with pyruvate (i.e., a cell wall-pyruvate-modifying enzyme) is suppressed or lost, or (b) the expression of at least one of an SLH domain-containing outer membrane protein and a cell wall-pyruvate-modifying enzyme is suppressed. Therefore, the bond (i.e., the amount and strength of the bond) between the SLH domain of the SLH domain-containing outer membrane protein in the outer membrane and the covalently bound glycans on the surface of the cell wall is reduced. As a result, the outer membrane is more likely to detach from the cell wall at areas where the bond between the outer membrane and the cell wall is weakened. This allows the modified cyanobacterium to perform at least one of secreting intracellular electrons or electron-containing substances or molecules extracellularly and taking extracellular electrons or electron-containing substances or molecules intracellularly, thereby improving the efficiency of extracellular electron transfer. Therefore, the electron carrier according to one embodiment of the present disclosure has improved electron transfer efficiency with the outside.

[0031] For example, in an electron carrier according to one embodiment of the present disclosure, the SLH domain-retaining outer membrane protein may be Slr1841 consisting of the amino acid sequence shown in SEQ ID NO: 1, NIES970_09470 consisting of the amino acid sequence shown in SEQ ID NO: 2, Anacy_3458 consisting of the amino acid sequence shown in SEQ ID NO: 3, or a protein having an amino acid sequence that is 50% or more identical to any of these SLH domain-retaining outer membrane proteins.

[0032] As a result, in the modified cyanobacterium, for example, (a) the function of any of the SLH domain-retaining outer membrane proteins set forth in SEQ ID NOs: 1 to 3 or a protein having an amino acid sequence that is 50% or more identical to that of any of these SLH domain-retaining outer membrane proteins is suppressed or lost, or (b) the expression of any of the SLH domain-retaining outer membrane proteins set forth in SEQ ID NOs: 1 to 3 or a protein having an amino acid sequence that is 50% or more identical to that of any of these SLH domain-retaining outer membrane proteins is suppressed. Therefore, in the modified cyanobacterium, (a) the function of an SLH domain-retaining outer membrane protein or a protein having a function equivalent to that of an SLH domain-retaining outer membrane protein in the outer membrane is suppressed or lost, or (b) the expression level of an SLH domain-retaining outer membrane protein or a protein having a function equivalent to that of an SLH domain-retaining outer membrane protein in the outer membrane is reduced. As a result, in the modified cyanobacterium, the binding amount and binding strength of the outer membrane binding domain (e.g., the SLH domain) to the cell wall is reduced, making the outer membrane more likely to partially detach from the cell wall. Therefore, the electron carrier according to one embodiment of the present disclosure has improved electron transfer efficiency with the outside.

[0033] For example, in an electron carrier according to one embodiment of the present disclosure, the cell wall-pyruvate modifying enzyme may be Slr0688 consisting of the amino acid sequence shown in SEQ ID NO: 4, Synpcc7942_1529 consisting of the amino acid sequence shown in SEQ ID NO: 5, Anacy_1623 consisting of the amino acid sequence shown in SEQ ID NO: 6, or a protein having an amino acid sequence that is 50% or more identical to any of these cell wall-pyruvate modifying enzymes.

[0034] As a result, in the modified cyanobacterium, for example, (a) the function of any of the cell wall-pyruvate modifying enzymes shown in SEQ ID NOs: 4 to 6 or a protein having an amino acid sequence that is 50% or more identical to that of any of these cell wall-pyruvate modifying enzymes is suppressed or lost, or (b) the expression of any of the cell wall-pyruvate modifying enzymes shown in SEQ ID NOs: 4 to 6 or a protein having an amino acid sequence that is 50% or more identical to that of any of these cell wall-pyruvate modifying enzymes is suppressed. Therefore, in the modified cyanobacterium, (a) the function of the cell wall-pyruvate modifying enzyme or a protein having a function equivalent to that enzyme is suppressed or lost, or (b) the expression level of the cell wall-pyruvate modifying enzyme or a protein having a function equivalent to that enzyme is reduced. As a result, covalently bonded sugar chains on the surface of the cell wall are less likely to be modified with pyruvate, thereby reducing the amount and strength of binding of the sugar chains on the cell wall to the SLH domain of the SLH domain-containing outer membrane protein in the outer membrane. As a result, in the modified cyanobacterium, the covalently bonded sugar chains on the surface of the cell wall are less likely to be modified with pyruvate, weakening the bond between the cell wall and the outer membrane and making it easier for the outer membrane to partially detach from the cell wall. Therefore, the electron carrier according to one embodiment of the present disclosure has improved electron transfer efficiency with the outside.

[0035] For example, in the electron carrier according to one embodiment of the present disclosure, in (i), a gene that expresses a protein involved in binding between the outer membrane and the cell wall may be deleted or inactivated.

[0036] As a result, in the modified cyanobacterium (i) above, the expression of a protein involved in the binding between the cell wall and the outer membrane is suppressed, or the function of the protein is suppressed or lost, resulting in a partial reduction in the binding between the cell wall and the outer membrane (i.e., the amount and strength of the binding). As a result, the outer membrane is more likely to detach from the cell wall in areas where the binding between the outer membrane and the cell wall is weakened. This allows the modified cyanobacterium (i) above to perform at least one of secreting intracellular electrons or electron-containing substances or molecules outside the cell, and taking extracellular electrons or electron-containing substances or molecules into the cell, thereby improving the efficiency of extracellular electron transfer. Therefore, the electron carrier according to one embodiment of the present disclosure improves the efficiency of electron transfer to the outside.

[0037] For example, in an electron carrier according to one embodiment of the present disclosure, the gene expressing a protein involved in the binding between the outer membrane and 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 modifying enzyme.

[0038] As a result, in the modified cyanobacterium, at least one gene encoding an SLH domain-retaining outer membrane protein and at least one gene encoding a cell wall-pyruvate-modifying enzyme is deleted or inactivated. Therefore, in the modified cyanobacterium, for example, (a) the expression of at least one of the SLH domain-retaining outer membrane protein and the cell wall-pyruvate-modifying enzyme is suppressed, or (b) the function of at least one of the SLH domain-retaining outer membrane protein and the cell wall-pyruvate-modifying enzyme is suppressed or lost. This reduces the bond (i.e., the amount and strength of bond) between the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane and the covalently bound sugar chains on the surface of the cell wall. As a result, in the modified cyanobacterium, the outer membrane is more likely to detach from the cell wall at areas where the bond between the outer membrane and the cell wall is weakened, making it easier for intracellular electrons or electron-containing substances or molecules to leak out of the cell. Therefore, the electron carrier according to one embodiment of the present disclosure allows intracellular electrons or substances or molecules containing electrons to more easily leak out of the cells in the modified cyanobacterium, thereby improving the efficiency of electron transfer with the outside.

[0039] In one embodiment of the electron carrier of the present disclosure, the gene encoding the SLH domain-retaining outer membrane protein may be slr1841 consisting of the base sequence shown in SEQ ID NO: 7, nies970_09470 consisting of the base sequence shown in SEQ ID NO: 8, anacy_3458 consisting of the base sequence shown in SEQ ID NO: 9, or a gene whose base sequence is 50% or more identical to any of these genes.

[0040] As a result, in the modified cyanobacterium, a gene encoding any of the SLH domain-retaining outer membrane proteins set forth in SEQ ID NOS: 7 to 9, or a gene having 50% or more identity to the nucleotide sequence of any of these genes, is deleted or inactivated. Therefore, in the modified cyanobacterium, (a) the expression of any of the SLH domain-retaining outer membrane proteins or a protein having a function equivalent to any of these proteins is suppressed, or (b) the function of any of the SLH domain-retaining outer membrane proteins or a protein having a function equivalent to any of these proteins is suppressed or lost. Therefore, in the modified cyanobacterium, the binding amount and strength of the binding domain (e.g., the SLH domain) that binds the outer membrane to the cell wall is reduced. As a result, the outer membrane is more likely to partially detach from the cell wall, which facilitates leakage of intracellular electrons or electron-containing substances or molecules outside the cell. This improves the extracellular electron transfer efficiency of the modified cyanobacterium, and therefore the electron carrier according to one embodiment of the present disclosure improves the efficiency of electron transfer to the outside.

[0041] For example, in an electron carrier according to one embodiment of the present disclosure, the gene encoding the cell wall-pyruvate modifying enzyme may be slr0688 consisting of the base sequence shown in SEQ ID NO: 10, synpcc7942_1529 consisting of the base sequence shown in SEQ ID NO: 11, anacy_1623 consisting of the base sequence shown in SEQ ID NO: 12, or a gene having a base sequence that is 50% or more identical to any of these genes.

[0042] As a result, in the modified cyanobacterium, a gene encoding any one of the cell wall-pyruvate-modifying enzymes shown in SEQ ID NOs: 10 to 12 or a gene that is 50% or more identical to the base sequence of a gene encoding any one of these enzymes is deleted or inactivated. Therefore, in the modified cyanobacterium, (a) the expression of any one of the cell wall-pyruvate-modifying enzymes or a protein having a function equivalent to any one of these enzymes is suppressed, or (b) the function of any one of the cell wall-pyruvate-modifying enzymes or a protein having a function equivalent to any one of these enzymes is suppressed or lost. This reduces the amount and strength of binding between the cell wall sugar chains and the SLH domain of the SLH domain-containing outer membrane protein in the outer membrane, as covalently bonded sugar chains on the cell wall surface are less likely to be modified with pyruvate. As a result, in the modified cyanobacterium, the amount of covalently bonded sugar chains on the cell wall surface that are modified with pyruvate is reduced, weakening the binding strength between the cell wall and the outer membrane and making the outer membrane more likely to partially detach from the cell wall. This allows the modified cyanobacterium to more easily release intracellular electrons or electron-containing substances or molecules outside the cell, thereby improving the efficiency of extracellular electron transfer. Therefore, the electron carrier according to one embodiment of the present disclosure improves the efficiency of electron transfer to the outside.

[0043] For example, in the electron carrier according to one embodiment of the present disclosure, in (ii), the channel protein that improves the protein permeability of the outer membrane may be CppS consisting of the amino acid sequence shown in SEQ ID NO: 13, CppF consisting of the amino acid sequence shown in SEQ ID NO: 14, or a protein having an amino acid sequence that is 50% or more identical to either of these channel proteins.

[0044] As a result, the modified cyanobacterium (ii) expresses CppS (SEQ ID NO: 13) or CppF (SEQ ID NO: 14), a channel protein that improves the protein permeability of the outer membrane, or a protein functionally equivalent to either of these channel proteins. Therefore, in the modified cyanobacterium (ii), the protein permeability of the outer membrane is improved, thereby improving the substance permeability of the outer membrane. As a result, the modified cyanobacterium (ii) can at least one of secrete intracellular electrons or electron-containing substances or molecules extracellularly and take up extracellular electrons or electron-containing substances or molecules intracellularly, thereby improving the efficiency of extracellular electron transfer. Therefore, the electron carrier according to one embodiment of the present disclosure improves the efficiency of electron transfer to the outside.

[0045] For example, in the electron carrier according to one embodiment of the present disclosure, in (ii) above, a gene encoding a channel protein that improves the protein permeability of the outer membrane may be introduced.

[0046] As a result, in the modified cyanobacterium (ii) above, a channel protein that improves the protein permeability of the outer membrane is expressed. Therefore, in the modified cyanobacterium (ii) above, the protein permeability of the outer membrane is improved, thereby improving the substance permeability of the outer membrane. As a result, the modified cyanobacterium (ii) above can at least one of secrete intracellular electrons or electron-containing substances or molecules to the outside of the cell, and take up extracellular electrons or electron-containing substances or molecules into the cell, thereby improving the efficiency of extracellular electron transfer. Therefore, the electron carrier according to one embodiment of the present disclosure improves the efficiency of electron transfer to the outside.

[0047] For example, in the electron carrier according to one embodiment of the present disclosure, the gene encoding the channel protein that improves the protein permeability of the outer membrane may be a chloroplast-derived gene, such as cppS consisting of the nucleotide sequence shown in SEQ ID NO: 15, cppF consisting of the nucleotide sequence shown in SEQ ID NO: 16, or a gene having a nucleotide sequence that is 50% or more identical to either of these genes.

[0048] As a result, in the modified cyanobacterium (ii), a gene encoding either of the channel proteins set forth in SEQ ID NO: 15 and SEQ ID NO: 16 or a gene having 50% or more identity to the nucleotide sequence of either of these genes is introduced. Therefore, in the modified cyanobacterium (ii), a protein having the function of improving protein permeability in the outer membrane or a protein having a function equivalent to that protein is expressed. This improves the protein permeability of the outer membrane in the modified cyanobacterium (ii), thereby improving the substance permeability of the outer membrane. As a result, the modified cyanobacterium (ii) can at least one of secrete intracellular electrons or electron-containing substances or molecules extracellularly and take up extracellular electrons or electron-containing substances or molecules intracellularly, thereby improving the efficiency of extracellular electron transfer. Therefore, an electron carrier according to one embodiment of the present disclosure has improved electron transfer efficiency with the outside.

[0049] Furthermore, a method for producing an electron carrier according to one aspect of the present disclosure includes producing a modified cyanobacterium in which at least one of (i) the function of a protein involved in binding between the outer membrane and the cell wall in the cyanobacterium is suppressed or lost, and (ii) a channel protein that improves the protein permeability of the outer membrane is expressed.

[0050] As a result, the produced modified cyanobacteria exhibit at least one of the following: (i) the bond between the cell wall and the outer membrane is partially weakened, making it easier for the outer membrane to partially detach from the cell wall; and (ii) the protein permeability of the outer membrane is improved, thereby improving the substance permeability of the outer membrane. Therefore, the modified cyanobacteria can at least one of secrete intracellular electrons or electron-containing substances or molecules extracellularly, and take up extracellular electrons or electron-containing substances or molecules intracellularly. As a result, the produced modified cyanobacteria exhibit improved extracellular electron transfer efficiency. Therefore, a method for producing an electron carrier according to one aspect of the present disclosure can provide an electron carrier with improved electron transfer efficiency to the outside.

[0051] An electron transfer method according to an embodiment of the present disclosure uses any one of the above electron carriers.

[0052] As a result, the electron carrier contains engineered cyanobacteria with improved extracellular electron transfer efficiency, allowing it to efficiently donate electrons to the outside (e.g., an external electrode) and efficiently accept electrons from the outside. Therefore, by using the electron carrier, the engineered cyanobacteria contained in the electron carrier can release intracellular electron carriers to the outside, efficiently supplying electrons to, for example, an external electrode and generating an electric current. Furthermore, for example, the engineered cyanobacteria contained in the electron carrier can accept electrons from the outside instead of light energy and perform photosynthesis or respiration. As a result, the engineered cyanobacteria contained in the electron carrier can also produce useful substances such as proteins within the cell and secrete them outside the cell.

[0053] Hereinafter, the embodiments will be specifically described with reference to the drawings.

[0054] The embodiments described below are all comprehensive or specific examples. The numerical values, materials, steps, and step orders shown in the following embodiments are merely examples and are not intended to limit the present disclosure. Furthermore, among the components in the following embodiments, components that are not described in the independent claims that represent the highest concepts are described as optional components.

[0055] In addition, the drawings are not necessarily strict illustrations, and in the drawings, substantially the same components are denoted by the same reference numerals, and overlapping descriptions may be omitted or simplified.

[0056] Furthermore, in the following, numerical ranges do not only represent strict meanings, but also include substantially equivalent ranges, for example, measuring the amount (e.g., number or concentration) of a protein or its range.

[0057] In this specification, the term "bacteria" and "cell" both refer to an individual cyanobacterium.

[0058] (Embodiments) [1. Definitions] In this specification, the identity of nucleotide sequences and amino acid sequences is calculated using the BLAST (Basic Local Alignment Search Tool) algorithm. Specifically, it is calculated by performing pairwise analysis using the BLAST program available on the NCBI (National Center for Biotechnology Information) website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Information on cyanobacterial and plant genes and the proteins encoded by these genes is publicly available, for example, in the above-mentioned NCBI database and Cyanobase (http: / / genome.microbedb.jp / cyanobase / ). The amino acid sequences of target proteins and the nucleotide sequences of the genes encoding those proteins can be obtained from these databases.

[0059] Cyanobacteria, also known as blue-green algae or cyanobacteria, are a group of prokaryotic organisms that perform photosynthesis by capturing light energy using chlorophyll and using the energy to induce charge separation in the reaction center chlorophyll, electrolyzing water and generating oxygen. Cyanobacteria are highly diverse, ranging from unicellular species such as Synechocystis sp. PCC 6803 to multicellular filamentous species such as Anabaena sp. PCC 7120. Cyanobacteria also vary in their habitats, ranging from thermophilic species such as Thermosynechococcus elongatus to marine species such as Synechococcus elongatus and freshwater species such as Synechocystis. Many species also possess unique characteristics, such as species with gas vesicles and toxins, such as Microcystis aeruginosa, and Gloeobacter violaceus, which lacks thylakoids and instead contains light-harvesting proteins called phycobilisomes in the plasma membrane.

[0060] In cyanobacteria, electrons are generated within the cell when water is split by photosynthesis and when organic compounds such as sugars synthesized by photosynthesis are metabolized as nutrients. The electrons generated by water splitting flow through the photosynthetic electron transport chain present on the thylakoid membrane, a membrane structure within the cytoplasm, generating a proton motive force that is used as a bioenergy source and ultimately is used in a reaction to reduce NADP+ and generate NADPH.

[0061] Meanwhile, electrons generated from organic compounds during their catabolism flow through the respiratory chains present on the cytoplasmic membrane and thylakoid membrane, generating the aforementioned proton motive force in the process, which is ultimately used in the reaction that reduces oxygen (O) to produce water (HO). It has been shown that the cytoplasmic membrane and thylakoid membrane are connected in cyanobacteria (Non-Patent Document 8: van de Meene et al., 2006, Arch. Microbiol., 184(5):259-270).

[0062] Figure 1 is a schematic diagram of the cell surface of a cyanobacterium. As shown in Figure 1, the cell surface of a cyanobacterium is composed of, from the inside out, a plasma membrane (also called inner membrane 1), peptidoglycan 2, and an outer membrane 5, a lipid membrane that forms the outermost layer of the cell. Sugar chains 3 composed of glucosamine, mannosamine, and the like are covalently bound to peptidoglycan 2, and pyruvate is bound to these covalently bound sugar chains 3 (Non-Patent Document 9: Jurgens and Weckesser, 1986, J. Bacteriol., 168:568-573). In this specification, the peptidoglycan 2 and the covalently bound sugar chains 3 are collectively referred to as the cell wall 4. The space between the plasma membrane (i.e., inner membrane 1) and the outer membrane 5 is called the periplasm, where various enzymes involved in protein degradation or three-dimensional structure formation, lipid or nucleic acid degradation, and extracellular nutrient uptake are present.

[0063] The enzyme that catalyzes the pyruvate modification of covalently linked glycan 3 on peptidoglycan 2 (hereafter referred to as cell wall-pyruvate-modifying enzyme 9) was identified in the Gram-positive bacterium Bacillus anthracis and named CsaB (Mesnage et al., 2000, EMBO J., 19:4473-4484). Among cyanobacteria whose genome sequences have been published, many species possess genes encoding proteins homologous to CsaB with an amino acid sequence identity of 30% or more. Examples include slr0688 possessed by Synechocystis sp. PCC 6803 and syn7502_03092 possessed by Synechococcus sp. 7502.

[0064] Furthermore, the cell wall of cyanobacteria is bound to an outer membrane protein 6 (slr1841 in the figure) that contains a surface layer homologous (SLH) domain 7 (Non-Patent Document 11: Kowata et al., 2017, J. Bacteriol., 199:e00371-17). It is known that the binding of this outer membrane protein to the cell wall is achieved by pyruvate modification of peptidoglycan-linked glycans (Non-Patent Document 12: Kojima et al., 2016, Biosci. Biotech. Biochem., 10:1954-1959).

[0065] Channel proteins are membrane proteins that form pathways (i.e., channels) that selectively allow specific substances to pass from the inside to the outside or from the outside to the inside of a lipid membrane (e.g., outer membrane 5). The outer membranes of common heterotrophic Gram-negative bacteria, such as Escherichia coli and Salmonella, contain large amounts of a channel protein called Porin, which selectively allows relatively low-molecular-weight nutrients, such as sugars and amino acids, to pass from the outside to the inside of the outer membrane and be taken up by the cells (Nikaido, 2003, Microbiol. Mol. Biol. Rev., 67(4):593-656). On the other hand, Porin is absent in the outer membrane 5 of cyanobacteria. Instead, ion channel proteins (e.g., SLH domain-containing outer membrane protein 6) that selectively allow only inorganic ions to pass through are abundant in the outer membrane 5. These ion channel proteins account for approximately 80% of the total protein content of the outer membrane 5 (Non-Patent Document 11). Therefore, in cyanobacteria, unless the properties of the outer membrane 5 are significantly modified 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 (i.e., outside the outer membrane 5).

[0066] Plant chloroplasts originated from cyanobacteria that lived symbiotically within primitive eukaryotic cells approximately 1.5 to 2 billion years ago and subsequently evolved into chloroplasts (Non-Patent Document 14: Ponce-Toledo et al., 2017, Curr. Biol., 27(3):386-391). Chloroplasts in glaucophytes, unicellular algae considered to be the most primitive plants, contain peptidoglycan and retain a surface structure similar to that of cyanobacteria. On the other hand, peptidoglycan is absent from the chloroplasts of seed plants, which are more evolved than unicellular algae. Furthermore, many of the outer membrane proteins of cyanobacteria were lost from the chloroplast outer membrane during evolution. Therefore, the outer membrane proteins of glaucophyte chloroplasts described above differ significantly from those of cyanobacteria. For example, the outer membrane 5 of cyanobacteria contains large amounts of ion channel proteins, such as Slr1841 (SLH domain-containing outer membrane protein 6), which allow inorganic substances to pass through. These ion channel proteins account for approximately 80% of the total protein in the outer membrane 5. Meanwhile, the outer membrane of glaucophyte chloroplasts contains large amounts of channel proteins named CppS and CppF, which allow organic substances to pass through (hereinafter also referred to as organic substance channel protein 18). These organic substance channel proteins account for more than 80% of the total protein in the outer membrane of glaucophyte chloroplasts (Non-Patent Document 15: Kojima et al., 2016, J. Biol. Chem., 291:20198-20209). CppS and CppF are channel proteins with a channel function that selectively allows the passage of relatively high molecular weight organic substances (e.g., biomolecules such as proteins) and are thought to function as a transport pathway connecting the interior of chloroplasts in plant cells with the plant cell cytoplasm. CppS and CppF are widely distributed in glaucoalgae. However, among bacteria, proteins similar to CppS and CppF are only found in bacteria belonging to the phylum Planctomycetes. Cyanobacteria do not possess CppS, CppF, or their analogous proteins (see Non-Patent Document 15).

[0067] 2. Electron Carrier Next, the electron carrier according to this embodiment will be described. Fig. 2 is a schematic diagram showing an example of an electron carrier 30 according to this embodiment.

[0068] The electron carrier 30 has the function of supplying electrons to the outside and taking in electrons from the outside. The outside is a substance or molecule that exists as an individual separate from the electron carrier 30, such as a redox substance involved in the transfer of electrons between substances or a molecule having a redox reactive group.

[0069] Here, "supplying electrons" refers not only to supplying electrons but also to supplying any substance or molecule having electrons, and "taking in electrons" refers not only to taking in electrons but also to taking in any substance or molecule having electrons.

[0070] The electron carrier 30 according to this embodiment includes a modified cyanobacterium 31 that is at least one of (i) a cyanobacterium (parent cyanobacterium, described below) in which the function of a protein involved in binding between the outer membrane 5 and the cell wall 4 has been suppressed or lost, and (ii) a channel protein (so-called organic channel protein 18) that improves the protein permeability of the outer membrane 5 has been expressed. The modified cyanobacterium 31 also performs at least one of supplying electrons to the outside of the electron carrier 30 and taking in electrons from the outside.

[0071] For example, the electron carrier 30 may be the modified cyanobacterium 31 described in (i) above, the modified cyanobacterium 31 described in (ii) above, or the modified cyanobacterium 31 described in both (i) and (ii) above.

[0072] Due to the above (i), in the modified cyanobacterium 31, the bond (e.g., the amount and strength of bond) between the cell wall 4 and the outer membrane 5 is partially reduced, making it easier for the outer membrane 5 to partially detach from the cell wall 4. Therefore, electrons or substances or molecules having electrons generated within the modified cyanobacterium 31 are more likely to leak out of the outer membrane 5, i.e., outside the cell. Furthermore, due to the above (ii), in the modified cyanobacterium 31, the protein permeability of the outer membrane 5 is improved, thereby improving the substance permeability of the outer membrane 5. This allows the modified cyanobacterium 31 to perform at least one of the following: secrete electrons or substances or molecules having electrons generated within the cell to the outside; and take up extracellular electrons or substances or molecules having electrons into the cell. As a result, the modified cyanobacterium 31 has improved extracellular electron transfer efficiency. Therefore, the electron carrier 30 according to this embodiment has improved electron transfer efficiency with the outside.

[0073] In addition, the electron carrier 30 may include at least one of the modified cyanobacteria 31 (i) and (ii) above, as well as an electron carrier substance 33, an electron mediator 35, and a conductive substance 37.

[0074] 2 , the electron carrier 30 may include the modified cyanobacterium 31, an electron carrier 33, an electron mediator 35, and an electrically conductive substance 37. This may further improve the efficiency of extracellular electron transfer in the modified cyanobacterium 31 in the electron carrier 30.

[0075] Here, the electron transfer substance 33 is a substance responsible for the electron transfer reaction, and is a so-called redox substance that includes an oxidized substance that receives electrons and a reduced substance that donates electrons. The electron transfer substance 33 is not particularly limited as long as it is a redox substance involved in the intracellular electron transfer system, and may be, for example, a peptide, a protein, a flavin, a quinone, heme iron, non-heme iron such as an iron-sulfur cluster, or a copper ion.

[0076] The electron mediator 35 is a substance, or so-called redox active species, that assists or promotes the electron transfer function of the electron transfer substance 33. The electron mediator 35 may be, for example, quinones, phenocene, ferricyanide, cytochromes, viologens, phenazines, phenoxazines, phenothiazines, ferredoxins, and derivatives thereof, or an appropriate substance may be selected depending on the type of the electron transfer substance 33.

[0077] The conductive material 37 is a material that allows electrons to move easily within the material, and may be selected from, for example, one or more materials selected from the group consisting of carbon-based materials, conductive polymers, semiconductors, and metals. Here, the term "carbon-based material" refers to a material containing carbon as a constituent component. For example, the carbon-based material may be carbon powder such as graphite, activated carbon, or carbon black; carbon fiber such as graphite felt, carbon wool, or carbon woven cloth; carbon nanotubes; carbon plates; carbon paper; or carbon disks.

[0078] Furthermore, a conductive polymer is a general term for a polymeric compound having electrical conductivity. The conductive polymer may be, for example, a polymer of a single monomer or two or more monomers having aniline, aminophenol, diaminophenol, pyrrole, thiophene, paraphenylene, fluorene, furan, acetylene, or a derivative thereof as a constituent unit. More specifically, the conductive polymer may be, for example, polyaniline, polyaminophenol, polydiaminophenol, polypyrrole, polythiophene, polyfuran, polyacetylene, etc.

[0079] Furthermore, the conductive material 37 may be a metal or a metal oxide, and from the viewpoint of further increasing the amount of current production, may be tungsten, tungsten oxide, copper, silver, platinum, gold, niobium, iron, cobalt, titanium, molybdenum, molybdenum oxide, tin, tin oxide, nickel, nickel oxide, or an alloy containing any of these, or an oxide thereof.

[0080] These configurations may be appropriately selected depending on the design of the electron carrier 30.

[0081] 3. Modified Cyanobacteria Next, a description will be given of the modified cyanobacteria 31. In this embodiment, the modified cyanobacteria 31 is included in the electron carrier 30.

[0082] For example, the modified cyanobacteria 31 generates electrons upon receiving light and releases the generated electrons to the outside of the outer membrane 5. As a result, the modified cyanobacteria 31 releases electrons or substances or molecules having electrons to the outside of the cell (i.e., outside the outer membrane 5) upon receiving light. Therefore, the electron carrier 30 can generate electrons internally upon receiving light and supply electrons or substances or molecules having electrons to the outside.

[0083] Furthermore, the modified cyanobacteria 31, for example, takes in electrons present outside the outer membrane 5 and places them inside the cell wall 4 (i.e., in the cytoplasm), where they are utilized. This allows the modified cyanobacteria 31 to take in electrons or electron-containing substances or molecules present outside the cell into the cell (into the cytoplasm), generating energy (ATP: adenosine triphosphate) during the process of flowing through the photosynthetic electron transport system. The modified cyanobacteria 31 then utilizes this energy to produce organic matter based on carbon dioxide. Therefore, the electron carrier 30 takes in electrons from the outside to generate energy, enabling the production of organic matter such as proteins.

[0084] Furthermore, the modified cyanobacteria 31 has at least one of the following characteristics: (i) the function of a protein (hereinafter also referred to as a binding-associated protein) involved in the binding between the outer membrane 5 and the cell wall 4 in cyanobacteria is suppressed or lost, and (ii) a channel protein (so-called organic channel protein 18) that improves the protein permeability of the outer membrane 5 is expressed.

[0085] First, the modified cyanobacterium 31 in (i) above will be explained.

[0086] In the above (i), the protein involved in the binding of the outer membrane 5 to 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. In this embodiment, the modified cyanobacterium 31 has the function of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 suppressed 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 modifying enzyme 9 may be suppressed 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 modifying enzyme 9) may be suppressed.

[0087] As a result, in the modified cyanobacterium 31, for example, (a) the function of at least one of the SLH domain-containing outer membrane protein 6 that binds to the cell wall and the enzyme that catalyzes the reaction of modifying the bound sugar chain on the surface of the cell wall with pyruvate (i.e., cell wall-pyruvate-modifying enzyme 9) is suppressed or lost, or (b) the expression of at least one of the SLH domain-containing outer membrane protein 6 and the cell wall-pyruvate-modifying enzyme 9 is suppressed. Therefore, the bond (i.e., the bond amount and bond strength) between the SLH domain of the SLH domain-containing outer membrane protein in the outer membrane 5 and the covalently bonded sugar chain 3 on the surface of the cell wall 4 is reduced. As a result, the outer membrane 5 is more likely to detach from the cell wall 4 at areas where the bond between the outer membrane 5 and the cell wall 4 is weakened. This allows the modified cyanobacterium 31 to perform at least one of secreting an intracellular electron transfer substance 33 outside the cell and taking up the extracellular electron transfer substance 33 into the cell, thereby improving the extracellular electron transfer efficiency. Therefore, the electron carrier 30 according to this embodiment improves the efficiency of electron transfer with the outside.

[0088] The parent microorganism of the modified cyanobacterium 31 in this embodiment may be (i) a cyanobacterium before the function of a protein involved in binding between the outer membrane 5 and the cell wall 4 is suppressed or lost, or (ii) a cyanobacterium before the channel protein (i.e., organic matter channel protein 18) that improves the protein permeability of the outer membrane 5 is expressed (hereinafter referred to as the parent cyanobacterium), and may be any type of cyanobacterium. For example, the parent cyanobacterium may be of the genus Syenechocystis, Synechococcus, Anabaena, or Thermosynechococcus, and may be Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, or Thermosynechococcus elongatus BP-1.

[0089] The amino acid sequences of (i) the proteins involved in the binding between the outer membrane 5 and the cell wall 4, and (ii) the channel protein (organic channel protein 18) that improves 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 these genes on the chromosomal DNA or plasmids can be confirmed in the above-mentioned NCBI database and Cyanobase.

[0090] In addition, the proteins involved in the binding between the outer membrane and the cell wall whose function is suppressed or lost in the modified cyanobacterium 31 in this embodiment may be from any parent cyanobacterium as long as they are possessed by the parent cyanobacterium, and are not limited by the location of the genes encoding them (e.g., on chromosomal DNA or on a plasmid).

[0091] For example, the SLH domain-containing outer membrane protein 6 may be Slr1841, Slr1908, or Slr0042 when the parent cyanobacterium is of the genus Synechocystis, NIES970_09470 when the parent cyanobacterium is of the genus Synechococcus, Anacy_5815 or Anacy_3458 when the parent cyanobacterium is of the genus Anabaena, A0A0F6U6F8_MICAE when the parent cyanobacterium is of the genus Microcystis, or A0A0F6U6F8_MICAE when the parent cyanobacterium is of the genus Cyanothese. When the parent cyanobacterium is of the genus Leptolyngbya, it may be A0A1Q8ZE23_9CYAN, etc. When the parent cyanobacterium is of the genus Calothrix, it may be A0A1Z4R6U0_9CYAN, when the parent cyanobacterium is of the genus Nostoc, it may be A0A1C0VG86_9NOSO, etc. When the parent cyanobacterium is of the genus Crocosphaera, it may be B1WRN6_CROS5, etc. When the parent cyanobacterium is of the genus Pleurocapsa, it may be K9TAE4_9CYAN, etc.

[0092] More specifically, the SLH domain-retaining outer membrane protein 6 may be, for example, Slr1841 (SEQ ID NO: 1) of Synechocystis sp. PCC 6803, NIES970_09470 (SEQ ID NO: 2) of Synechococcus sp. NIES-970, or Anacy_3458 (SEQ ID NO: 3) of Anabaena cylindrica PCC 7122. Alternatively, the protein may have an amino acid sequence that is 50% or more identical to these SLH domain-retaining outer membrane proteins 6.

[0093] As a result, in the modified cyanobacterium 31, for example, (a) the function of any one of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOs: 1 to 3 or a protein having an amino acid sequence that is 50% or more identical to any one of these SLH domain-retaining outer membrane proteins 6 may be suppressed or lost, or (b) the expression of any one of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOs: 1 to 3 or a protein having an amino acid sequence that is 50% or more identical to any one of these SLH domain-retaining outer membrane proteins 6 may be suppressed. Therefore, in the modified cyanobacterium 31, (a) the function of the SLH domain-retaining outer membrane protein 6 or a protein having a function equivalent to the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 is suppressed or lost, or (b) the expression amount of the SLH domain-retaining outer membrane protein 6 or a protein having a function equivalent to the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 is reduced. As a result, in the modified cyanobacterium 31, the binding amount and binding strength of the binding domain (e.g., SLH domain 7) of the outer membrane 5 to the cell wall 4 are reduced, making it easier for the outer membrane 5 to partially detach from the cell wall 4. Therefore, the electron carrier 30 according to this embodiment has improved electron transfer efficiency with the outside.

[0094] In general, if a protein has an amino acid sequence that is 30% or more identical to that of the other protein, the homology of the three-dimensional structure of the protein is high, and therefore the protein is likely to have the same function as the other protein. Therefore, the SLH domain-retaining outer membrane protein 6 whose function is suppressed or eliminated may be, for example, a protein or polypeptide that has an amino acid sequence that is 40% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and still more preferably 90% or more identical to the amino acid sequence of any of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOS: 1 to 3, and that has the function of binding to the covalently bound sugar chains 3 of the cell wall 4.

[0095] Furthermore, for example, cell wall-pyruvate modifying enzyme 9 may be Slr0688 or the like when the parent cyanobacterium is of the genus Synechocystis, Syn7502_03092 or Synpcc7942_1529 or the like when the parent cyanobacterium is of the genus Synechococcus, ANA_C20348 or Anacy_1623 or the like when the parent cyanobacterium is of the genus Anabaena, or CsaB when the parent cyanobacterium is of the genus Microcystis. (NCBI access ID: TRU80220), etc., and when the parent cyanobacterium is of the genus Cyanothese, it may be CsaB (NCBI access ID: WP_107667006.1), etc., and when the parent cyanobacterium is of the genus Spirulina, it may be CsaB (NCBI access ID: WP_026079530.1), etc., and when the parent cyanobacterium is of the genus Calothrix, it may be CsaB (NCBI access ID: WP_09665 8142.1) or the like, and when the parent cyanobacterium is of the genus Nostoc, it may be CsaB (NCBI access ID: WP_099068528.1) or the like, when the parent cyanobacterium is of the genus Crocosphaera, it may be CsaB (NCBI access ID: WP_012361697.1) or the like, and when the parent cyanobacterium is of the genus Pleurocapsa, it may be CsaB (NCBI access ID: WP_036798735) or the like.

[0096] More specifically, cell wall-pyruvate-modifying enzyme 9 may be, for example, Slr0688 (SEQ ID NO: 4) of Synechocystis sp. PCC 6803, Synpcc7942_1529 (SEQ ID NO: 5) of Synechococcus sp. PCC 7942, or Anacy_1623 (SEQ ID NO: 6) of Anabaena cylindrica PCC 7122. Alternatively, the cell wall-pyruvate-modifying enzyme 9 may be a protein having an amino acid sequence that is 50% or more identical to that of these cell wall-pyruvate-modifying enzymes 9.

[0097] As a result, in the modified cyanobacterium 31, for example, (a) the function of any of the cell wall-pyruvate modifying enzymes 9 shown in SEQ ID NOs: 4 to 6 or a protein having an amino acid sequence that is 50% or more identical to that of any of these cell wall-pyruvate modifying enzymes 9 is suppressed or lost, or (b) the expression of any of the cell wall-pyruvate modifying enzymes 9 shown in SEQ ID NOs: 4 to 6 or a protein having an amino acid sequence that is 50% or more identical to that of any of these cell wall-pyruvate modifying enzymes 9 is suppressed. Therefore, in the modified cyanobacterium 31, (a) the function of the cell wall-pyruvate modifying enzyme 9 or a protein having a function equivalent to that enzyme is suppressed or lost, or (b) the expression level of the cell wall-pyruvate modifying enzyme 9 or a protein having a function equivalent to that enzyme is reduced. As a result, the covalently bonded sugar chains 3 on the surface of the cell wall 4 are less likely to be modified with pyruvate, thereby reducing the amount and strength of binding of the sugar chains 3 on the cell wall 4 to the SLH domain 7 of the SLH domain-containing outer membrane protein 6 in the outer membrane 5. As a result, in the modified cyanobacterium 31, the covalently bonded sugar chains 3 on the surface of the cell wall 4 are less likely to be modified with pyruvic acid, weakening the binding force between the cell wall 4 and the outer membrane 5, making it easier for the outer membrane 5 to partially detach from the cell wall 4. Therefore, the electron carrier 30 according to this embodiment has improved electron transfer efficiency with the outside.

[0098] As mentioned above, it is said that if a protein shares 30% or more of its amino acid sequence with another protein, it is likely to have the same function as that protein. Therefore, the cell wall-pyruvate modifying enzyme 9 whose function is inhibited or eliminated may be, for example, a protein or polypeptide having an amino acid sequence that shares 40% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and still more preferably 90% or more identity with the amino acid sequence of any of the cell wall-pyruvate modifying enzymes 9 shown in SEQ ID NOS: 4 to 6, and having the function of catalyzing the reaction of modifying the covalently bonded sugar chain 3 of peptidoglycan 2 in the cell wall 4 with pyruvate.

[0099] In this specification, inhibiting or eliminating the function of the SLH domain-retaining outer membrane protein 6 means inhibiting or eliminating the ability of the protein to bind to the cell wall 4, inhibiting or eliminating the transport of the protein to the outer membrane 5, or inhibiting or eliminating the ability of the protein to function while embedded in the outer membrane 5.

[0100] In addition, suppressing or eliminating the function of the cell wall-pyruvate modifying enzyme 9 means suppressing or eliminating the function of the protein to modify the covalently bonded sugar chain 3 on the cell wall 4 with pyruvate.

[0101] The means for inhibiting or eliminating the function of these proteins is not particularly limited as long as it is a means commonly used for inhibiting or eliminating the function of a protein, and may be, for example, deleting or inactivating the gene encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9, inhibiting the transcription of these genes, inhibiting the translation of the transcription products of these genes, or administering an inhibitor that specifically inhibits these proteins.

[0102] In this embodiment, in the above (i), the modified cyanobacterium 31 may have a deleted or inactivated gene that expresses a protein involved in the binding between the outer membrane 5 and the cell wall 4.

[0103] As a result, in the modified cyanobacterium 31 described in (i) above, the expression of a protein involved in the bonding between the cell wall 4 and the outer membrane 5 is suppressed, or the function of the protein is suppressed or lost, resulting in a partial reduction in the bonding between the cell wall 4 and the outer membrane 5 (i.e., the amount and strength of bonding). As a result, the outer membrane 5 is more likely to detach from the cell wall 4 in areas where the bonding between the outer membrane 5 and the cell wall 4 is weakened. This allows the modified cyanobacterium 31 described in (i) above to perform at least one of secreting intracellular electrons or electron-containing substances or molecules outside the cell, and taking extracellular electrons or electron-containing substances or molecules into the cell, thereby improving the efficiency of extracellular electron transfer. Therefore, the electron carrier 30 according to this embodiment improves the efficiency of electron transfer with the outside.

[0104] The gene expressing a protein involved in the binding between the outer membrane 5 and the cell wall 4 may be at least one of a gene encoding an SLH domain-retaining outer membrane protein 6 and a gene encoding a cell wall-pyruvate modifying enzyme 9. In the modified cyanobacterium 31, at least one of the genes encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9 is deleted or inactivated. Therefore, in the modified cyanobacterium 31, for example, (a) the expression of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 is suppressed, or (b) the function of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 is suppressed or lost. Therefore, the binding (i.e., the binding amount and binding strength) between the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 and the covalently bound glycan 3 on the surface of the cell wall 4 is reduced. As a result, in the modified cyanobacterium 31, the outer membrane 5 is more likely to detach from the cell wall 4 at areas where the bond between the outer membrane 5 and the cell wall 4 is weakened, which makes it easier for intracellular electrons or substances or molecules having electrons to leak out of the cell. Therefore, the electron carrier 30 of this embodiment makes it easier for intracellular electrons or substances or molecules having electrons to leak out of the cell in the modified cyanobacterium 31, thereby improving the efficiency of electron transfer with the outside.

[0105] In this embodiment, in order to suppress or eliminate the function of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 in cyanobacteria, for example, the transcription of at least one of the genes encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9 may be suppressed.

[0106] For example, the gene encoding the SLH domain-containing outer membrane protein 6 may be slr1841, slr1908, or slr0042, etc., when the parent cyanobacterium is of the genus Synechocystis; nies970_09470, etc., when the parent cyanobacterium is of the genus Synechococcus; anacy_5815 or anacy_3458, etc., when the parent cyanobacterium is of the genus Anabaena; A0A0F6U6F8_MICAE, etc., when the parent cyanobacterium is of the genus Microcystis; and A0A0F6U6F8_MICAE, etc., when the parent cyanobacterium is of the genus Cyanothese. A0A3B8XX12_9CYAN, etc., and when the parent cyanobacterium is of the genus Leptolyngbya, it may be A0A1Q8ZE23_9CYAN, etc., when the parent cyanobacterium is of the genus Calothrix, it may be A0A1Z4R6U0_9CYAN, etc., when the parent cyanobacterium is of the genus Nostoc, it may be A0A1C0VG86_9NOSO, etc., when the parent cyanobacterium is of the genus Crocosphaera, it may be B1WRN6_CROS5, etc., and when the parent cyanobacterium is of the genus Pleurocapsa, it may be K9TAE4_9CYAN, etc. The nucleotide sequences of these genes can be obtained from the above-mentioned NCBI database or Cyanobase.

[0107] More specifically, the gene encoding the SLH domain-retaining outer membrane protein 6 may be slr1841 (SEQ ID NO: 7) of Synechocystis sp. PCC 6803, nies970_09470 (SEQ ID NO: 8) of Synechococcus sp. NIES-970, anacy_3458 (SEQ ID NO: 9) of Anabaena cylindrica PCC 7122, or a gene having an amino acid sequence that is 50% or more identical to these genes.

[0108] As a result, in the modified cyanobacterium 31, a gene encoding any one of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOS: 7 to 9, or a gene having 50% or more identity to the base sequence of any one of these genes, is deleted or inactivated. Therefore, in the modified cyanobacterium 31, (a) the expression of any one of the SLH domain-retaining outer membrane proteins 6 or a protein having a function equivalent to any one of these proteins is suppressed, or (b) the function of any one of the SLH domain-retaining outer membrane proteins 6 or a protein having a function equivalent to any one of these proteins is suppressed or lost. Therefore, in the modified cyanobacterium 31, the binding amount and binding strength of the binding domain (e.g., the SLH domain 7) that binds the outer membrane 5 to the cell wall 4 is reduced. As a result, the outer membrane 5 is more likely to partially detach from the cell wall 4, which makes it easier for the intracellular electron carrier 33 to leak out of the cell. This improves the extracellular electron transfer efficiency of the modified cyanobacterium 31, and therefore the electron carrier 30 according to this embodiment improves the electron transfer efficiency with the outside.

[0109] As described above, it is said that if a protein has an amino acid sequence that is 30% or more identical to that protein, it is likely to have a function equivalent to that protein. Therefore, if the nucleotide sequence of a gene encoding a protein is 30% or more identical, it is likely that a protein with a function equivalent to that protein will be expressed. Therefore, a gene encoding an SLH domain-retaining outer membrane protein 6 whose function is suppressed or eliminated may be, for example, a gene consisting of a nucleotide sequence that is 40% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and even more preferably 90% or more identical to the nucleotide sequence of any of the genes encoding the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOS: 7 to 9, and may be a gene encoding a protein or polypeptide that has the function of binding to the covalently bound sugar chain 3 of the cell wall 4.

[0110] Furthermore, for example, the gene encoding cell wall-pyruvate modifying enzyme 9 may be slr0688 or the like when the parent cyanobacterium is of the genus Synechocystis, may be syn7502_03092 or synpcc7942_1529 or the like when the parent cyanobacterium is of the genus Synechococcus, may be ana_C20348 or anacy_1623 or the like when the parent cyanobacterium is of the genus Anabaena, and may be csaB or the like when the parent cyanobacterium is of the genus Microcystis. (NCBI access ID: TRU80220), etc., and when the parent cyanobacterium is of the genus Cynahothese, it may be csaB (NCBI access ID: WP_107667006.1), etc., and when the parent cyanobacterium is of the genus Spirulina, it may be csaB (NCBI access ID: WP_026079530.1), etc., and when the parent cyanobacterium is of the genus Calothrix, it may be csaB (NCBI access ID: WP_0966 58142.1), etc., and when the parent cyanobacterium is of the genus Nostoc, it may be csaB (NCBI access ID: WP_099068528.1), etc., when the parent cyanobacterium is of the genus Crocosphaera, it may be csaB (NCBI access ID: WP_012361697.1), etc., and when the parent cyanobacterium is of the genus Pleurocapsa, it may be csaB (NCBI access ID: WP_036798735), etc. The nucleotide sequences of these genes can be obtained from the above-mentioned NCBI database or Cyanobase.

[0111] More specifically, the gene encoding cell wall-pyruvate-modifying enzyme 9 may be slr0688 (SEQ ID NO: 10) of Synechocystis sp. PCC 6803, synpcc7942_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 one having a nucleotide sequence that is 50% or more identical to these genes.

[0112] As a result, in the modified cyanobacterium 31, a gene encoding any one of the cell wall-pyruvate-modifying enzymes 9 shown in SEQ ID NOs: 10 to 12 or a gene that is 50% or more identical to the base sequence of a gene encoding any one of these enzymes is deleted or inactivated. Therefore, in the modified cyanobacterium 31, (a) the expression of any one of the cell wall-pyruvate-modifying enzymes 9 or a protein having a function equivalent to any one of these enzymes is suppressed, or (b) the function of any one of the cell wall-pyruvate-modifying enzymes 9 or a protein having a function equivalent to any one of these enzymes is suppressed or lost. As a result, the covalently bonded sugar chains 3 on the surface of the cell wall 4 are less likely to be modified with pyruvate, thereby reducing the amount and strength of binding between the sugar chains 3 on the cell wall 4 and the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5. As a result, in the modified cyanobacterium 31, the amount of covalently bonded sugar chains 3 on the surface of the cell wall 4 modified with pyruvic acid is reduced, weakening the binding force between the cell wall 4 and the outer membrane 5 and making it easier for the outer membrane 5 to partially detach from the cell wall 4. This makes it easier for intracellular electrons or electron-containing substances or molecules to leak out of the cell in the modified cyanobacterium 31, improving the efficiency of extracellular electron transfer. Therefore, the electron carrier 30 according to this embodiment improves the efficiency of electron transfer with the outside.

[0113] As described above, if the base sequence of a gene encoding a protein is 30% or more identical, it is considered highly likely that a protein having equivalent function to the protein will be expressed. Therefore, a gene encoding cell wall-pyruvate-modifying enzyme 9 whose function is suppressed or eliminated may be, for example, a gene having a base sequence that is 40% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and still more preferably 90% or more identical to the base sequence of any of the genes encoding cell wall-pyruvate-modifying enzyme 9 shown in SEQ ID NOs: 10 to 12, and encoding a protein or polypeptide that has the function of catalyzing a reaction to modify covalently bonded sugar chain 3 of peptidoglycan 2 in cell wall 4 with pyruvate.

[0114] Next, the modified cyanobacterium 31 in (ii) above will be described.

[0115] The modified cyanobacterium 31 (ii) above expresses an organic channel protein 18 that improves the protein permeability of the outer membrane 5 .

[0116] Here, expressing the organic matter channel protein 18 in the outer membrane 5 of a cyanobacterium means inserting a gene encoding the organic matter channel protein 18 into the chromosomal DNA or plasmid of a cyanobacterium, and transporting the synthesized organic matter channel protein 18 to the outer membrane 5 through transcription and translation of the gene, thereby expressing a channel function that selectively permeates proteins 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 type of promoter base sequence for transcription activation, ribosome binding sequence for translation, or signal sequence for transport to the outer membrane 5.

[0117] In this embodiment, the organic matter channel protein 18 expressed in the outer membrane 5 of the cyanobacterium may be an outer membrane channel protein derived from chloroplasts. The organic matter channel protein 18 may be, for example, CppS (SEQ ID NO: 13) or CppF (SEQ ID NO: 14) of the glaucoalgia Cyanophora paradoxa (hereinafter also referred to as C. paradoxa). Furthermore, the organic matter channel protein 18 may be a protein having an amino acid sequence that is 50% or more identical to that of CppS or CppF. The protein having an amino acid sequence that is 50% or more identical to that of CppS or CppF is not limited to a protein derived from chloroplasts, and may be, for example, a protein similar to CppS or CppF derived from a microorganism such as a bacterium.

[0118] As a result, the modified cyanobacterium 31 of (ii) expresses CppS (SEQ ID NO: 13) or CppF (SEQ ID NO: 14), which is an organic substance channel protein 18 that improves the protein permeability of the outer membrane 5, or a protein having a function equivalent to either of these organic substance channel proteins 18. Therefore, the modified cyanobacterium 31 of (ii) improves the protein permeability of the outer membrane 5, thereby improving the substance permeability of the outer membrane 5. As a result, the modified cyanobacterium 31 of (ii) can at least one of secrete intracellular electrons or electron-containing substances or molecules outside the cell and take up extracellular electrons or electron-containing substances or molecules into the cell, thereby improving the efficiency of extracellular electron transfer. Therefore, the electron carrier 30 of this embodiment improves the efficiency of electron transfer with the outside.

[0119] Generally, if the amino acid sequence of a protein is 30% or more identical, the homology of the three-dimensional structure of the protein is high, and it is therefore believed that the protein is likely to have the same function as the protein. Therefore, the organic channel protein 18 may be, for example, a protein or polypeptide having an amino acid sequence that is 40% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and still more preferably 90% or more identical to the amino acid sequence of either of the proteins shown in SEQ ID NO: 13 and SEQ ID NO: 14, and having the function of improving the permeability of the protein through the outer membrane 5.

[0120] Furthermore, in this embodiment, in (ii) above, the modified cyanobacterium 31 may be introduced with a gene encoding an organic channel protein 18 that improves the protein permeability of the outer membrane 5. As a result, the modified cyanobacterium 31 of (ii) above expresses the organic channel protein 18 that improves the protein permeability of the outer membrane 5. Therefore, in the modified cyanobacterium 31 of (ii) above, the protein permeability of the outer membrane 5 is improved, thereby improving the substance permeability of the outer membrane 5. As a result, the modified cyanobacterium 31 of (ii) above can at least one of secrete intracellular electrons or electron-containing substances or molecules outside the cell and take up extracellular electrons or electron-containing substances or molecules into the cell, thereby improving the efficiency of extracellular electron transfer. Therefore, the electron carrier 30 of this embodiment improves the efficiency of electron transfer with the outside.

[0121] The gene may be, for example, a chloroplast-derived gene. The gene encoding the chloroplast-derived organic matter channel protein 18 may be, for example, cppS (SEQ ID NO: 15) or cppF (SEQ ID NO: 16) of the glaucophyte Cyanophora paradoxa. The organic matter channel protein 18 may also be a gene having 50% or more identical base sequence to either of these genes. Thus, in the modified cyanobacterium 31 of (ii) above, a gene encoding either of the organic matter channel proteins 18 shown in SEQ ID NO: 15 and SEQ ID NO: 16 or a gene having 50% or more identical base sequence to either of these genes is introduced. Therefore, in the modified cyanobacterium 31 of (ii) above, a protein having the function of improving protein permeability in the outer membrane 5 or a protein having a function equivalent to that protein is expressed. As a result, in the modified cyanobacterium 31 of (ii) above, the protein permeability of the outer membrane 5 is improved, thereby improving the substance permeability of the outer membrane 5. As a result, the modified cyanobacterium 31 of (ii) above can at least one of secrete intracellular electrons or electron-containing substances or molecules outside the cell and take up extracellular electrons or electron-containing substances or molecules into the cell, thereby improving the efficiency of extracellular electron transfer. Therefore, the electron carrier 30 of this embodiment improves the efficiency of electron transfer with the outside.

[0122] The gene encoding the organic channel protein 18 is not limited to a 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, even more preferably 70% or more, still more preferably 80% or more, and still more preferably 90% or more identity to the base sequence of either of the above-mentioned genes cppS (SEQ ID NO: 15) and cppF (SEQ ID NO: 16), and encoding a protein or polypeptide having the function of improving the protein permeability of the outer membrane 5.

[0123] [4. Method for Producing Electron Carrier] Next, a method for producing the electron carrier 30 according to this embodiment will be described. The electron carrier 30 includes a step of producing a modified cyanobacterium 31 (hereinafter referred to as a step of producing the modified cyanobacterium 31) that exhibits at least one of the following characteristics: (i) the function of a protein involved in binding between the outer membrane 5 and the cell wall 4 in the cyanobacterium is suppressed or lost, and (ii) an organic channel protein 18 that improves the protein permeability of the outer membrane 5 is expressed.

[0124] The steps for producing the modified cyanobacterium 31 are described below. The steps for producing the modified cyanobacterium 31 include at least one of the following steps: (i) suppressing or eliminating the function of a protein involved in binding between the outer membrane 5 and the cell wall 4 in the cyanobacterium; and (ii) expressing an organic channel protein 18 that improves the protein permeability of the outer membrane 5.

[0125] 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 an SLH domain-containing outer membrane protein 6 and a cell wall-pyruvate modifying enzyme 9.

[0126] The means for suppressing or eliminating the function of the protein is not particularly limited, but 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 an inhibitor that specifically inhibits these proteins.

[0127] The means for deleting or inactivating the gene may be, for example, introducing a mutation into one or more bases in the base sequence of the gene, substituting or inserting another base sequence into the base sequence, or deleting part or all of the base sequence of the gene.

[0128] The means for inhibiting transcription of the gene may be, for example, introducing a mutation into the promoter region of the gene, inactivating the promoter by substituting or inserting another base sequence, or CRISPR interference (Non-Patent Document 16: Yao et al., ACS Synth. Biol., 2016, 5:207-212), etc. Specific techniques for introducing the mutation or substituting or inserting the base sequence may be, for example, ultraviolet irradiation, site-specific mutagenesis, or homologous recombination.

[0129] Furthermore, the means for inhibiting the translation of the transcription product of the gene may be, for example, RNA (ribonucleic acid) interference.

[0130] Any of the above means may be used to suppress or eliminate the function of a protein involved in the binding between the outer membrane 5 and the cell wall 4 in cyanobacteria, thereby producing a modified cyanobacterium 31. As a result, the binding between the cell wall 4 and the outer membrane 5 (i.e., the amount and strength of binding) is partially reduced in the modified cyanobacterium 31 produced in step (i) above, making it easier for the outer membrane 5 to partially detach from the cell wall 4. Therefore, in the modified cyanobacterium 31, intracellular electrons or substances or molecules having electrons are more likely to leak out of the cell, improving the efficiency of extracellular electron transfer.

[0131] 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 may be CppS consisting of the amino acid sequence shown in SEQ ID NO: 13 or CppF consisting of the amino acid sequence shown in SEQ ID NO: 14. Furthermore, the organic channel protein 18 may be a protein having an amino acid sequence that is 50% or more identical to that of any of these channel proteins.

[0132] 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 a plasmid of a cyanobacterium. The organic channel protein 18 synthesized through transcription and translation of the gene is then transported to the outer membrane 5 and expresses its channel function in the outer membrane 5 of the cyanobacterium. Note that 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 type of promoter base sequence for transcription activation, ribosome binding sequence for translation, or signal sequence for transport to the outer membrane 5.

[0133] As described above, the modified cyanobacteria 31 may be produced by expressing the organic channel protein 18 that improves the protein permeability of the outer membrane 5. As a result, the modified cyanobacteria 31 produced in step (ii) above has improved protein permeability in the outer membrane 5, thereby improving the substance permeability of the outer membrane 5. As a result, the modified cyanobacteria 31 can at least one of secrete intracellular electrons or electron-containing substances or molecules outside the cell, and take up extracellular electrons or electron-containing substances or molecules into the cell, thereby improving the efficiency of extracellular electron transfer.

[0134] By the above procedure, a modified cyanobacterium 31 may be produced that has at least one of the following: (i) the function of a protein involved in the binding between the outer membrane 5 and the cell wall 4 in the cyanobacterium is suppressed or lost, and (ii) an organic channel protein 18 that improves the protein permeability of the outer membrane 5 is expressed.

[0135] As a result, the modified cyanobacterium 31 produced by the above method can at least one of secrete intracellular electrons or electron-containing substances or molecules outside the cell and take up extracellular electrons or electron-containing substances or molecules into the cell, thereby improving the efficiency of extracellular electron transfer. Therefore, the method for producing the electron carrier 30 according to this embodiment can provide an electron carrier 30 with improved electron transfer efficiency with the outside.

[0136] 5. Electron Transfer Method The electron transfer method according to this embodiment uses an electron carrier 30 including any of the modified cyanobacteria 31 described above.

[0137] The electron carrier 30 includes a modified cyanobacterium 31 that is at least one of the above (i) in which the function of a protein involved in binding between the outer membrane 5 and the cell wall 4 in the cyanobacterium is suppressed or lost, and (ii) in which an organic channel protein 18 that improves the 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) taking in electrons from the outside.

[0138] As described above, in this embodiment, electrons refer to electrons or substances or molecules having electrons. Supplying electrons not only refers to supplying electrons but also refers to supplying any substance or molecule having electrons. Taking in electrons not only refers to taking in electrons but also refers to taking in any substance or molecule having electrons.

[0139] The electron carrier 30 may also be a modified cyanobacterium 31 that is at least one of (i) and (ii) above, and may include, in addition to the modified cyanobacterium 31, at least one of an electron carrier 33, an electron mediator 35, and a conductive substance 37, as shown in Figure 2.

[0140] First, the case (I) of supplying electrons to the outside will be described. Supplying electrons to the outside by the modified cyanobacterium 31 means, for example, that the modified cyanobacterium 31 receives light and produces electrons or a substance or molecule that has electrons. The substance or molecule that has electrons may be, for example, an electron carrier 33.

[0141] The modified cyanobacteria 31 may also release the generated electrons or electron-containing substances or molecules outside the outer membrane. For example, the modified cyanobacteria 31 may release a portion of the electron transfer substance 33 involved in the photosynthetic electron transport chain outside the cell (i.e., outside the outer membrane 5). The released electron transfer substance 33 may, for example, be taken up into the cell of another modified cyanobacterium 31 and participate in the generation of a bioenergy source, or electron transfer may occur via multiple other modified cyanobacteria 31. The released electron transfer substance 33 may then supply electrons to an electrode through a redox reaction with the outside (e.g., an external electrode). Electron transfer can be confirmed by measuring the current value. For example, if the modified cyanobacteria 31 are cultured, an electrode is placed in a cell suspension (so-called culture solution), and an external potential is applied, electron transfer between the cells and the electrode occurs with high efficiency, generating a current.

[0142] Next, the above-mentioned case (II) of taking up electrons from the outside will be described. Taking up electrons from the outside by the modified cyanobacteria 31 means, for example, taking up electrons or electron-containing substances or molecules present outside the outer membrane into the cell wall 4. Furthermore, the modified cyanobacteria 31 may utilize electrons or electron-containing substances or molecules inside the cell wall 4 (in the cytoplasm). As described in the prior art section, cyanobacteria generally have high photosynthetic ability and produce various organic substances within the cells. Similar to cyanobacteria, the modified cyanobacteria 31 also produces various organic substances within the cells (in the cell wall and thylakoids). In this case, the modified cyanobacteria 31 may take up electrons or electron-containing substances or molecules (which may be part of the electron carrier 33) present outside the outer membrane 5 into the cell wall 4, and then accept electrons from the electron carrier 33 inside the cell wall 4 (i.e., in the cytoplasm) to use for substance production. Furthermore, the modified cyanobacteria 31 may use electrons taken in from the outside for respiration (catabolism of organic matter). In this way, the modified cyanobacteria 31 can use electrical energy instead of light energy, making it less likely that the intracellular reducing power will be insufficient, even in environments with insufficient sunlight. Therefore, the modified cyanobacteria 31 can use both light energy and electrical energy, improving the efficiency of intracellular substance production.

[0143] Furthermore, for example, the modified cyanobacteria 31 contained in the electron carrier 30 can use electrical energy instead of light energy to stably generate the energy and reducing power required within the cell, catabolize substances, and produce substances.

[0144] As described above, the electron carrier 30 according to the present embodiment includes the modified cyanobacteria 31 with improved extracellular electron transfer efficiency, and therefore can efficiently donate electrons to the outside (e.g., an external electrode) and efficiently accept electrons from the outside. Therefore, for example, when the electron carrier 30 is used, the modified cyanobacteria 31 contained in the electron carrier 30 can release intracellular electron mediators to the outside, thereby efficiently supplying electrons to, for example, an external electrode and generating a current.

[0145] EXAMPLES Hereinafter, the electron carrier, the method for producing the electron carrier, and the electron transfer method of the present disclosure will be specifically explained in examples, but the present disclosure is not limited to the following examples in any way.

[0146] In the following examples, (i) modified cyanobacteria were prepared by suppressing or eliminating the function of a protein involved in the binding between the outer membrane and the cell wall, thereby partially detaching the outer membrane from the cell wall (Examples 1 and 2), and (ii) modified cyanobacteria were prepared by expressing a channel protein in the outer membrane that improves protein permeability, thereby improving the permeability of the outer membrane (Example 3). These three types of modified cyanobacteria were evaluated for their ability to secrete intracellular electron transfer substance 33 and perform at least one of the following functions: supplying electrons to the outside of the cell and / or transporting electrons from the outside into the cell; that is, for their ability to improve the efficiency of extracellular electron transfer between the cell and an external electrode. Evaluation was performed by quantifying and identifying the proteins secreted extracellularly by these modified cyanobacteria and measuring the intracellular current generated by photosynthesis.

[0147] The cyanobacteria species used in this example was Synechocystis sp. PCC 6803 (hereinafter simply referred to as "cyanobacteria").

[0148] Example 1 In Example 1, modified cyanobacteria were produced in which the expression of the slr1841 gene (SEQ ID NO: 7), which encodes an SLH domain-containing outer membrane protein, was suppressed.

[0149] (1) Construction of a cyanobacterial engineered strain with suppressed slr1841 gene expression. We used CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference to suppress the expression of the slr1841 gene (SEQ ID NO: 7) by introducing the gene encoding the dCas9 protein (hereafter referred to as the dCas9 gene) and the slr1841_sgRNA (single-guide ribonucleic acid) gene into the chromosomal DNA of the cyanobacterium.

[0150] The mechanism by which gene expression is suppressed by this method is as follows.

[0151] First, a Cas9 protein lacking 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.

[0152] This complex then recognizes the slr1841 gene on the chromosomal DNA of cyanobacteria and binds specifically to it. This binding creates steric hindrance, inhibiting transcription of the slr1841 gene. As a result, expression of the slr1841 gene in cyanobacteria is suppressed.

[0153] Below, we will specifically explain how to introduce each of the above two genes into the chromosomal DNA of cyanobacteria.

[0154] (1-1) Introduction of the dCas9 Gene Using the chromosomal DNA of Synechocystis LY07 strain (hereinafter also referred to as LY07 strain) (see Non-Patent Document 16) as a template, the dCas9 gene, an operator gene for regulating dCas9 gene expression, and a spectinomycin resistance marker gene serving as a marker for gene introduction were amplified by PCR (polymerase chain reaction) using the primers psbA1-Fw (SEQ ID NO: 17) and psbA1-Rv (SEQ ID NO: 18) listed in Table 1. In the LY07 strain, the above three genes are inserted in a linked state into the psbA1 gene on the chromosomal DNA, and therefore can be amplified as a single DNA fragment by PCR. Here, the resulting DNA fragment is referred to as the "psbA1::dCas9 cassette." Using the In-Fusion PCR cloning method (registered trademark), the psbA1::dCas9 cassette was inserted into the pUC19 plasmid to obtain the pUC19-dCas9 plasmid.

[0155]

[0156] One microgram of the resulting pUC19-dCas9 plasmid was mixed with a cyanobacterial culture medium (cell density OD730 = approximately 0.5), and the pUC19-dCas9 plasmid was introduced into the cyanobacterial cells by natural transformation. Transformed cells were selected by growing them on BG-11 agar medium containing 20 μg / mL spectinomycin. In the selected cells, homologous recombination occurred between the psbA1 gene on the chromosomal DNA and the psbA1 upstream and downstream fragment regions on the pUC19-dCas9 plasmid. This resulted in the generation of Synechocystis dCastis strains carrying the dCas9 cassette inserted into the psbA1 gene region. The composition of the BG-11 medium used is shown in Table 2.

[0157]

[0158] (1-2) Introduction of slr1841_sgRNA gene In CRISPR interference, a sequence of approximately 20 bases complementary to the target sequence is introduced into a region called the protospacer on the sgRNA gene, allowing the sgRNA to specifically bind to the target gene. The protospacer sequence used in this example is shown in Table 3.

[0159]

[0160] In the Synechocystis LY07 strain, the sgRNA gene (excluding the protospacer region) is linked to a kanamycin resistance marker gene and inserted into the slr2030-slr2031 gene on the chromosomal DNA. Therefore, by adding a protospacer sequence (SEQ ID NO: 33) complementary to the slr1841 gene (SEQ ID NO: 7) to the primers used to amplify the sgRNA gene by PCR, an sgRNA (slr1841_sgRNA) that specifically recognizes slr1841 can be easily obtained.

[0161] First, using the chromosomal DNA of the LY07 strain as a template, two DNA fragments were amplified by PCR using a set of primers listed in Table 1: slr2030-Fw (SEQ ID NO: 19) and sgRNA_slr1841-Rv (SEQ ID NO: 20), and a set of primers listed in Table 1: sgRNA_slr1841-Fw (SEQ ID NO: 21) and slr2031-Rv (SEQ ID NO: 22).

[0162] Next, the mixed solution of the above DNA fragments was used as a template and amplified by PCR using primers slr2030-Fw (SEQ ID NO: 19) and slr2031-Rv (SEQ ID NO: 22) listed in Table 1. (i) slr2030 gene fragment, (ii) slr1841_sgRNA, (iii) kanamycin resistance marker gene, (iv) a DNA fragment (slr2030-2031::slr1841_sgRNA) in which the slr2031 gene fragment was linked in order was obtained. 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.

[0163] The pUC19-slr1841_sgRNA plasmid was introduced into the Synechocystis dCas9 strain using the same method as in (1-1) above, and transformed cells were selected on BG-11 agar medium containing 30 μg / mL kanamycin. This resulted in a Synechocystis dCas9 slr1841_sgRNA transformant strain (hereinafter also referred to as the slr1841-suppressed strain) in which the slr1841_sgRNA was inserted into the slr2030-slr2031 gene on the chromosomal DNA.

[0164] (1-3) Suppression of the slr1841 gene The promoter sequences of the dCas9 gene and slr1841_sgRNA gene were designed so that their expression was induced in the presence of anhydrotetracycline (aTc). In this example, expression of the slr1841 gene was suppressed by adding aTc to the medium at a final concentration of 1 μg / mL.

[0165] Example 2 In Example 2, a modified cyanobacterium in which the expression of the slr0688 gene encoding a cell wall-pyruvate modifying enzyme was suppressed was obtained by the following procedure.

[0166] (2) Construction of a cyanobacterial modified strain in which expression of the slr0688 gene is suppressed Using the same procedure as in (1-2) above, an sgRNA gene containing a protospacer sequence (SEQ ID NO: 34) complementary to the slr0688 gene (SEQ ID NO: 10) was introduced into the Synechocystis dCas9 strain to obtain the Synechocystis dCas9 slr0688_sgRNA strain. The same conditions as in (1-2) above were used, except that the set of primers slr2030-Fw (SEQ ID NO: 19) and sgRNA_slr0688-Rv (SEQ ID NO: 23) and the set of primers sgRNA_slr0688-Fw (SEQ ID NO: 24) and slr2031-Rv (SEQ ID NO: 22) listed in Table 1 were used, and a DNA fragment (slr2030-2031::slr0688_sgRNA) in which (i) the slr2030 gene fragment, (ii) slr0688_sgRNA, (iii) a kanamycin resistance marker gene, and (iv) a slr2031 gene fragment were linked in this order was inserted into the pUC19 plasmid using the In-Fusion PCR cloning method (registered trademark) to obtain the pUC19-slr0688_sgRNA plasmid.

[0167] Furthermore, the expression of the slr0688 gene was suppressed by the same procedure as in (1-3) above.

[0168] (Comparative Example 1) In Comparative Example 1, the Synechocystis dCas9 strain was obtained by the same procedure as in Example 1 (1-1).

[0169] Next, the state of the cell surface was observed for each of the strains obtained in Example 1, Example 2, and Comparative Example 1. Details are described below.

[0170] (3) Observation of the state of the cell surface of the bacterial strains Ultrathin sections were prepared from the modified cyanobacterium Synechocystis dCas9 slr1841_sgRNA strain (the so-called slr1841-suppressed strain) obtained in Example 1, the modified cyanobacterium Synechocystis dCas9 slr0688_sgRNA strain (hereinafter also referred to as the slr0688-suppressed strain) obtained in Example 2, and the modified cyanobacterium Synechocystis dCas9 strain (hereinafter referred to as the control strain) obtained in Comparative Example 1, and the state of the cell surface (in other words, the outer membrane structure) was observed using an electron microscope.

[0171] (3-1) Culturing of Bacterial Strains The slr1841-suppressed strain of Example 1 was inoculated into BG-11 medium containing 1 μg / mL aTc to an initial cell concentration of OD730 = 0.05, and cultured with shaking for 5 days at a light intensity of 100 μmol / m2 / s and 30°C. The slr0688-suppressed strain of Example 2 and the control strain of Comparative Example 1 were also cultured under the same conditions as in Example 1.

[0172] (3-2) Preparation of Ultrathin Sections of Strains The culture medium obtained in (3-1) above was centrifuged at 2,500 g for 10 minutes at room temperature to recover cells of the slr1841-suppressed strain of Example 1. The cells were then rapidly frozen in liquid propane at -175°C and fixed for two days at -80°C using an ethanol solution containing 2% glutaraldehyde and 1% tannic acid. The fixed cells were dehydrated with ethanol, infiltrated with propylene oxide, and then submerged in a resin (Quetol-651) solution. The cells were then left to stand at 60°C for 48 hours to harden the resin, and the cells were embedded in the resin. The cells in the resin were sliced ​​to a thickness of 70 nm using an ultramicrotome (Ultracut) to prepare ultrathin sections. These ultrathin sections were stained with a 2% uranium acetate and 1% lead citrate solution to prepare a transmission electron microscope specimen of the slr1841-suppressed strain of Example 1. The same procedure was also carried out for the slr0688 suppressed strain of Example 2 and the control strain of Comparative Example 1, and samples for transmission electron microscopy were prepared.

[0173] (3-3) Observation by Electron Microscope The ultrathin sections obtained in (3-2) above were observed using a transmission electron microscope (JEOL JEM-1400Plus) at an accelerating voltage of 100 kV. The observation results are shown in Figures 3 to 8.

[0174] First, the slr1841-suppressed strain of Example 1 will be described. Figure 3 is a TEM (Transmission Electron Microscope) image of the slr1841-suppressed strain of Example 1. Figure 4 is an enlarged image of the dashed-line region A in Figure 3. Figure 4(a) is an enlarged TEM image of the dashed-line region A in Figure 3, and Figure 4(b) is a depiction of the enlarged TEM image of Figure 4(a).

[0175] As shown in FIG. 3, in the slr1841 suppressed strain of Example 1, the outer membrane was partially detached from the cell wall (that is, the outer membrane was partially peeled off) and the outer membrane was partially flexed.

[0176] To confirm the state of the cell surface in more detail, the dashed-line area A was magnified and observed. As shown in Figures 4(a) and 4(b), a portion where the outer membrane had partially peeled off (the dashed-dotted areas a1 and a2 in the figures) was confirmed. A portion where the outer membrane had significantly flexed was also confirmed near the dashed-dotted area a1. This portion is thought to be where the bond between the outer membrane and the cell wall was weakened, and the outer membrane expanded outward and flexed as the culture medium penetrated through the outer membrane into the periplasm.

[0177] Next, the slr0688-suppressed strain of Example 2 will be described. Figure 5 is a TEM image of the slr0688-suppressed strain of Example 2. Figure 6 is an enlarged image of the dashed-line region B in Figure 5. Figure 6(a) is an enlarged TEM image of the dashed-line region B in Figure 5, and Figure 6(b) is a depiction of the enlarged TEM image of Figure 6(a).

[0178] As shown in Figure 5, the outer membrane was partially detached from the cell wall and partially bent in the slr0688-suppressed strain of Example 2. It was also confirmed that the outer membrane was partially detached from the cell wall in the slr0688-suppressed strain.

[0179] To confirm the state of the cell surface in more detail, the dashed-line region B was magnified and observed. As shown in Figures 6(a) and 6(b), a portion where the outer membrane was significantly bent (dotted-dash-line region b1) and portions where the outer membrane had partially peeled off (dotted-dash-line regions b2 and b3) were confirmed. In addition, portions where the outer membrane had detached from the cell wall were confirmed near each of the dashed-dotted-line regions b1, b2, and b3.

[0180] Next, the control strain of Comparative Example 1 will be described. Fig. 7 is a TEM image of the control strain of Comparative Example 1. Fig. 8 is an enlarged image of the dashed line area C in Fig. 7. Fig. 8(a) is an enlarged TEM image of the dashed line area C in Fig. 7, and Fig. 8(b) is a depiction of the enlarged TEM image of Fig. 8(a).

[0181] 7, the cell surface of the control strain in Comparative Example 1 was intact, with the inner membrane, cell wall, outer membrane, and S layer remaining laminated in that order. In other words, the control strain did not show any areas where the outer membrane had detached from the cell wall, areas where the outer membrane had peeled off (i.e., fallen off) from the cell wall, or areas where the outer membrane had sagged, as in Examples 1 and 2.

[0182] (Example 3) In Example 3, a Synechocystis cppS tetR strain (hereinafter also referred to as a cppS-introduced strain) was obtained by introducing the chloroplast outer membrane channel protein CppS (SEQ ID NO: 13) contained in the glaucophyte Cyanophora paradoxa into the outer membrane of a cyanobacterium by the following procedure.

[0183] (4) Introduction of the chloroplast outer membrane channel protein CppS into the cyanobacterial outer membrane (4-1) Construction of a cppS gene expression cassette A gene cassette containing the cppS gene, a promoter region (PL22) for controlling the expression of the cppS gene, a cyanobacterial outer membrane localization signal sequence (slr0042-signal), and a kanamycin resistance marker gene (KmR) serving as a marker for gene introduction was constructed as follows.

[0184] First, the slr0042 gene was obtained by PCR amplification using cyanobacterial chromosomal DNA as a template and the primers slr0042-Fw (SEQ ID NO: 25) and slr0042-Rv (SEQ ID NO: 26) listed in Table 1. Next, the chromosomal DNA of Synechocystis LY07 strain (Non-Patent Document 16) was used as a template and amplified by PCR using a set of primers slr2030-Fw (SEQ ID NO: 19) and PL22-Rv (SEQ ID NO: 27) and a set of primers KmR-Fw (SEQ ID NO: 28) and slr2031-Rv (SEQ ID NO: 22) listed in Table 1, to obtain PL22 and KmR. In the LY07 strain, these genes are inserted into the slr2030-slr2031 gene on the chromosome, and PCR amplification using the four primers above results in the slr2030 gene fragment linked to the 5' end of PL22 and the slr2031 gene fragment linked to the 3' end of KmR. Next, using the mixture of the slr0042 gene, PL22, and KmR obtained above as a template, PCR amplification was performed using the four primers listed in Table 1 (SEQ ID NOS: 19, 22, 27, and 28) to obtain a gene cassette (slr2030-2031::slr0042-KmR cassette) in which the slr2030 gene fragment, PL22, slr0042 gene, KmR, and slr2031 gene fragment are linked in order from the 5' end. Using the In-Fusion PCR cloning method®, the slr2030-2031::slr0042-KmR cassette was inserted into the pUC19 plasmid, resulting in the pUC19-slr0042 plasmid.

[0185] In parallel with the above procedure, total cDNA was prepared from the glaucoalga C. paradoxa NIES-547 using the SMART cDNA Library Synthesis Kit (Clontech). This cDNA was used as a template for PCR amplification using the primers cppS-Fw (SEQ ID NO: 29) and cppS-Rv (SEQ ID NO: 30) listed in Table 1 to obtain the cppS gene (SEQ ID NO: 13). The cppS gene (SEQ ID NO: 13) was inserted into the pUC19-slr0042 plasmid using the In-Fusion PCR Cloning Method (registered trademark) to obtain the pUC19-CppS plasmid. This procedure inserted the cppS gene linked to the 3' end of the outer membrane localization signal sequence of the slr0042 gene, and removed the rest of the slr0042 gene except for the outer membrane localization signal sequence, replacing the cppS coding region.

[0186] (4-2) Construction of a PL22 promoter activity control cassette (tetR). The PL22 promoter activity is induced only in the presence of anhydrotetracycline (aTc) through the TetR repressor. Therefore, the tetR gene for controlling PL22 activity must be introduced into the engineered cyanobacterium.

[0187] First, the tetR gene and the spectinomycin resistance marker gene (SpcR), which serves as a marker for gene introduction, were obtained by PCR amplification using the chromosomal DNA of the LY07 strain as a template and a set of primers, psbA1-Fw (SEQ ID NO: 17) and tetR-Rv (SEQ ID NO: 31), and a set of primers, tetR-Fw (SEQ ID NO: 32) and psbA1-Rv (SEQ ID NO: 18), listed in Table 1. In the LY07 strain, these genes are inserted into the psbA1 gene on the chromosome. Therefore, when PCR was performed using the primers, the upstream fragment of the psbA1 gene was amplified in a form linked to the 5' end of the tetR gene, and the downstream fragment of the psbA1 gene was amplified in a form linked to the 3' end of SpcR. Next, a mixture of the tetR gene and SpcR was used as a template for PCR amplification using primers psbA1-Fw (SEQ ID NO: 17) and psbA1-Rv (SEQ ID NO: 18) listed in Table 1 to obtain a gene cassette (psbA1::tetR cassette) in which the upstream fragment of the psbA1 gene, tetR, SpcR, and the downstream fragment of the psbA1 gene were linked in this 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.

[0188] (4-3) Introduction of the cppS Gene Expression Cassette and tetR Cassette: 1 μg of the pUC19-cppS plasmid obtained by the above procedure was mixed with a cyanobacterial culture medium (cell density OD730 = approximately 0.5), and the plasmid was introduced into the cells by natural transformation. Transformed cells were selected by growing them on BG-11 agar medium containing 30 μg / mL kanamycin. In the selected cells, homologous recombination occurred between the slr2030-2031 gene on the chromosome and the slr2030 and slr2031 gene fragment regions on the pUC19-cppS plasmid. This resulted in a Synechocystis cppS strain with the cppS gene expression cassette inserted into the slr2030-2031 gene fragment region. The composition of the BG-11 medium used is shown in Table 2.

[0189] Next, 1 μg of the pUC19-tetR plasmid was mixed with Synechocystis cppS culture medium (cell density OD730 = approximately 0.5) and the plasmid was introduced into the cells by natural transformation. Transformed cells were selected by growing them on BG-11 agar medium containing 30 μg / mL kanamycin and 20 μg / mL spectinomycin, and a Synechocystis cppS tetR strain was obtained. As above, this strain contained the tetR cassette inserted into the psbA1 gene on its chromosomal DNA.

[0190] (4-4) Induction of cppS Gene Expression The expression of the cppS gene in the Synechocystis cppS tetR strain is induced in the presence of anhydrotetracycline (aTc). In this example, aTc was added to a BG-11 medium at a final concentration of 1 μg / mL to induce cppS gene expression.

[0191] (5) Test for Protein Secretion Productivity Whether or not proteins present in the periplasm (the space between the outer membrane and the inner membrane) are secreted outside the cells was confirmed by the following method.

[0192] More specifically, the slr1841-suppressed strain of Example 1, the slr0688-suppressed strain of Example 2, the cppS-introduced strain of Example 3, and the control strain of Comparative Example 1 were each cultured, and the amount of protein secreted outside the cells (hereinafter also referred to as secreted protein amount) was measured. The protein secretion productivity of each of the above strains was evaluated based on the amount of protein in the culture medium. Note that protein secretion productivity refers to the ability to produce a protein by secreting a protein produced inside the cell outside the cell. Specific methods are described below.

[0193] (5-1) Culturing of Strains The slr1841-suppressed strain of Example 1 was cultured in the same manner as in (3-1) above. The culture was carried out three times independently. The strains of Examples 2, 3, and Comparative Example 1 were also cultured under the same conditions as the strain of Example 1.

[0194] (5-2) Quantification of Extracellularly Secreted Proteins The culture medium obtained in (5-1) above was centrifuged at 2,500 g for 10 minutes at room temperature to obtain a culture supernatant. The resulting culture supernatant was filtered using a 0.22 μm pore-size membrane filter to completely remove the cells of the slr1841-suppressed strain of Example 1. The total protein content of the filtered culture supernatant was quantified using the BCA (bicinchoninic acid) method. This series of procedures was performed for each of three independently cultured cultures, and the average and standard deviation of the protein content secreted by the slr1841-suppressed strain of Example 1 were calculated. The protein content of the three cultures obtained in (5-1) above was also quantified under similar conditions for the strains of Examples 2, 3, and Comparative Example 1, and the average and standard deviation of the protein content in the three cultures were calculated.

[0195] The results are shown in Figure 9. Figure 9 is a graph showing the protein amounts (n=3, error bars=SD) in the culture solutions of the modified cyanobacteria of Examples 1 to 3 and Comparative Example 1.

[0196] As shown in Figure 9, the amount of protein (mg / L) secreted into the culture supernatant of each of the slr1841-suppressed strain of Example 1, the slr0688-suppressed strain of Example 2, and the cppS-introduced strain of Example 3 was approximately 25-fold higher than that of the control strain of Comparative Example 1.

[0197] Although the data are not shown here, the absorbance (730 nm) of the culture medium was measured and the amount of secreted protein per gram of dry cell weight (mg protein / g cell dry weight) was calculated. As a result, the amount of secreted protein per gram of dry cell weight (mg protein / g cell dry weight) was approximately 36-fold higher for the slr1841-suppressed strain of Example 1, the slr0688-suppressed strain of Example 2, and the cppS-introduced strain of Example 3 compared to the control strain of Comparative Example 1.

[0198] Furthermore, comparing the amounts of secreted protein (mg / L) between the "modified cyanobacteria in which the cyanobacterial outer membrane was partially detached from the cell wall" in Example 1 and Example 2 in Figure 9, the amount of protein secreted into the culture supernatant was greater for the slr0688-suppressed strain in Example 2, in which expression of the gene encoding the cell wall-pyruvate-modifying enzyme (slr0688) was suppressed, than for the slr1841-suppressed strain in Example 1, in which expression of the gene encoding the SLH domain-retaining outer membrane protein (slr1841) was suppressed. This is thought to be related to the fact that the number of covalently bound glycans on the cell wall surface is greater than the number of SLH domain-retaining outer membrane proteins (Slr1841) in the outer membrane. In other words, the amount of protein secreted from the slr0688-suppressed strain in Example 2 was greater than that of the slr1841-suppressed strain in Example 1, likely because the amount and strength of binding between the outer membrane and the cell wall were reduced more than those of the slr1841-suppressed strain in Example 1.

[0199] Furthermore, in Figure 9, when 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 matter channel protein CppS was expressed in the outer membrane of the cyanobacterium, the amount of secreted protein of the slr0688-suppressed strain of Example 2 was slightly (approximately 20 mg / L) higher than that of the cppS-introduced strain of Example 3.

[0200] These results confirmed that inhibiting the function of proteins involved in the binding between the outer membrane and the cell wall partially weakens the binding between the outer membrane and the cell wall of the cyanobacterium, causing the outer membrane to partially detach from the cell wall. It was also confirmed that weakening the binding between the outer membrane and the cell wall facilitates extracellular secretion of proteins produced within the cyanobacterial cell. Therefore, the modified cyanobacteria disclosed herein demonstrate significantly improved protein secretion productivity.

[0201] (5-3) Identification of Secreted Proteins Next, the proteins contained in the culture supernatant obtained in (5-2) above were identified by LC-MS / MS. The method is described below.

[0202] (5-3-1) Sample Preparation: Eight volumes of cold acetone were added to the culture supernatant, and the mixture was incubated at 20°C for 2 hours. The mixture was then centrifuged at 20,000 g for 15 minutes to obtain a protein precipitate. 100 mM Tris pH 8.5, 0.5% sodium dodecanoate (SDoD) was added to the precipitate, and the protein was dissolved using a closed-loop ultrasonicator. After adjusting the protein concentration to 1 μg / mL, dithiothreitol (DTT) was added to a final concentration of 10 mM and the mixture was incubated at 50°C for 30 minutes. Subsequently, iodoacetamide (IAA) was added to a final concentration of 30 mM and the mixture was incubated at room temperature (protected from light) for 30 minutes. To stop the IAA reaction, cysteine ​​was added to a final concentration of 60 mM and the mixture was incubated at room temperature for 10 minutes. 400 ng of trypsin was added and the mixture was incubated overnight at 37°C to obtain peptide fragments. After adding 5% TFA (trifluoroacetic acid), the mixture was centrifuged at 15,000 g for 10 minutes at room temperature to obtain the supernatant. This process removed SDoD. After desalting using a C18 spin column, the sample was dried using a centrifugal evaporator. 3% acetonitrile and 0.1% formic acid were then added, and the sample was dissolved using a closed-type ultrasonic homogenizer. The peptide concentration was adjusted to 200 ng / μL.

[0203] (5-3-2) LC-MS / MS Analysis The sample obtained in (5-3-1) above was analyzed using an LC-MS / MS device (UltiMate 3000 RSLCnano LC System) under the following conditions.

[0204] Sample injection volume: 200 ng Column: CAPCELL CORE MP 75 μm × 250 mm Solvent: Solvent A is 0.1% formic acid aqueous solution, Solvent B is 0.1% formic acid + 80% acetonitrile Gradient program: 8% B solvent 4 minutes after sample injection, 44% B solvent 27 minutes later, 80% B solvent 28 minutes later, and measurement completed 34 minutes later

[0205] (5-3-3) Data Analysis The obtained data was analyzed under the following conditions to identify proteins and peptides and calculate quantitative values.

[0206] Software: Scaffold DIA Database: UniProtKB / Swiss Prot database (Synechocystis sp. PCC 6803) Fragmentation: HCD Precursor Tolerance: 8 ppm Fragment Tolerance: 1.0 ppm Data Acquisition Type: Overlapping DIA Peptide Length: 8-70 Peptide Charge: 2-8 Max Missed Cleavages: 1 Fixed Modification: Carbamidomethylation Peptide FDR: ≤ 1%

[0207] Table 4 shows the 10 identified proteins in descending order of relative quantification value.

[0208]

[0209] All 10 types of proteins were contained in the culture supernatants of Examples 1 to 3. Periplasmic (referring to the gap between the outer membrane and inner membrane) localization signals were retained in all of these proteins. These results confirmed that in the slr1841-suppressed strain of Example 1 and the slr0688-suppressed strain of Example 2, the binding between the outer membrane and the cell wall was weakened, causing the outer membrane to partially detach from the cell wall, facilitating leakage of proteins in the periplasm outside the outer membrane (i.e., outside the bacterial cell). Furthermore, in the cppS-introduced strain of Example 3, expression of the cppS gene improved the protein permeability of the outer membrane, making it easier for proteins in the periplasm to permeate the channel protein CppS and be secreted outside the outer membrane (i.e., outside the bacterial cell). Therefore, the modified strains of Examples 1 to 3 were shown to have improved outer membrane permeability.

[0210] (6) Evaluation of Extracellular Electron Transfer Efficiency of Modified Cyanobacteria 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 solutions of these modified strains were irradiated with light, and the intracellular current generated by water decomposition during photosynthesis was detected with an external electrode to evaluate the extracellular electron transfer efficiency. The equipment used and the procedure for measuring the current are described below.

[0211] (6-1) Apparatus for Electrochemical Measurements First, the configuration of the apparatus for electrochemical measurements 100 will be described with reference to the drawings. Fig. 10 is an exploded perspective view that schematically shows an example of the configuration of the apparatus for electrochemical measurements 100. Fig. 11 is a schematic cross-sectional view taken along the XI-XI cross-sectional line in Fig. 10.

[0212] 10 and 11 , the electrochemical measurement device 100 includes a measurement unit 10 and a light irradiation unit 20. The measurement unit 10 includes a reaction vessel 12 having a storage unit 11 for storing a cyanobacterial culture solution 40, a first electrode 13 installed inside the reaction vessel 12 so as to be in contact with the culture solution 40 in the storage unit 11, a second electrode 14 installed inside the reaction vessel 12 so as to be in contact with the culture solution 40 in the storage unit 11, a potentiostat 15 for controlling the potential of the first electrode 13, and a reference electrode 16 installed inside the reaction vessel 12 so as to be in contact with the culture solution 40 in the storage unit 11.

[0213] The reaction vessel 12 is electrically insulating and impermeable to the culture solution 40. The reaction vessel 12 is 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.

[0214] For example, the measurement unit 10 applies a voltage or passes a current between the first electrode 13 and the second electrode 14, and measures the current or potential corresponding to the voltage or current. Here, the measurement unit 10 applies a voltage between the first electrode 13 and the second electrode 14 and measures the current.

[0215] The first electrode 13 is a so-called working electrode, and is an electrode that sensitively responds electrochemically to trace amounts of substances on the electrode surface in the culture solution 40. The second electrode 14 is a so-called counter electrode, and is an electrode that sets a potential difference between itself and the working electrode (first electrode 13) or that passes a current.

[0216] The first electrode 13 and the second electrode 14 are made of a conductive material. Examples of the conductive material include carbon materials, conductive polymer materials, semiconductors, and metals. For example, the carbon material may include carbon nanotubes, ketjen black, glassy carbon, graphene, fullerene, carbon fiber, carbon fabric, and carbon aerogel. For example, the conductive polymer material may include polyaniline, polyacetylene, polypyrrole, poly(3,4-ethylenedioxythiophene), poly(p-phenylene vinylene), polythiophene, and poly(p-phenylene sulfide). For example, the semiconductor may include silicone, germanium, indium tin oxide (ITO), titanium oxide, copper oxide, and silver oxide. For example, the metal may include gold, platinum, silver, titanium, aluminum, tungsten, copper, iron, and palladium. Here, the first electrode 13 is an indium tin oxide (ITO) electrode, and the second electrode 14 is a platinum electrode. Note that the conductive material is not particularly limited as long as it is not decomposed by its own oxidation reaction.

[0217] The reference electrode 16 is an electrode that does not react with substances in the culture solution 40 and maintains a constant potential, and is used by the potentiostat 15 to control the potential difference between the first electrode 13 and the reference electrode 16 to a constant value. In this example, the reference electrode 16 is a silver / silver chloride electrode.

[0218] 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.

[0219] The light irradiation unit 20 includes a light source 21 and a housing 22 that holds the light source 21. Although detailed configuration is not shown, the light source 21 includes, for example, one or more light emitters (e.g., LEDs (light emitting diodes)) and a reflective surface that surrounds the light emitters. The light irradiation unit 20 may be disposed a predetermined distance away from the measurement unit 10 in the positive direction of the Z axis.

[0220] 11 illustrates a configuration in which the first electrode 13, the second electrode 14, and the reference electrode 16 are each extracted below the reaction vessel 12 (i.e., in the negative Z-axis direction), but they may be configured so as not to protrude from the reaction vessel 12 as long as they can be electrically connected to the potentiostat 15. Furthermore, the first electrode 13, the second electrode 14, and the reference electrode 16 may each be extracted to the side of the reaction vessel 12.

[0221] (6-2) Measurement of Current The amount of current produced by each of the three strains, the control strain of Comparative Example 1, the slr0688-suppressed strain of Example 2, and the cppS-introduced strain of Example 3, was measured by the following procedure.

[0222] The electrochemical measurement apparatus 100 used had the following configuration.

[0223] First electrode 13: Indium tin oxide electrode (surface area: 3.14 cm 2 ) Second electrode 14: Platinum electrode Reference electrode 16: Silver / silver chloride electrode Light source 21: Approximately 120 μmol / m 2 / s white light source

[0224] (6-2-1) Preparation of culture solution First, the control strain of Comparative Example 1 was inoculated into BG-11 medium containing 1 μg / mL aTc so that the initial bacterial cell concentration OD730 = 0.1, and the culture was incubated under a light intensity of 100 μmol / m 2 The strain was cultured with shaking for 3 days at 1 / s and 30° C. The slr0688-suppressed strain of Example 2 and the cppS-introduced strain of Example 3 were also cultured under the same conditions.

[0225] (6-2-2) Measurement of Current After 4 mL of the culture solution 40 of the Control strain of Comparative Example 1 obtained in (6-2-1) above was poured into the reaction vessel 12, the potential of the first electrode 13 relative to the reference electrode 16 was controlled by the potentiostat 15 to be +0.0 V. Then, approximately 120 μmol / m 2 / s white light was irradiated for a certain period of time, and the current flowing between the first electrode 13 and the second electrode 14 was measured. Next, the potential of the first electrode 13 relative to the reference electrode 16 was changed in the order of +0.1 V, +0.2 V, +0.25 V, and +0.3 V, and the current flowing between the first electrode 13 and the second electrode 14 was similarly measured when the potential was controlled at each potential. The results are shown in Figure 12. Figure 12 shows the results of measuring the current flowing when the culture solution 40 of the control strain in Comparative Example 1 was irradiated with light.

[0226] As shown in Figure 12, in the culture solution 40 of the control strain in Comparative Example 1, when the potential of the first electrode 13 relative to the reference electrode 16 was +0.0 V, +0.1 V, +0.2 V, +0.25 V, and +0.3 V, the measured current values ​​(maximum values ​​after baseline correction) were approximately +0.0 nA, approximately +0.0 nA, approximately +0.0 nA, approximately +0.0 nA, and approximately +1.0 nA, respectively.

[0227] Next, current values ​​were measured for the slr0688-suppressed strain of Example 2 and the cppS-introduced strain of Example 3 in the same manner as in Comparative Example 1. The results are shown in Figures 13 and 14. Figure 13 shows the results of measuring the current flowing when culture solution 40 of the slr0688-suppressed strain of Example 2 was irradiated with light. Figure 14 shows the results of measuring the current flowing when culture solution 40 of the cppS-introduced strain of Example 3 was irradiated with light.

[0228] As shown in Figure 13, in the culture solution 40 of the slr0688-suppressed strain of Example 2, when the potential of the first electrode 13 relative to the reference electrode 16 was +0.0 V, +0.1 V, +0.2 V, +0.25 V, and +0.3 V, the measured current values ​​(maximum values ​​after baseline correction) were approximately +90 nA, approximately +250 nA, approximately +560 nA, approximately +750 nA, and approximately +1100 nA, respectively.

[0229] Furthermore, as shown in Figure 14, 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 +0.0 V, +0.1 V, +0.2 V, +0.25 V, and +0.3 V, the measured current values ​​(maximum values ​​after baseline correction) were approximately +10 nA, approximately +20 nA, approximately +70 nA, approximately +150 nA, and approximately +260 nA, respectively.

[0230] As shown in FIGS. 13 and 14, the current value increased in a potential-dependent manner.

[0231] From the above, when the potential of the first electrode 13 relative to the reference electrode 16 was controlled to +0.3 V, the current values ​​of the culture solution 40 of all three strains mentioned above could be measured, and these current values ​​were extracted and compared in Table 5.

[0232]

[0233] As shown in Table 5, when the potential of the first electrode 13 relative to the reference electrode 16 was controlled to +0.3 V, the current flowing in the culture solution 40 due to light irradiation (hereinafter referred to as photocurrent) was 1,000 times higher in the culture solution 40 of the slr0688-suppressed strain of Example 2 than in the culture solution 40 of the control strain of Comparative Example 1. Furthermore, when the potential of the first electrode 13 relative to the reference electrode 16 was controlled to +0.3 V in the culture solution 40 of the cppS-introduced strain of Example 3, the photocurrent was about 300 times higher than in the culture solution 40 of the control strain of Comparative Example 1.

[0234] The above results show that the modified cyanobacteria of this embodiment have an extracellular electron transport efficiency that is improved by approximately 300 to 1,000 times by partially detaching the outer membrane of the cyanobacterium from the cell wall or by improving the permeability of the outer membrane of the cyanobacterium to substances.

[0235] (7) Discussion Based on the above results, it is believed that in modified cyanobacteria in which the bond between the outer membrane and cell wall of the cyanobacterium was weakened to facilitate leakage of intracellular proteins, as in Examples 1 and 2, the outer membrane was torn and peeled off, which facilitated leakage of organic substances other than proteins (e.g., quinones) outside the cell. Therefore, it is believed that the photocurrent was approximately five times higher than that of modified cyanobacteria in which the protein permeability of the outer membrane of the cyanobacterium was improved, as in Example 3.

[0236] Therefore, in order to further improve the efficiency of electron transfer between the modified cyanobacteria and the outside, it was found that having part of the outer membrane peeled off is more effective than expressing an organic matter channel protein.

[0237] The electron carrier of the present disclosure is also highly beneficial in that it improves the protein production efficiency of the engineered cyanobacteria and allows the engineered cyanobacteria to be reused after protein recovery, thereby enabling the outer membrane to be appropriately detached and peeled off. Furthermore, based on the results of the above protein identification, it is also possible to cause the engineered cyanobacteria to secrete desired proteins by modifying the genes encoding the proteins in the periplasm. When the electron carrier of the present disclosure is used in the production of such useful substances, useful substances can be produced stably and efficiently by providing electrical energy in addition to sunlight.

[0238] The electron carrier, the method for producing an electron carrier, and the electron transfer method according to the present disclosure have been described above based on the embodiments, but the present disclosure is not limited to these embodiments. As long as they do not deviate from the gist of the present disclosure, various modifications that would occur to a person skilled in the art and other forms constructed by combining some of the components of the embodiments are also included in the scope of the present disclosure.

[0239] In the above embodiment, an example has been described in which the efficiency of extracellular electron transfer is improved by weakening the bond between the outer membrane and the cell membrane in cyanobacteria by suppressing or eliminating the function of a protein involved in the bond between the outer membrane and the cell wall, but this is not limiting. For example, the bond between the outer membrane and the cell wall may be weakened or the outer membrane may be weakened by applying an external force to the cyanobacteria. Alternatively, the outer membrane may be weakened by adding an enzyme or a drug to the cyanobacterial culture solution.

[0240] The electron carrier, the method for producing the electron carrier, and the electron carrier method of the present disclosure can efficiently donate electrons to the outside of the electron carrier, thereby enabling sludge or wastewater treatment and power generation to be performed simultaneously. Furthermore, the present disclosure enables the electron carrier to efficiently donate and receive electrons from the outside, allowing the electron carrier to utilize electrical energy when the amount of sunlight is insufficient. Therefore, the present disclosure enables efficient production of useful substances in the food, pharmaceutical, or chemical fields, soil improvement, wastewater treatment, power generation, and the like.

[0241] REFERENCE SIGNS LIST 1 Inner membrane 2 Peptidoglycan 3 Sugar chain 4 Cell wall 5 Outer membrane 6 SLH domain-containing outer membrane protein 7 SLH domain 8, 18 Organic channel protein 9 Cell wall-pyruvate modifying enzyme 10 Measuring unit 11 Storage unit 12 Reaction vessel 13 First electrode 14 Second electrode 15 Potentiostat 16 Reference electrode 20 Light irradiation unit 21 Light source 22 Housing 30 Electron carrier 31 Modified cyanobacterium 33 Electron carrier 35 Electron mediator 37 Conductive material 40 Culture solution 100 Electrochemical measurement device

Claims

1. An electron carrier comprising at least one of: (i) a modified cyanobacterium in which the function of a protein involved in binding between the outer membrane and the cell wall is suppressed or lost; and (ii) a channel protein that improves the protein permeability of the outer membrane is expressed, wherein the modified cyanobacterium performs at least one of supplying electrons to the outside and taking in electrons from the outside.

2. The electron carrier according to claim 1, wherein the modified cyanobacterium receives light to produce electrons and releases the produced electrons outside the outer membrane.

3. The electron carrier described in claim 1 or 2, wherein the modified cyanobacterium takes up electrons present outside the outer membrane into the inside of the cell wall and utilizes the electrons inside the cell wall.

4. The electron carrier according to claim 1, wherein in (i), the protein involved in binding between the outer membrane and the cell wall is at least one of an SLH (Surface Layer Homology) domain-containing outer membrane protein and a cell wall-pyruvate modifying enzyme.

5. The electron carrier described in claim 4, wherein the SLH domain-retaining outer membrane protein is Slr1841 consisting of the amino acid sequence shown in SEQ ID NO: 1, NIES970_09470 consisting of the amino acid sequence shown in SEQ ID NO: 2, Anacy_3458 consisting of the amino acid sequence shown in SEQ ID NO: 3, or a protein having an amino acid sequence that is 50% or more identical to any of these SLH domain-retaining outer membrane proteins.

6. The electron carrier according to claim 4, wherein the cell wall-pyruvate modifying enzyme is Slr0688 consisting of the amino acid sequence shown in SEQ ID NO: 4, Synpcc7942_1529 consisting of the amino acid sequence shown in SEQ ID NO: 5, Anacy_1623 consisting of the amino acid sequence shown in SEQ ID NO: 6, or a protein having an amino acid sequence that is 50% or more identical to any of these cell wall-pyruvate modifying enzymes.

7. The electron carrier according to claim 1, wherein in (i), a gene that expresses a protein involved in binding between the outer membrane and the cell wall is deleted or inactivated.

8. The electron carrier according to claim 7, wherein the gene expressing a protein involved in the binding between the outer membrane and the cell wall is at least one of a gene encoding an SLH domain-containing outer membrane protein and a gene encoding a cell wall-pyruvate modifying enzyme.

9. The electron carrier described in claim 8, wherein the gene encoding the SLH domain-retaining outer membrane protein is slr1841 having the base sequence shown in SEQ ID NO: 7, nies970_09470 having the base sequence shown in SEQ ID NO: 8, anacy_3458 having the base sequence shown in SEQ ID NO: 9, or a gene having a base sequence that is 50% or more identical to any of these genes.

10. The electron carrier described in claim 8, wherein the gene encoding the cell wall-pyruvate modifying enzyme is slr0688 consisting of the base sequence shown in SEQ ID NO: 10, synpcc7942_1529 consisting of the base sequence shown in SEQ ID NO: 11, anacy_1623 consisting of the base sequence shown in SEQ ID NO: 12, or a gene having a base sequence that is 50% or more identical to any of these genes.

11. The electron carrier according to claim 1, wherein in (ii), the channel protein that improves the protein permeability of the outer membrane is CppS consisting of the amino acid sequence shown in SEQ ID NO: 13, CppF consisting of the amino acid sequence shown in SEQ ID NO: 14, or a protein having an amino acid sequence that is 50% or more identical to any of these channel proteins.

12. The electron carrier according to claim 1, wherein in (ii) a gene encoding a channel protein that improves protein permeability of the outer membrane is introduced.

13. The electron carrier according to claim 12, wherein the gene encoding a channel protein that increases the protein permeability of the outer membrane is a gene derived from chloroplasts.

14. The electron carrier according to claim 12 or 13, wherein the gene encoding a channel protein that improves the protein permeability of the outer membrane is cppS having the nucleotide sequence shown in SEQ ID NO: 15, cppF having the nucleotide sequence shown in SEQ ID NO: 16, or a gene having a nucleotide sequence that is 50% or more identical to any of these genes.

15. A method for producing an electron carrier, comprising the step of producing a modified cyanobacterium having at least one of the following: (i) a function of a protein involved in binding between the outer membrane and the cell wall in the cyanobacterium is suppressed or lost, and (ii) a channel protein that improves the protein permeability of the outer membrane is expressed.

16. A method for electron transfer, comprising using the electron carrier according to any one of claims 1 to 14.

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