Method for determining exfoliation of outer membrane of cyanobacteria, device for determining exfoliation of outer membrane of cyanobacteria, and program

Abstract

The present disclosure provides a method for determining exfoliation of an outer membrane of cyanobacteria capable of easily determining whether or not the outer membrane has been exfoliated from a cell wall. A method for determining exfoliation of an outer membrane of cyanobacteria according to the present invention comprises: a measurement step (S01) for measuring the concentration of at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine and phenylalanine in a culture supernatant of cyanobacteria; and a determination step (S02) for determining whether or not the outer membrane of the cyanobacteria has been peeled off from the cell wall on the basis of the concentration of the at least one type of amino acid measured in the measurement step.

Description

Technical Field

[0001] The present disclosure relates to a method for determining the outer membrane peeling of cyanobacteria, a device for determining the outer membrane peeling of cyanobacteria, and a program. Background Art

[0002] Photosynthetic microorganisms such as cyanobacteria and algae have attracted much attention as tools for realizing a new generation of material production systems with low environmental load. Material production using photosynthetic microorganisms is carried out under normal temperature and pressure, using light as energy and carbon dioxide (CO2) in water and air. Furthermore, through the development of genetic manipulation technology in recent years, it is possible to produce a wide variety of compounds using genetically recombinant photosynthetic microorganisms, so material production using photosynthetic microorganisms is expected to be a new generation technology that can achieve carbon neutrality.

[0003] As a material production using such photosynthetic microorganisms, for example, a technology for improving the productivity of a material by using a genetically modified strain obtained by transforming a gene that participates in the metabolism of photosynthetic microorganisms or the biosynthesis of a material is disclosed. By this technology, for example, the productivity of sucrose (non-patent literature 1), isobutanol (non-patent literature 2), fatty acids (non-patent literature 3), amino acids (non-patent literature 4) and proteins (non-patent literature 5) etc. is improved. In addition, for example, a technology (patent literature 1) for improving the productivity of a protein by using a genetically modified strain that peels off the outer membrane of a cyanobacterium from the cell wall is also disclosed.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: International Publication No. 2021 / 100640

[0007] Non-patent literature

[0008] Non-patent document 1: Daniel C Ducat et al., "Rerouting Carbon Flux To Enhance Photosynthetic Productivity", Applied and Environmental Microbiology, American Society for Microbiology, April 2012, Vol.78, No.8, pp.2660-2668

[0009] Non-patent document 2: Shota Atsumi et al., “Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde”, Nature Biotechnology, Nature Publishing Group, November 2009, Vol. 27, No. 12, pp. 1177-1180

[0010] Non-patent document 3: Xinyao Liu et al., "Fatty acid production in genetically modified cyanobacteria", The Proceedings of the Natural Academy of Sciences (PNAS), National Academy of Sciences, April 2011, Vol. 108, No. 17, pp. 6899-6904

[0011] Non-patent document 4: Arnav Deshpande et al., "Combining random mutagenesis and metabolic engineering for enhanced tryptophan production in Synechocystissp.strain PCC 6803", Applied Environmental Microbiology, May 2020, Vol.86, No.9, e02816-19

[0012] Non-patent document 5: James A.Gregory et al., "Alga-Produced Cholera Toxin-Pfs25Fusion Proteins as Oral Vaccines", Applied and Environmental Microbiology, American Society for Microbiology, June 2013, Vol.79, No.13, pp.3917-3925 Summary of the invention

[0013] Problems to be solved by the invention

[0014] However, in the above-mentioned prior art, it is necessary to use, for example, electron microscope observation of cells or complex biochemical analysis methods to determine whether the state of cells of genetically modified strains of photosynthetic microorganisms is suitable for efficient substance production, which takes time and labor.

[0015] Therefore, the present disclosure provides: a method for determining outer membrane peeling of cyanobacteria, which can easily determine whether the outer membrane has peeled off from the cell wall as a state suitable for efficient substance production of cyanobacterial cells; an apparatus for determining outer membrane peeling of cyanobacteria; and a program.

[0016] Means for solving problems

[0017] A method for determining whether the outer membrane of a cyanobacterium has been peeled off involves the following steps: a measuring step of measuring the concentration of at least one amino acid selected from isoleucine, leucine, tyrosine and phenylalanine in a culture supernatant of the cyanobacterium; and a determining step of determining whether the outer membrane of the cyanobacterium has been peeled off from the cell wall based on the concentration of the at least one amino acid measured in the measuring step.

[0018] In addition, one embodiment of the present disclosure relates to a device for determining whether the outer membrane of cyanobacteria has been peeled off: a measuring unit for measuring the concentration of at least one amino acid selected from isoleucine, leucine, tyrosine and phenylalanine in the culture supernatant of cyanobacteria; and a determining unit for determining whether the outer membrane of the cyanobacterium has been peeled off from the cell wall based on the concentration of the at least one amino acid measured by the measuring unit.

[0019] In addition, a program according to one aspect of the present disclosure is a program for causing a computer to execute a method of determining whether an outer membrane of a cyanobacterium has been detached from a cell wall based on the concentration of at least one amino acid selected from isoleucine, leucine, tyrosine, and phenylalanine in a culture supernatant of the cyanobacterium.

[0020] Effects of the Invention

[0021] According to the method for determining the detachment of the outer membrane of cyanobacteria, the device and program for determining the detachment of the outer membrane of cyanobacteria disclosed in the present invention, it is possible to easily determine whether the outer membrane of cyanobacteria has detached from the cell wall. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a block diagram showing an example of the functional configuration of a device for determining whether the outer membrane of cyanobacteria has been peeled off according to an embodiment.

[0023] Figure 2A This is a flowchart showing an example of the flow of a method for determining outer membrane peeling of cyanobacteria according to an embodiment.

[0024] Figure 2B Yes Display Figure 2AFlow chart of the detailed process of step S02.

[0025] Figure 3 is a schematic diagram showing the cell surface layers of cyanobacteria.

[0026] Figure 4 This is a transmission electron microscope observation image of an ultrathin section of the modified cyanobacteria of Example 1.

[0027] Figure 5 yes Figure 4 A magnified image of the dashed area A.

[0028] Figure 6 It is a transmission electron microscope image of the ultrathin section of the modified cyanobacteria of Example 2.

[0029] Figure 7 yes Figure 6 A magnified image of the dashed area B.

[0030] Figure 8 This is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Comparative Example 1.

[0031] Fig. 9 yes Figure 8 Magnified view of the dashed area C.

[0032] Fig.10 It is a graph showing the amount of protein in the culture supernatant of the modified cyanobacteria of Example 1, Example 2 and Comparative Example 1 (n=3, error bar=SD).

[0033] Fig.11 It is a graph showing the results of amino acid analysis of culture supernatants of two types of cyanobacteria by LC-MS / MS.

[0034] Fig.12 Displays serial number 1 to serial number 3.

[0035] Fig.13 Serial number 4 to 6 are displayed.

[0036] Fig.14 Displays the serial number 7.

[0037] Fig.15 Displays the serial number 8.

[0038] Fig.16 Displays the serial number 9.

[0039] Fig.17 Displays the serial number 10.

[0040] Fig.18 Displays the serial number 11.

[0041] Fig.19Serial numbers 12 to 18 are displayed.

[0042] Fig. 20 Serial number 19 to serial number 22 are displayed. DETAILED DESCRIPTION

[0043] (Knowledge that forms the basis of this disclosure)

[0044] Photosynthetic microorganisms such as cyanobacteria and algae have attracted much attention as tools for realizing a next-generation material production system with low environmental impact. Among them, in material production using cyanobacteria, methods for evaluating and managing cyanobacterial cells and their culture states are required to be efficient and simple.

[0045] Therefore the inventor, as the photosynthetic microorganism that uses in the production of material, has set his sights on cyanobacteria.Cyanobacteria (also referred to as cyanobacteria or cyanobacteria) is a group of true bacteria, produces oxygen by photosynthesis with water decomposition, fixes with the CO2 in the air by the energy of gained.In addition, cyanobacteria can also fix nitrogen (N2) in the air according to the different kinds of cyanobacteria.In addition, as the characteristic of cyanobacteria, known growth and reproduction is fast, light utilization efficiency is high, and compared with other algae species, genetic manipulation is easy in addition, therefore in photosynthetic microorganism, the utilization about cyanobacteria has been carried out active research and development.As mentioned above, as the example of the material production utilizing cyanobacteria (more specifically transforming cyanobacteria), the production of sucrose (non-patent literature 1), isobutanol (non-patent literature 2), lipid acid (non-patent literature 3), amino acid (non-patent literature 4) and protein (non-patent literature 5) etc. has been reported.

[0046] For example, Non-Patent Document 1 discloses that a genetically modified strain of Synechococcus elongatus in which a gene involved in a sucrose biosynthesis pathway is modified has improved sucrose productivity compared to a wild-type strain.

[0047] Furthermore, for example, Non-Patent Document 2 discloses that the isobutanol productivity of a genetically modified strain overexpressing ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) prepared by genetic manipulation of Synechococcus elongatus PCC7942 is improved compared to a wild-type strain.

[0048] Furthermore, for example, Non-Patent Document 3 discloses that the fatty acid productivity of a genetically modified strain into which an acyl-acyl transporter thioesterase gene was introduced into Synechocystis sp. PCC6803 was improved compared with that of a wild-type strain.

[0049] Furthermore, Non-Patent Document 4 discloses that tryptophan productivity of a tryptophan-overproducing strain isolated by random mutagenesis of a wild-type strain of Synechocystis sp. PCC 6803 and selection using amino acids themselves is improved compared to that of the wild-type strain.

[0050] In addition, non-patent document 5 discloses that the chloroplasts of Chlamydomonas reinhardtii, a type of algae, are genetically manipulated so that a chimeric protein (CtxB-Pfs25) composed of a 25 kDa Plasmodium falciparum (Plasmodium falciparum) surface protein (Pfs25) fused to the β subunit of cholera toxin (CtxB) is produced in the cells of genetically modified algae, which can be used as an oral vaccine for malaria.

[0051] However, for the above-mentioned prior art, even if the target substance is produced in the cell of the genetically modified strain of photosynthetic microorganisms, it is not secreted outside the cell and accumulates or is not easily secreted outside the cell effectively, so it is necessary to crush the cells and recycle the target substance, and the production of the substance takes time and labor. In addition, since there are various substances in the cell, it is sometimes necessary to remove these substances and purify the target substance, and the recovery rate of the target substance decreases. In addition, since it is necessary to prepare a new genetically modified strain each time the target substance is produced, it takes time and labor, and the production cost also increases. In this way, for the above-mentioned prior art, the production efficiency of the substance using photosynthetic microorganisms is still low, and it is expected to develop a technology with higher production efficiency.

[0052] The structure of the cell wall and cell membrane of cyanobacteria makes the permeability of substances produced in the cell low, and it is not easy to artificially modify the structure of the cell membrane and cell wall to improve the secretion and production capacity of substances. In particular, substances with large molecular weights such as proteins (also called macromolecular compounds) are not easily secreted outside the cell, unlike substances with relatively small molecular weights such as amino acids (also called low molecular weight compounds).

[0053] However, it is recorded that if the slr1841 gene or slr0688 gene, which is involved in the adhesion between the outer membrane and the cell wall of cyanobacteria and contributes to the structural stability of the cell surface, is deleted, the proliferation ability of cyanobacterial cells is lost (Non-patent Document 6: Hikaru Kiwata, Studies on molecular basis of cyanobacterial outer membrane function and its evolutionary relationship with primitive chloroplasts, doctoral dissertation, [online], 2018.03.27, Internet:<URL:http: / / hdl.handle.net / 10097 / 00122689> ; and non-patent document 7: Seiji Kojima, Explanation and application of membrane stabilization mechanism and substance permeation mechanism of origin of chloroplast surface membrane function, bacterial origin, scientific research funds, [online], 2018.04.23, Internet:<URL:https: / / kaken.nii.ac.jp / grant / KAKENHI-PROJECT-18H02117> ).

[0054] As the technology for solving the above-mentioned problem, in patent documentation 1, disclose the function of the outer membrane that participates in cyanobacteria and the protein of the combination of cell wall (hereinafter, also referred to as in conjunction with associated protein) is suppressed or lost transformation cyanobacteria and the protein manufacture method that adopts this transformation cyanobacteria.In the technology put down in writing in patent documentation 1, this transformation cyanobacteria is owing to under the state of the proliferation ability of maintenance cell, the outer membrane of cyanobacteria is peeled off from cell wall, therefore by culturing cell, thereby the protein that produces in cell is secreted to the extracellular, can manufacture protein efficiently.

[0055] However, in the above-mentioned prior art, it is necessary to use electron microscope observation of cells or complicated biochemical analysis methods to determine whether the state of cells of transformed cyanobacteria is suitable for effective substance production, therefore, it takes time and labor.

[0056] Furthermore, it is known that if the outer membrane of cyanobacteria is peeled off from the cell wall, the productivity of a substance is improved. However, whether the outer membrane has been peeled off requires observation using an electron microscope and complex biochemical analysis methods, and a simple method has not yet been developed.

[0057] Therefore, the present inventor is the state that is suitable for efficient substance production as the state of the cell of cyanobacteria, and has studied in depth the method that simply judges whether adventitia peels off from cell wall.Its result found that, can simply judge whether the adventitia of cyanobacteria peels off from cell wall based on the concentration of the specific amino acid in the culture supernatant of cyanobacteria.Therefore, according to the disclosure, can judge whether to be the cyanobacteria that is suitable for effective substance production, so can improve the substance productivity utilizing cyanobacteria.

[0058] (Overview of the present disclosure)

[0059] An outline of one aspect of the present disclosure is as follows.

[0060] A method for determining whether the outer membrane of a cyanobacterium has been peeled off according to one embodiment of the present invention comprises: a measuring step of measuring the concentration of at least one amino acid selected from isoleucine, leucine, tyrosine and phenylalanine in a culture supernatant of the cyanobacterium; and a determining step of determining whether the outer membrane of the cyanobacterium has been peeled off from the cell wall based on the concentration of the at least one amino acid measured in the measuring step.

[0061] Thus, the method for determining the outer membrane exfoliation of cyanobacteria only requires collecting the culture supernatant of cyanobacteria and measuring the concentration of at least one amino acid selected from the above four amino acids in the culture supernatant, so the outer membrane exfoliation of cyanobacteria can be determined simply.

[0062] For example, a method for determining whether the outer membrane of cyanobacteria has been peeled off according to one embodiment of the present invention may include, in the measuring step, determining the concentrations of four amino acids, namely, isoleucine, leucine, tyrosine and phenylalanine, and in the determining step, determining whether the outer membrane of the cyanobacterium has been peeled off from the cell wall based on the concentrations of the four amino acids.

[0063] Thus, the method for determining outer membrane exfoliation of cyanobacteria measures the concentrations of the above four amino acids in the culture supernatant of cyanobacteria and determines outer membrane exfoliation of cyanobacteria based on the concentrations of these amino acids, and thus outer membrane exfoliation of cyanobacteria can be determined with higher accuracy.

[0064] For example, a method for determining whether the outer membrane of a cyanobacterium has been peeled off according to one embodiment of the present disclosure may, in the determination step, determine that the outer membrane of the cyanobacterium has been peeled off from the cell wall when at least one of the concentrations of the at least one amino acid is above a threshold value, and determine that the outer membrane of the cyanobacterium has not been peeled off from the cell wall when all of the concentrations of the at least one amino acid are less than the threshold value.

[0065] Thus, the method for determining outer membrane exfoliation of cyanobacteria determines that the outer membrane of cyanobacteria has been exfoliated as long as the concentration of at least one of the measured amino acids exceeds a threshold value. Even if ambiguity occurs due to the culture state of the cyanobacteria, the outer membrane exfoliation of cyanobacteria can be determined with high accuracy.

[0066] For example, in the method for determining outer membrane exfoliation of cyanobacteria according to one embodiment of the present disclosure, the threshold value is 100 nM.

[0067] Thus, the method for determining outer membrane exfoliation of cyanobacteria can simply and quantitatively determine outer membrane exfoliation of cyanobacteria by determining whether the concentration of at least one amino acid selected from the above four amino acids contained in the culture supernatant of cyanobacteria whose outer membrane has been exfoliated from the cell wall exceeds 100 nM.

[0068] In addition, an apparatus for determining whether the outer membrane of cyanobacteria has been peeled off according to one embodiment of the present disclosure comprises: a measuring unit for measuring the concentration of at least one amino acid selected from isoleucine, leucine, tyrosine and phenylalanine in a culture supernatant of cyanobacteria; and a determining unit for determining whether the outer membrane of the cyanobacterium has been peeled off from the cell wall based on the concentration of the at least one amino acid measured by the measuring unit.

[0069] Therefore, the device for determining the outer membrane peeling of cyanobacteria only needs to measure the concentration of at least one amino acid selected from the above four amino acids in the culture supernatant of cyanobacteria, and thus the outer membrane peeling of cyanobacteria can be determined simply.

[0070] In addition, a program according to one aspect of the present disclosure is a program for causing a computer to execute a method of determining whether an outer membrane of a cyanobacterium has been detached from a cell wall based on the concentration of at least one amino acid selected from isoleucine, leucine, tyrosine, and phenylalanine in a culture supernatant of the cyanobacterium.

[0071] Thus, the program causes the computer to determine whether the outer membrane of the cyanobacterium has been peeled off from the cell wall based on the concentration of at least one amino acid selected from the above four amino acids in the culture supernatant of the cyanobacterium, so that the computer can easily determine whether the outer membrane of the cyanobacterium has been peeled off.

[0072] Hereinafter, the embodiments will be described in detail with reference to the drawings.

[0073] In addition, the embodiments described below all represent comprehensive or specific examples. The numerical values, materials, steps, and the order of the steps shown in the following embodiments are examples and do not limit the subject matter of the present disclosure. In addition, among the constituent elements in the following embodiments, constituent elements not recorded in the independent claims representing the highest concept are described as arbitrary constituent elements.

[0074] In addition, each figure does not necessarily illustrate strictly. In each figure, the same code|symbol is attached|subjected to the substantially same structure, and the repeated description may be abbreviated or simplified.

[0075] In the following, the numerical range does not only have a strict meaning but also includes a range in which substantially the same amount of protein (eg, number or concentration) or a range thereof is measured.

[0076] In addition, in this specification, both bacterial body and cell refer to one individual of cyanobacteria.

[0077] (Implementation Method)

[0078] [1. Definition]

[0079] In this specification, the identity of the base sequence and the amino acid sequence is calculated by the BLAST (Basic Local Alignment Search Tool) algorithm. Specifically, it is calculated by performing a pairwise analysis using the BLAST algorithm that can be used on the website of the NCBI (National Center for Biotechnology Information) website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Information about the genes of cyanobacteria and the proteins encoded by the genes is disclosed, for example, in the above-mentioned NCBI database and Cyanobase (http: / / genome.microbedb.jp / cyanobase / ). The amino acid sequence of the target protein and the base sequence of the genes encoding these proteins can be obtained from these databases.

[0080] [2. Device for determining the outer membrane peeling of cyanobacteria]

[0081] Next, a determination device for outer membrane peeling of cyanobacteria (hereinafter, also simply referred to as a determination device) according to the present embodiment will be described. Figure 1 This is a block diagram showing an example of the functional configuration of a device for determining whether the outer membrane of cyanobacteria has been peeled off according to the present embodiment.

[0082] like Figure 1 As shown, the determination device 100 includes, for example, a measurement unit 110, a control unit 120, a storage unit 130, an input receiving unit 140, and a display unit 150. The control unit 120 includes, for example, a determination unit 122.

[0083] The determination unit 110, for example, measures the concentration of at least one amino acid selected from isoleucine, leucine, tyrosine and phenylalanine in the culture supernatant of cyanobacteria. The determination unit 110 can be an existing known amino acid analyzer as long as it can measure the concentration of the amino acid in the culture supernatant. The determination unit 110, for example, is a liquid chromatography (HPLC), a liquid chromatography mass spectrometer (LC / MS), or a liquid chromatography tandem mass spectrometer (LC-MS / MS) etc.

[0084] Although not shown in the figure, the measuring unit 110 includes, for example, a control unit that controls the operation of the measuring unit 110, and the control unit controls the operation of the measuring unit 110 according to a control signal output from the control unit 120 of the determination device 100. Figure 1 In the example of , the determination device 100 includes the measuring unit 110, but may not include the measuring unit 110. In this case, the measuring unit 110 is a measuring device, and the determination device 100 and the measuring device are connected via communication.

[0085] The control unit 120 performs information processing for controlling the operation of the determination device 100. The control unit 120 is implemented by, for example, a microcomputer, but may also be implemented by a processor or a dedicated circuit. Specifically, the control unit 120 includes a determination unit 122. The determination unit 122 is implemented by executing a program for the processor to perform the above-mentioned information processing.

[0086] The determination unit 122 determines whether the outer membrane of the cyanobacteria is peeled off from the cell wall based on the concentration of at least one amino acid measured by the determination unit 110. For example, the function of the determination unit 122 is realized by the CPU (central processing unit) executing a program stored in the storage unit 130. The specific function of the determination unit 122 is described in the next item.

[0087] The storage unit 130 is a storage device that stores a control program and the like executed by the control unit 120. The storage unit 130 is implemented by, for example, a semiconductor memory.

[0088] The input receiving unit 140 receives an operation input from the user. Specifically, the input receiving unit 140 is implemented by a mouse, a microphone, a touch panel, or the like.

[0089] The display unit 150 is a display device that displays information presented to the user based on the control of the control unit 120. The display unit 150 is implemented by a liquid crystal panel or an organic EL (Electro Luminescence) panel.

[0090] [3. Method for determining the outer membrane peeling of cyanobacteria]

[0091] Next, regarding the method for determining the outer membrane peeling of cyanobacteria according to the present embodiment, refer to Figure 2A and Figure 2B Provide explanation. Figure 2A This is a flowchart showing an example of the flow of a method for determining outer membrane peeling of cyanobacteria according to an embodiment. Figure 2B Yes Display Figure 2A Flow chart of the detailed process of step S02.

[0092] The determination method is implemented by the above-mentioned determination device. Figure 2A As shown, the determination unit of the determination device measures at least one amino acid concentration (S01) selected from isoleucine, leucine, tyrosine and phenylalanine in the culture supernatant of the cyanobacteria that is imported into the determination unit. The culture supernatant can be sampled at the interval (for example, in days) of regulation after the main cultivation begins. Sampling can be carried out by manual operation by the user, or it can be carried out automatically. Isoleucine, leucine, tyrosine and phenylalanine are characteristically secreted into the culture supernatant when the cell that the outer membrane of culturing cyanobacteria has been peeled off.

[0093] Next, the determination unit of the determination device determines whether the outer membrane of the cyanobacterium is peeled off from the cell wall based on the concentration of at least one amino acid measured by the determination unit (S02). More specifically, Figure 2B As shown, in step S02, the determination unit determines whether the concentration of at least one amino acid measured by the measuring unit is all less than a threshold value (S11). When it is determined that the concentration of at least one amino acid measured is all less than the threshold value (Yes in S11), it is determined that the outer membrane of the cyanobacterium is not peeled off from the cell wall (S12).

[0094] On the other hand, when at least one of the concentrations of at least one amino acid measured by the measuring unit is equal to or higher than the threshold value (No in S11), the determining unit determines that the outer membrane of the cyanobacterium has been peeled off from the cell wall (S13).

[0095] In addition, in the method for determining whether the outer membrane of cyanobacteria has been stripped, the concentrations of isoleucine, leucine, tyrosine and phenylalanine in the culture supernatant of cyanobacteria are measured (in other words, quantitatively) to determine whether the outer membrane of cyanobacteria has been stripped from the cell wall, but the amino acids and biomolecules (e.g., intracellular metabolites) that are the objects of measurement are not limited to these.

[0096] In addition, the measurement method performed in step S01 can be any method that can measure amino acids or biological molecules. For example, it can be a method performed by liquid chromatography (HPLC), liquid chromatography mass spectrometer (LC / MS), or liquid chromatography tandem mass spectrometer (LC-MS / MS). In addition, for example, the measurement method performed in step S01 can also be an enzyme measurement for detecting specific amino acids or biological molecules.

[0097] [4. Cyanobacteria]

[0098] Cyanobacteria, also known as blue algae or blue bacteria, are a group of prokaryotes that capture light energy with chlorophyll and perform photosynthesis while electrolyzing water to produce oxygen using the energy obtained. Cyanobacteria are rich in diversity. For example, in terms of cell shape, there are single-celled species such as Synechocystis sp. PCC 6803 and multi-celled filamentous species such as Anabaena sp. PCC 7120. Regarding the growth and reproduction environment, there are also thermophilic species such as Thermosynechococcus elongatus, marine species such as Synechococcuse longatus, and freshwater species such as Synechocystis. In addition, there are many species such as Microcystis aeruginosa that have air sacs and produce toxins, and species such as Gloeobacterviolaceus that do not have thylakoids but have proteins called phycobilisomes as light-harvesting antennas in the plasma membrane that have unique characteristics.

[0099] Figure 3 is a schematic diagram showing the cell surface layers of cyanobacteria. Figure 3 As shown, the cell surface layer of cyanobacteria is composed of a protoplasmic membrane (also referred to as an inner membrane 1), a peptidoglycan 2, and a lipid membrane 5 forming the outermost layer of the cell in order from the inside. Peptidoglycan 2 is covalently bonded with a sugar chain 3 composed of glucosamine and mannosamine, etc., and pyruvic acid is bound to these covalently bonded sugar chains 3 (non-patent literature 8: Jurgens and Weckesser, 1986, J. Bacteriol., 168: 568-573). In this specification, the cell wall 4 is referred to as the cell wall 4, including the peptidoglycan 2 and the covalently bonded sugar chain 3. In addition, the gap between the protoplasmic membrane (i.e., the inner membrane 1) and the outer membrane 5 is called the periplasm, and there are various enzymes involved in the decomposition of proteins or the formation of a three-dimensional structure, the decomposition of lipids or nucleic acids, or the uptake of nutrients outside the cell.

[0100] SLH domain-retaining outer membrane proteins (e.g., Slr1841 in the figure) are composed of a C-terminal region integrated into the lipid membrane (also referred to as outer membrane 5) and an N-terminal SLH domain 7 protruding from the lipid membrane, and are widely distributed in bacteria belonging to the Negativicutes class (Firmicutes), which is a group of cyanobacteria and Gram-negative bacteria (Non-patent document 9: Kojima et al., 2016, Biosci. Biotech. Biochem., 10: 1954-1959). The region integrated into the lipid membrane (i.e., outer membrane 5) forms a channel for the outer membrane to allow hydrophilic substances to pass through, while the SLH domain 7 has the function of binding to the cell wall 4 (Non-patent document 10: Kowata et al., 2017, J. Bacteriol., 199: e00371-17). In order for the SLH domain 7 to bind to the cell wall 4, the covalently bonded sugar chain 3 in the peptidoglycan 2 needs to be modified with pyruvic acid (Non-patent document 11: Kojima et al., 2016, J. Biol. Chem., 291: 20198-20209). Examples of genes encoding SLH domain-retaining outer membrane proteins 6 include slr1841 or slr1908 retained by Synechocystis sp. PCC 6803, or oprB retained by Anabaenasp. 90.

[0101] An enzyme that catalyzes the pyruvate modification reaction of the covalently bonded sugar chain 3 in the peptidoglycan 2 (hereinafter referred to as cell wall-pyruvate modification enzyme 9) was identified in Bacillus anthracis, a Gram-positive bacterium, and was named CsaB (Non-patent document 12: Mesnage et al., 2000, EMBO J., 19: 4473-4484). Among cyanobacteria whose genome base sequences have been published, many species retain genes encoding homologous proteins having an amino acid sequence identity of 30% or more with CsaB. Examples include slr0688 retained by Synechocystis sp. PCC 6803 and synpcc7502_03092 retained by Synechococcus sp. 7502.

[0102] In cyanobacteria, CO2 fixed by photosynthesis is converted into various amino acids through multi-stage enzyme reactions. Using these as raw materials, proteins are synthesized in the cytoplasm of cyanobacteria. Some of these proteins function in the cytoplasm, while others are transported from the cytoplasm to the periplasm and function in the periplasm. However, cases of active secretion of proteins outside the cell have not been reported in cyanobacteria so far.

[0103] Since cyanobacteria have high photosynthesis ability, they do not necessarily need to take in organic matter from the outside as nutrients. Therefore, in cyanobacteria, the outer membrane 5 only has very little Figure 3 For example, in Synechocystis sp. PCC 6803, the organic matter channel protein 8 that permeates organic matter is only about 4% of the total protein in the outer membrane 5. On the other hand, in order to efficiently take in inorganic ions required for growth and reproduction into the cell, cyanobacteria have a large number of organic matter channel proteins such as Slr1270 in the outer membrane 5. Figure 3 Ion channel proteins that only permeate inorganic ions, such as the SLH domain-retaining outer membrane protein 6 (e.g., Slr1841) of Synechocystis sp. PCC 6803, for example, account for about 80% of the total proteins in the outer membrane 5.

[0104] Like this, in cyanobacteria, owing to make the passage that organic matter such as protein in the outer membrane 5 is seen through very few, therefore can think that it is difficult to be secreted outside the thalline protein produced in the thalline actively.In addition, although the structure of the cell wall of cyanobacteria and cell membrane is about protein permeability, it is not easy to improve protein secretion production capacity artificially by transforming cell membrane and cell wall structure.For example, in non-patent literature 6 and non-patent literature 7, record if make participate in the bonding of outer membrane and cell wall, contribute to the structural stability of cell surface layer slr1841 gene or slr0688 gene defect, then the proliferation ability of cell is lost.

[0105] [5. Transformation of cyanobacteria]

[0106] Next, for the cyanobacteria involved in this embodiment (hereinafter referred to as modified cyanobacteria), refer to Figure 3 In addition, the following description will take protein as an example of a substance produced using modified cyanobacteria.

[0107] In the modified cyanobacteria that the present embodiment relates to, the function of the protein (so-called binding related protein) participating in the combination of the outer membrane 5 and the cell wall 4 in the cyanobacteria is suppressed or lost. More specifically, for example, the total amount of the protein (that is, binding related protein) participating in the combination of the outer membrane 5 and the cell wall 4 in the modified cyanobacteria is suppressed to more than 30% and less than 70% of the total amount of the protein in the parent strain (that is, the parent cyanobacteria). Here, for example, the so-called "the total amount of binding related protein is suppressed to 30% of the total amount of the protein in the parent strain" refers to the state that 70% of the total amount of the protein in the parent strain is lost and 30% remains. Thus, in the modified cyanobacteria, the combination (for example, binding amount and binding force) of the outer membrane 5 and the cell wall 4 is partially reduced, so that the outer membrane 5 becomes easy to partially break away from the cell wall 4. Therefore, the secretion productivity of the protein produced in the thalline by the modified cyanobacteria to the protein outside the thalline is improved. In addition, since it is not necessary to break the thalline and recycle the protein, even after reclaiming the protein, the modified cyanobacteria can be reused to produce protein. In this specification, the production of proteins in the modified cyanobacteria is referred to as production, and the secretion of the produced proteins outside the bacteria is referred to as secretory production.

[0108] The protein involved in the binding of the outer membrane 5 to the cell wall 4 can be, for example, at least one of the SLH domain-maintained outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9. In the present embodiment, the function of at least one protein of the modified cyanobacteria, for example, the SLH domain-maintained outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9 is inhibited. For example, in the modified cyanobacteria, (i) the function of at least one of the SLH domain-maintained outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9 can be inhibited, and (ii) the expression of the SLH domain-maintained outer membrane protein 6 bound to the cell wall 4 and the expression of the enzyme (i.e., the cell wall-pyruvate modification enzyme 9) that catalyzes the pyruvate modification reaction of the bound sugar chain on the surface of the cell wall 4 can be inhibited. As a result, the binding (i.e., the binding amount and binding strength) of the SLH domain 7 of the SLH domain-maintained outer membrane protein 6 in the outer membrane 5 and the covalently bonded sugar chain 3 on the surface of the cell wall 4 is reduced. Therefore, the outer membrane 5 becomes easy to separate from the cell wall 4 at these weakly bonded portions. As the outer membrane 5 is partially separated from the cell wall 4, proteins present in the cells of the modified cyanobacteria, especially in the periplasm, become easy to leak out of the cells (outside the outer membrane 5).

[0109] As mentioned above, cyanobacteria is owing to having high photosynthetic ability, thereby may not need to take in organic matter from the outside as nutrient.Therefore, as long as have light, air, water and trace inorganic matter in the cultivation of cyanobacteria, cyanobacteria, by the ion channel of outer membrane 5, take in inorganic matter to the cell, produce protein in the cell.Particularly in the periplasm of the space between outer membrane 5 and cell wall 4, there are various proteins.In the transformation cyanobacteria that the present embodiment relates to, the protein function that participates in the combination of outer membrane 5 and cell wall 4 is suppressed.As a result, outer membrane 5 becomes easy to peel off from cell wall 4 partly.Then, the protein in the periplasm leaks into the culture fluid from the peeling part of outer membrane 5.Thus, the secretion productivity of the protein secreted to the protein outside the thalline that transformation cyanobacteria will produce in the thalline improves.

[0110] The following describes in more detail a cyanobacterium modified such that the outer membrane 5 is partially separated from the cell wall 4 by suppressing the function of at least one of the binding-related proteins of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9.

[0111] The type of cyanobacteria (referred to as "parent strain" or "parent cyanobacteria" in this specification) that becomes the parent microorganism of the modified cyanobacteria involved in the present embodiment and suppresses at least one of the expression of SLH domain-retaining outer membrane protein 6 and the expression of cell wall-pyruvate modifying enzyme 9 is not particularly limited, and can be all types of cyanobacteria. For example, the parent cyanobacteria can be Synechocystis, Synechococcus, Anabaena, or Thermosynechococcus, wherein it can be Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, or Thermosynechococcus elongatus (slender thermophilic synechococcus) BP-1. The parent strain may be wild-type or modified cyanobacteria as long as the total amount of binding-related proteins is suppressed to 30% or more and 70% or less, or may be a cyanobacterium having the same binding-related proteins as wild-type cyanobacteria.

[0112] The amino acid sequences of the SLH domain-retaining outer membrane protein 6 and the enzyme that catalyzes the cell wall-pyruvate modification reaction (i.e., cell wall-pyruvate modification enzyme 9) in these parental cyanobacteria, the base sequences of the genes encoding these binding-related proteins, and the locations of the genes on the chromosomal DNA or plasmid can be confirmed in the above-mentioned NCBI database and Cyanobase.

[0113] In addition, the SLH domain-retaining outer membrane protein 6 and cell wall-pyruvate modifying enzyme 9 whose functions are inhibited in the modified cyanobacteria involved in this embodiment can be substances of any parent cyanobacteria as long as they are retained by the parent cyanobacteria, and are not restricted by the location of the genes encoding them (for example, on chromosomal DNA or on a plasmid).

[0114] For example, when the parent cyanobacteria is Synechocystis, the SLH domain-maintaining outer membrane protein 6 may be Slr1841, Slr1908, or Slr0042, etc., when the parent cyanobacteria is Synechococcus, it may be NIES970_09470, etc., when the parent cyanobacteria is Anabaena, it may be Anacy_5815 or Anacy_3458, etc., when the parent cyanobacteria is Microcystis, it may be A0A0F6U6F8_MICAE, etc., when the parent cyanobacteria is Cyanothece, It can be A0A3B8XX12_9CYAN etc., when the parent cyanobacteria is Leptolyngbya, it can be A0A1Q8ZE23_9CYAN etc., when the parent cyanobacteria is Calothrix, A0A1Z4R6U0_9CYAN can be mentioned, when the parent cyanobacteria is Nostoc, it can be A0A1C0VG86_9NOSO etc., when the parent cyanobacteria is Crocosphaera, it can be B1WRN6_CROS5 etc., when the parent cyanobacteria is Pleurocapsa, it can be K9TAE4_9CYAN etc.

[0115] More specifically, the SLH domain-retaining outer membrane protein 6 may be, for example, Slr1841 (sequence number 1) of Synechocystis sp. PCC6803, NIES970_09470 (sequence number 2) of Synechococcus sp. NIES-970, or Anacy_3458 (sequence number 3) of Anabaena cylindrica PCC 7122. In addition, it may be a protein having an amino acid sequence that is 50% or more identical to these SLH domain-retaining outer membrane proteins 6.

[0116] Thus, in the modified cyanobacteria, for example, (i) the function of any of the SLH domain-retaining outer membrane proteins 6 shown in the above sequence numbers 1 to 3 or a protein having an amino acid sequence 50% or more identical to any of the above SLH domain-retaining outer membrane proteins 6 can be inhibited, and (ii) the expression of any of the SLH domain-retaining outer membrane proteins 6 shown in the above sequence numbers 1 to 3 or a protein having an amino acid sequence 50% or more identical to any of the above SLH domain-retaining outer membrane proteins 6 can be inhibited. Therefore, in the modified cyanobacteria, (i) the function of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 or a protein having the same function as the SLH domain-retaining outer membrane protein 6 is inhibited, or (ii) the expression level of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 or a protein having the same function as the SLH domain-retaining outer membrane protein 6 is reduced. Therefore, in the modified cyanobacteria involved in this embodiment, the binding amount and binding force of the binding domain (e.g., SLH domain 7) of the outer membrane 5 for binding to the cell wall 4 are reduced, so that the outer membrane 5 becomes easy to partially detach from the cell wall 4.

[0117] Generally, if the amino acid sequence of a protein is more than 30% identical, the homology of the three-dimensional structure of the protein is high, so it can be said that the possibility of having the same function as the protein is high. Therefore, as a function-inhibited SLH domain-retaining outer membrane protein 6, for example, it can be composed of an amino acid sequence that has an identity of more than 40%, preferably more than 50%, more preferably more than 60%, further preferably more than 70%, further more preferably more than 80%, and preferably more than 90% with any of the amino acid sequences of the SLH domain-retaining outer membrane protein 6 shown in the above sequence numbers 1 to 3, and has a function of binding to the covalently bonded sugar chain 3 of the cell wall.

[0118] In addition, for example, cell wall-pyruvate modification enzyme 9 can be Slr0688 when the parent cyanobacterium is Synechocystis, can be Syn7502_03092 or Synpcc7942_1529 when the parent cyanobacterium is Synechococcus, can be ANA_C20348 or Anacy_1623 when the parent cyanobacterium is Anabaena, can be CsaB (NCBI access ID: TRU80220) when the parent cyanobacterium is Microcystis, can be CsaB (NCBI access ID: WP_107667006.1) when the parent cyanobacterium is Cyanothece, can be CsaB (NCBI access ID: WP_107667006.1) when the parent cyanobacterium is Cyanothece, can be CsaB (NCBI access ID: WP_107667006.1) when the parent cyanobacterium is Anabaena. When the parent cyanobacterium is Spirulina, it can be CsaB (NCBI access ID: WP_026079530.1) and the like; when the parent cyanobacterium is Calothrix, it can be CsaB (NCBI access ID: WP_096658142.1) and the like; when the parent cyanobacterium is Nostoc, it can be CsaB (NCBI access ID: WP_099068528.1) and the like; when the parent cyanobacterium is Crocosphaera, it can be CsaB (NCBI access ID: WP_012361697.1) and the like; when the parent cyanobacterium is Pleurocapsa, it can be CsaB (NCBI access ID: WP_036798735) and the like.

[0119] More specifically, the cell wall-pyruvate modification 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. In addition, it may be a protein having an amino acid sequence 50% or more identical to that of these cell wall-pyruvate modification enzymes 9.

[0120] Thus, in the modified cyanobacteria, for example, (i) the function of any of the cell wall-pyruvate modification enzymes 9 shown in the above sequence numbers 4 to 6 or a protein having 50% or more of the same amino acid sequence as any of the cell wall-pyruvate modification enzymes 9 is inhibited, or (ii) the expression of any of the cell wall-pyruvate modification enzymes 9 shown in the above sequence numbers 4 to 6 or a protein having 50% or more of the same amino acid sequence as any of the cell wall-pyruvate modification enzymes 9 is inhibited. Therefore, in the modified cyanobacteria, (i) the function of the cell wall-pyruvate modification enzyme 9 or a protein having the same function as the enzyme is inhibited, or (ii) the expression level of the cell wall-pyruvate modification enzyme 9 or a protein having the same function as the enzyme is reduced. Thus, the covalently bonded sugar chain 3 on the surface of the cell wall 4 becomes less susceptible to modification by pyruvate, and thus the binding amount and binding force of the sugar chain 3 of the cell wall 4 to the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 are reduced. Therefore, in the modified cyanobacterium according to the present embodiment, the covalently bonded sugar chains 3 on the surface of the cell wall 4 are less likely to be modified by pyruvic acid, so that the binding force between the cell wall 4 and the outer membrane 5 is weak, and the outer membrane 5 is easily partially detached from the cell wall 4 .

[0121] In addition, as described above, if the amino acid sequence of a protein is 30% or more identical, it can be said that there is a high possibility that it has the same function as the protein. Therefore, as the cell wall-pyruvate modification enzyme 9 whose function is inhibited, it can be, for example, a protein or polypeptide composed of an amino acid sequence having an identity of 40% or more, preferably 50% or more, more preferably 60% or more, further preferably 70% or more, further more preferably 80% or more, and further preferably 90% or more to any of the amino acid sequences of the cell wall-pyruvate modification enzyme 9 shown in the above sequence numbers 4 to 6, and having a function of catalyzing a reaction in which the covalently bonded sugar chain 3 of the peptidoglycan 2 of the cell wall 4 is modified with pyruvic acid.

[0122] In the present specification, the so-called inhibition of the function of the SLH domain-retaining outer membrane protein 6 is to inhibit the ability of the protein to bind to the cell wall 4, thereby inhibiting or losing the transport of the protein to the outer membrane 5, or inhibiting the ability of the protein to be integrated into the outer membrane 5 and function.

[0123] Furthermore, the function of inhibiting the cell wall-pyruvate modification enzyme 9 refers to the function of inhibiting the protein from modifying the covalently bonded sugar chain 3 of the cell wall 4 with pyruvate.

[0124] The means for inhibiting the functions of these proteins are not particularly limited as long as they are means commonly used in inhibiting the functions of proteins. The means may be, for example, deletion or inactivation of the gene encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9, inhibition of transcription of these genes, inhibition of translation of the transcription products of these genes, or administration of inhibitors that specifically inhibit these proteins.

[0125] In the present embodiment, transformation cyanobacteria can express gene deletion or the inactivation of the protein that participates in the combination of adventitia 5 and cell wall 4.Thus, transformation cyanobacteria is suppressed owing to the protein expression that participates in the combination of cell wall 4 and adventitia 5, or because the function of this protein is suppressed, thereby the combination (so-called binding capacity and bonding force) of cell wall 4 and adventitia 5 partly reduces.As a result, in transformation cyanobacteria, because adventitia 5 becomes easy to partly break away from cell wall 4, thereby the protein that produces in thalline becomes easy to leak outside adventitia 5, i.e. outside thalline.Therefore, the protein that the transformation cyanobacteria that the present embodiment relates to will produce in thalline secretes to the secretion productivity of protein outside thalline to improve.In addition, owing to not needing to break thalline and reclaim protein, thereby even after having reclaimed protein, also can reuse transformation cyanobacteria and produce protein.

[0126] The gene expressing the protein involved in the combination of the outer membrane 5 and the cell wall 4 can be, for example, at least one of the genes encoding the SLH domain-maintaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9. In the modified cyanobacteria, at least one of the genes encoding the SLH domain-maintaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9 is deleted or inactivated. Therefore, in the modified cyanobacteria, for example, the expression of at least one of the SLH domain-maintaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 (i) is inhibited, or the function of at least one of the SLH domain-maintaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 (ii) is inhibited. Therefore, the SLH domain 7 of the SLH domain-maintaining outer membrane protein 6 in the outer membrane 5 and the covalently bonded sugar chain 3 on the surface of the cell wall 4 (i.e., the binding amount and binding force) are reduced. Thus, in the part where the outer membrane 5 is weakly bound to the cell wall 4, the outer membrane 5 becomes easy to detach from the cell wall 4. Therefore, according to the modified cyanobacterium of the present embodiment, since the outer membrane 5 becomes easy to partially separate from the cell wall 4 due to the reduction of the bonding between the outer membrane 5 and the cell wall 4, the protein produced in the bacteria becomes easy to leak out of the bacteria.

[0127] In this embodiment, in order to inhibit the function of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9 in cyanobacteria, for example, the transcription of at least one of the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modification enzyme 9 can be inhibited.

[0128] For example, the gene encoding the SLH domain-maintaining outer membrane protein 6 can be slr1841, slr1908, or slr0042 when the parent cyanobacterium is Synechocystis, can be nies970_09470 when the parent cyanobacterium is Anabaena, can be anacy_5815 or anacy_3458 when the parent cyanobacterium is Microcystis, can be A0A0F6U6F8_MICAE when the parent cyanobacterium is Cyanothece, can be For example, A0A3B8XX12_9CYAN, A0A1Q8ZE23_9CYAN, A0A1Z4R6U0_9CYAN, A0A1C0VG86_9NOSO, B1WRN6_CROS5, and K9TAE4_9CYAN when the parent cyanobacteria is Crocosphaera, and Pleurocapsa, etc. The base sequences of these genes can be obtained from the above-mentioned NCBI database or Cyanobase.

[0129] More specifically, the gene encoding SLH domain-retaining outer membrane protein 6 can be slr1841 (sequence number 7) of Synechocystis sp. PCC 6803, nies970_09470 (sequence number 8) of Synechococcus sp. NIES-970, anacy_3458 (sequence number 9) of Anabaena cylindrica PCC 7122, or a gene whose base sequence is more than 50% identical to these genes.

[0130] Thus, in the modified cyanobacteria, any gene encoding the SLH domain-maintaining outer membrane protein 6 shown in the above sequence numbers 7 to 9 or a gene with a base sequence more than 50% identical to any of the above genes is deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the above SLH domain-maintaining outer membrane proteins 6 or a protein having the same function as any of the above proteins is inhibited, or (ii) the function of any of the above SLH domain-maintaining outer membrane proteins 6 or a protein having the same function as any of the above proteins is inhibited. Therefore, in the modified cyanobacteria involved in the present embodiment, the binding amount and binding force of the binding domain (e.g., SLH domain 7) used for the outer membrane 5 to bind to the cell wall 4 are reduced, so that the outer membrane 5 becomes easy to partially detach from the cell wall 4.

[0131] As described above, if the amino acid sequence of a protein is more than 30% identical, it can be said that there is a high possibility that it has the same function as the protein. Therefore, if the base sequence of a gene encoding a protein is more than 30% identical, it can be considered that there is a high possibility that a protein with the same function as the protein is expressed. Therefore, as a gene encoding a functionally inhibited SLH domain-retaining outer membrane protein 6, it can be, for example, a gene composed of a base sequence having an identity of more than 40%, preferably more than 50%, more preferably more than 60%, further preferably more than 70%, further more preferably more than 80%, and preferably more than 90% with any of the base sequences of the genes encoding the SLH domain-retaining outer membrane protein 6 shown in the above-mentioned sequence numbers 7 to 9, and encoding a protein or polypeptide having a function of binding to a covalently bonded sugar chain 3 of a cell wall 4.

[0132] In addition, for example, the gene encoding cell wall-pyruvate modification enzyme 9 may be slr0688 when the parent cyanobacterium is Synechocystis, syn7502_03092 or synpcc7942_1529 when the parent cyanobacterium is Synechococcus, ana_C20348 or anacy_1623 when the parent cyanobacterium is Anabaena, csaB (NCBI access ID: TRU80220) when the parent cyanobacterium is Microcystis, csaB (NCBI access ID: WP_107667006.1) when the parent cyanobacterium is Cyanothece, and csaC (NCBI access ID: WP_107667006.2) when the parent cyanobacterium is Cyanothece. When the cyanobacteria is Spirulina, it may be csaB (NCBI access ID: WP_026079530.1), etc., when the parent cyanobacteria is Calothrix, it may be csaB (NCBI access ID: WP_096658142.1), etc., when the parent cyanobacteria is Nostoc, it may be csaB (NCBI access ID: WP_099068528.1), etc., when the parent cyanobacteria is Crocosphaera, it may be csaB (NCBI access ID: WP_012361697.1), etc., when the parent cyanobacteria is Pleurocapsa, it may be csaB (NCBI access ID: WP_036798735), etc. The base sequences of these genes can be obtained from the above-mentioned NCBI database or Cyanobase.

[0133] More specifically, the gene encoding cell wall-pyruvate modification enzyme 9 may be slr0688 (SEQ ID NO: 10) of Synechocystis sp. PCC6803, synpcc7942_1529 (SEQ ID NO: 11) of Synechococcus sp. PCC 7942, or anacy_1623 (SEQ ID NO: 12) of Anabaena cylindrica PCC 7122. In addition, the gene may be one having a base sequence 50% or more identical to these genes.

[0134] Thus, in the modified cyanobacteria, any gene encoding the cell wall-pyruvate modification enzyme 9 shown in the above sequence numbers 10 to 12 or a gene with a base sequence more than 50% identical to that of the gene encoding any of the above enzymes is deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the above cell wall-pyruvate modification enzymes 9 or a protein having the same function as any of the above enzymes is inhibited, or (ii) the function of any of the above cell wall-pyruvate modification enzymes 9 or a protein having the same function as any of the above enzymes is inhibited. Thus, the covalently bonded sugar chain 3 on the surface of the cell wall 4 becomes less susceptible to modification by pyruvic acid, thereby reducing the binding amount and binding force of the sugar chain 3 of the cell wall 4 and the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5. Therefore, in the modified cyanobacterium involved in this embodiment, the amount of sugar chain 3 modified by pyruvic acid for binding of the cell wall 4 to the outer membrane 5 is reduced, so the binding force between the cell wall 4 and the outer membrane 5 is weak, and the outer membrane 5 becomes easy to partially detach from the cell wall 4.

[0135] As described above, if the base sequence of the gene encoding the protein is 30% or more identical, it can be considered that there is a high possibility that a protein having the same function as the protein is expressed. Therefore, as a gene encoding the cell wall-pyruvate modification enzyme 9 whose function is suppressed, for example, it can be composed of a base sequence having an identity of 40% or more, preferably 50% or more, more preferably 60% or more, further preferably 70% or more, further more preferably 80% or more, and preferably 90% or more with any of the base sequences of the genes encoding the cell wall-pyruvate modification enzyme 9 shown in the above-mentioned sequence numbers 10 to 12, and encoding a protein or polypeptide having a function of catalyzing a reaction in which the covalently bonded sugar chain 3 of the peptidoglycan 2 of the cell wall 4 is modified with pyruvic acid.

[0136] [6. Method for producing modified cyanobacteria]

[0137] Next, the method for producing the modified cyanobacteria according to the present embodiment will be described. The method for producing the modified cyanobacteria includes the step of inhibiting the function of a protein involved in the binding of the outer membrane 5 and the cell wall 4 in the cyanobacteria.

[0138] In the present embodiment, the protein involved in the binding between the outer membrane 5 and the cell wall 4 may be, for example, at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 .

[0139] In addition, the means for inhibiting the function of the protein is not particularly limited and can be, for example, deleting or inactivating the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9, inhibiting the transcription of these genes, inhibiting the translation of the transcription products of these genes, or administering inhibitors that specifically inhibit these proteins.

[0140] The means for deleting or inactivating the above-mentioned gene can be, for example, the introduction of mutations into one or more bases in the base sequence of the gene, the replacement of the base sequence with other base sequences or the insertion of other base sequences, or the deletion of part or all of the base sequence of the gene.

[0141] The means for inhibiting the transcription of the above-mentioned gene can be, for example, introduction of mutations into the promoter region of the gene, inactivation of the promoter caused by substitution to other base sequences or insertion of other base sequences, or CRISPR interference method (non-patent document 13: Yao et al., ACS Synth. Biol., 2016, 5: 207-212), etc. The specific method of the above-mentioned introduction of mutations or substitution or insertion of base sequences can be, for example, ultraviolet irradiation, site-directed mutagenesis, or recombination method, etc.

[0142] In addition, means for inhibiting the translation of the transcription products of the above genes include, for example, RNA (ribonucleic acid) interference method and the like.

[0143] By using any of the above means, the function of the protein involved in the binding of the outer membrane 5 and the cell wall 4 in the cyanobacteria can be inhibited, thereby producing a modified cyanobacterium.

[0144] Thus, in the transformation cyanobacteria made by this preparation method, the combination (that is, binding capacity and bonding force) of cell wall 4 and adventitia 5 partially reduces, thereby adventitia 5 becomes easy to partially break away from cell wall 4.Therefore, in the transformation cyanobacteria, the protein produced in thalline becomes easy to leak outside adventitia 5 (that is, outside thalline).Therefore, according to the preparation method of the transformation cyanobacteria involved in the present embodiment, the transformation cyanobacteria that the secretion productivity of protein has improved can be provided.

[0145] In addition, the transformation cyanobacteria made by this preparation method is owing to the protein that produces in thalline easily leaks out to outside thalline, thereby does not need to be broken thalline for the recovery of protein.For example, as long as the transformation cyanobacteria is cultivated under suitable condition, then reclaim the protein that is secreted in the nutrient solution, thereby also can cultivate the protein that reclaims in the nutrient solution on one side of transformation cyanobacteria.Therefore, if use the transformation cyanobacteria that obtains by this preparation method, then can implement efficient microbiology protein production.Therefore, even can provide the transformation cyanobacteria that the utilization efficiency that also can reuse is high after having reclaimed protein according to the preparation method of the transformation cyanobacteria that this embodiment relates to.

[0146] In addition, the transformation cyanobacteria made by this preparation method for example, the protein group that is mainly originally present in the periplasm such as peptase or phosphatase is secreted into the extracellular.In the present embodiment, for example, also can be by the genetic modification of the protein that the protein group that will be present in the periplasm of coding produces in the cell of cyanobacteria like this, replace with the gene of other proteins of encoding, thereby make the transformation cyanobacteria produce desired protein.Therefore, according to the preparation method of the transformation cyanobacteria that the present embodiment relates to, the transformation cyanobacteria that can easily and efficiently produce desired protein can also be provided.

[0147] [7. Method for producing protein using modified cyanobacteria]

[0148] Next, a method for producing a protein using the modified cyanobacteria according to the present embodiment will be described. The method for producing a protein using the modified cyanobacteria according to the present embodiment includes the step of culturing the modified cyanobacteria described above.

[0149] The cultivation of cyanobacteria can generally be implemented based on the liquid culture or its transformation method that has used BG-11 substratum (with reference to Table 2). Therefore, the cultivation of transformation cyanobacteria can also be implemented in the same manner. In addition, as the incubation time of the cyanobacteria for producing protein, as long as it is the time that can be carried out in the mode of accumulating protein at high concentration under the condition that thalline fully proliferates, for example, it can be 1~3 days, it can also be 4~7 days. In addition, the culture method, for example, can be aeration stirring culture or shaking culture.

[0150] By cultivating under the above-mentioned conditions, thereby transforming cyanobacteria and producing protein in thalline, this protein is secreted in the culture fluid.When reclaiming the protein that is secreted in the culture fluid, can by filtration of the culture fluid or centrifugation etc., thereby remove solid components such as cell (so-called thalline), reclaim culture supernatant.According to the protein production method of the transforming cyanobacteria that utilizes the present embodiment to relate to, because protein is secreted into the cell outside of transforming cyanobacteria, so do not need to reclaim broken cells for protein.Therefore, the remaining transforming cyanobacteria after protein recovery can be reused, produce protein.

[0151] In addition, the recovery method of the protein secreted into the culture fluid is not limited to the above-mentioned example, and the protein in the culture fluid can also be recovered while cultivating and transforming cyanobacteria. For example, the protein that has penetrated the membrane can be recovered by using a permeation membrane that penetrates the protein. In this case, useful microorganisms such as lactic acid bacteria can be cultivated as a nutrient source using the protein that has penetrated the membrane. Like this, the protein in the culture fluid can be recovered while cultivating and transforming cyanobacteria, thereby removing the processing of the thalline of the transformed cyanobacteria from the culture fluid becomes unnecessary. Therefore, it is possible to produce protein more simply and efficiently.

[0152] In addition, the recovery process of the bacterial cell from the culture solution and the fragmentation process of the bacterial cell become unnecessary, thereby can reduce the damage and the stress that the transformation cyanobacteria is subjected to. Therefore, the secretion productivity of the protein of the transformation cyanobacteria becomes difficult for it, can use the transformation cyanobacteria for a longer time.

[0153] As described above, according to the method for producing proteins using the modified cyanobacteria involved in the present embodiment, enzymes used for food ingredient raw materials or compound manufacturing, diagnostic enzymes or therapeutic enzymes in the medical field, or feed enzymes in the agricultural, aquatic and livestock fields, etc. can be easily and efficiently obtained.

[0154] [Example]

[0155] Hereinafter, the method for identifying the outer membrane peeling of cyanobacteria and the device for identifying the outer membrane peeling of cyanobacteria of the present disclosure will be specifically described through examples, but the present disclosure is not limited to the following examples and is not limited in any way by the following examples.

[0156] In the following examples, as a method for partially separating the outer membrane of cyanobacteria from the cell wall, the expression of the slr1841 gene encoding the SLH domain-maintaining outer membrane protein (Example 1) and the expression of the slr0688 gene encoding the cell wall-pyruvate modifying enzyme (Example 2) were suppressed, and two kinds of modified cyanobacteria were made. Then, the secretion productivity of the protein of these modified cyanobacteria was measured and the identification of the secreted protein was carried out. In addition, the cyanobacterial species used in this example is Synechocystis sp.PCC 6803 (hereinafter, only referred to as "cyanobacteria").

[0157] (Example 1)

[0158] In Example 1, a modified cyanobacterium in which the expression of the slr1841 gene encoding the SLH domain-retaining outer membrane protein was suppressed was prepared.

[0159] (1) Construction of a cyanobacterial strain in which the expression of the slr1841 gene is suppressed

[0160] As a gene expression inhibition method, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference method is used. In this method, the gene encoding dCas9 protein (hereinafter referred to as dCas9 gene) and slr1841_sgRNA (single-guide Ribonucleic Acid) gene are introduced into the chromosomal DNA of cyanobacteria, thereby inhibiting the expression of slr1841 gene. In addition, by controlling the transcriptional activity of slr1841_sgRNA, the degree of inhibition of slr1841 gene can be controlled.

[0161] The construction of gene expression inhibition using this method is as follows.

[0162] First, a complex is formed between a Cas9 protein lacking nuclease activity (dCas9) and an sgRNA (slr1841_sgRNA) that complementarily binds to the base sequence of the slr1841 gene.

[0163] Then, the complex recognizes the slr1841 gene on the chromosomal DNA of cyanobacteria and specifically binds to the slr1841 gene. The combination forms a steric hindrance, thereby suppressing the transcription of the slr1841 gene. As a result, the expression of the slr1841 gene of cyanobacteria is suppressed. In addition, by controlling the transcriptional activity of slr1841_sgRNA, the degree of suppression of the slr1841 gene can be controlled.

[0164] Hereinafter, the method for introducing the above two genes into the chromosomal DNA of cyanobacteria will be specifically described.

[0165] (1-1) Introduction of dCas9 gene

[0166] Using the chromosomal DNA of Synechocystis LY07 strain (hereinafter also referred to as LY07 strain) (refer to non-patent literature 13) as a template, the primers psbA1-Fw (sequence number 13) and psbA1-Rv (sequence number 14) described in Table 1 were used to amplify the dCas9 gene and the operating element gene for controlling the expression of the dCas9 gene, as well as the spectinomycin resistance marker gene that serves as a mark for gene introduction by the PCR (Polymerase chain reaction) method. In addition, in the LY07 strain, the above three genes are inserted into the psbA1 gene on the chromosomal DNA in a connected state, so that they can be amplified as one DNA fragment by the PCR method. Here, the resulting DNA fragment is described as a "psbA1::dCas9 box". Using the In-Fusion PCR cloning method (registered trademark), the psbA1::dCas9 box is inserted into the pUC19 plasmid to obtain the pUC19-dCas9 plasmid.

[0167]

[0168] The obtained pUC19-dCas9 plasmid 1 μg was mixed with a cyanobacterial culture solution (cell concentration OD730 = about 0.5), and the pUC19-dCas9 plasmid was introduced into the cells of cyanobacteria by natural transformation. The transformed cells were grown and propagated on a BG-11 agar medium containing 20 μg / mL of spectinomycin for screening. In the screened cells, homologous recombination occurred between the psbA1 gene on the chromosomal DNA and the psbA1 upstream fragment region and the psbA1 downstream fragment region on the pUC19-dCas9 plasmid. Thus, a Synechocystis dCas9 strain with a dCas9 box inserted in the psbA1 gene region was obtained. In addition, the composition of the BG-11 medium used is shown in Table 2.

[0169] Table 2

[0170] Element Content (mg / L) EDTA-Na 1 Ammonium Ferric Citrate 6 <![CDATA[NaNO3]]> 1500 <![CDATA[MgSO4]]> 75 <![CDATA[K2HPO4]]> 39 <![CDATA[CaCl2]]> 28.6 <![CDATA[H3BO4]]> 2.8 <![CDATA[MnCl2]]> 1.8 <![CDATA[ZnSO4]]> 0.2 <![CDATA[CuSO4]]> 0.08 <![CDATA[Na2MoO4]]> 0.02 <![CDATA[Co(NO3)2]]> 0005 TES-KOH (pH 7.5) 4580

[0171] (1-2) Introduction of slr1841_sgRNA gene

[0172] In the CRISPR interference method, a sequence of about 20 bases complementary to the target sequence is introduced into a region called protospacer on the sgRNA gene, so that the sgRNA specifically binds to the target gene. The protospacer sequence used in this example is shown in Table 3.

[0173] Table 3

[0174] Protospacer target genes Base sequence Serial Number slr1841 5′-ACAGCAGGGCTCACTAACA-3′ 21 slr0688 5′-TGCAGCAAACAGACTAAAA-3′ 22

[0175] In Synechocystis LY04, LY05, or LY07, the sgRNA gene (except the protospacer region) and the kanamycin resistance marker gene are connected to the slr2030-slr2031 gene on the chromosomal DNA (see non-patent document 13). Therefore, by adding a protospacer sequence (sequence number 21) complementary to the slr1841 gene (sequence number 7) to the primers used when amplifying the sgRNA gene by PCR, it is possible to easily obtain an sgRNA (slr1841_sgRNA) that specifically recognizes slr1841. In addition, by controlling the transcriptional activity of slr1841_sgRNA, the degree of inhibition of the slr1841 gene can be controlled.

[0176] First, using the chromosomal DNA of the LY07 strain as a template, the two DNA fragments were amplified by PCR using the set of primers slr2030-Fw (sequence number 15) and sgRNA_slr1841-Rv (sequence number 16) and the set of sgRNA_slr1841-Fw (sequence number 17) and slr2031-Rv (sequence number 18) recorded in Table 1.

[0177] Next, the mixed solution of the above DNA fragments was used as a template and amplified by PCR using the primers slr2030-Fw (SEQ ID NO: 15) and slr2031-Rv (SEQ ID NO: 18) described in Table 1, thereby obtaining a DNA fragment (slr2030-2031::slr1841_sgRNA) in which (i) the slr2030 gene fragment, (ii) slr1841_sgRNA, (iii) the kanamycin resistance marker gene, and (iv) the slr2031 gene fragment were sequentially connected. Using the In-Fusion PCR cloning method (registered trademark), slr2030-2031::slr1841_sgRNA was inserted into the pUC19 plasmid to obtain the pUC19-slr1841_sgRNA plasmid.

[0178] The pUC19-slr1841_sgRNA plasmid was introduced into the Synechocystis dCas9 strain by the same method as described above (1-1), and the transformed cells were screened on BG-11 agar culture containing 30 μg / mL kanamycin. Thus, a transformant Synechocystis dCas9 slr1841_sgRNA strain (hereinafter also referred to as slr1841 suppression strain) in which slr2030-slr2031 gene on chromosomal DNA was inserted with slr1841_sgRNA was obtained.

[0179] (1-3) Suppression of slr1841 gene

[0180] The promoter sequences of the dCas9 gene and the slr1841_sgRNA gene were designed so that expression was induced in the presence of anhydrotetracycline (aTc). In this example, the expression of the slr1841 gene was inhibited by adding aTc to a final concentration of 1 μg / mL during culture.

[0181] As described above, in Example 1, the total amount of proteins involved in the binding of the outer membrane to the cell wall in cyanobacteria was suppressed to about 30% compared with the amount of the protein in the parent strain (Synechocystis dCas9 strain, Comparative Example 1 described later) without damaging the proliferation ability of the cells. The modified cyanobacterium Synechocystis dCas9slr1841_sgRNA strain (so-called slr1841 inhibition strain) is obtained. Here, the proteins involved in the binding of the outer membrane to the cell wall are slr1841, slr1908 and slr0042.

[0182] (Example 2)

[0183] In Example 2, a modified cyanobacterium in which the expression of the slr0688 gene encoding a cell wall-pyruvate modification enzyme was suppressed was obtained through the following steps.

[0184] (2) Construction of a cyanobacterial strain in which the expression of the slr0688 gene was suppressed

[0185] By the same steps as above (1-2), the sgRNA gene containing the protospacer sequence (sequence number 22) complementary to the slr0688 gene (sequence number 4) was introduced into the Synechocystis dCas9 strain to obtain the Synechocystis dCas9slr0688_sgRNA strain. In addition, using the set of primers slr2030-Fw (SEQ ID NO: 15) and sgRNA_slr0688-Rv (SEQ ID NO: 19) and the set of sgRNA_slr0688-Fw (SEQ ID NO: 20) and slr2031-Rv (SEQ ID NO: 18) described in Table 1, and using the In-Fusion PCR cloning method (registered trademark), a DNA fragment (slr2030-2031::slr0688_sgRNA) in which (i) the slr2030 gene fragment, (ii) slr0688_sgRNA, (iii) kanamycin resistance marker gene, and (iv) slr2031 gene fragment were sequentially connected was inserted into the pUC19 plasmid to obtain the pUC19-slr0688_sgRNA plasmid, except that the same conditions as those in the above (1-2) were used. In addition, by controlling the transcriptional activity of slr0688_sgRNA, the degree of repression of the slr0688 gene can be controlled.

[0186] Furthermore, the expression of the slr0688 gene was suppressed by the same procedure as in the above (1-3).

[0187] As described above, in Example 2, the total amount of proteins involved in the binding of the outer membrane to the cell wall in cyanobacteria was suppressed to about 50% compared with the amount of the protein in the parent strain (Synechocystis dCas9 strain, Comparative Example 1 described later) without damaging the proliferation ability of the cells. The modified cyanobacterium Synechocystis dCas9 slr0688_sgRNA strain (hereinafter also referred to as slr0688 inhibition strain) was obtained. Here, the protein involved in the binding of the outer membrane to the cell wall is slr0688.

[0188] (Comparative Example 1)

[0189] In Comparative Example 1, the Synechocystis dCas9 strain was obtained by the same procedure as in Example 1 (1-1).

[0190] Next, observation of the cell surface state and protein secretion productivity test were performed on the strains obtained in Example 1, Example 2 and Comparative Example 1. The details are described below.

[0191] (3) Observation of the cell surface status of the strain

[0192] Ultrathin sections were prepared for the modified cyanobacterium Synechocystis dCas9 slr1841_sgRNA strain obtained in Example 1 (so-called slr1841 inhibition strain), the modified cyanobacterium Synechocystis dCas9 slr0688_sgRNA strain obtained in Example 2 (so-called slr0688 inhibition strain), and the modified cyanobacterium Synechocystis dCas9 strain obtained in Comparative Example 1 (hereinafter also referred to as control strain), and the state of the cell surface (in other words, the outer membrane structure) was observed using an electron microscope.

[0193] (3-1) Cultivation of strains

[0194] The slr1841 inhibitor strain of Example 1 was inoculated into 12 mL of BG-11 medium (see Table 2) containing 1 μg / mL aTc so that the initial bacterial cell concentration OD730 was 0.05, and the slr1841 inhibitor strain was inoculated into 12 mL of BG-11 medium (see Table 2) containing 1 μg / mL aTc. 2 / s, and shaking culture was performed for 5 days at 30°C. In addition, the slr0688-inhibited strain of Example 2 and the control strain of Comparative Example 1 were also cultured under the same conditions as in Example 1.

[0195] (3-2) Preparation of Ultrathin Sections of Strain

[0196] The culture solution obtained in (3-1) above was centrifuged at 2,500g for 10 minutes at room temperature to recover the cells of the slr1841 inhibitor strain of Example 1. Next, the cells were quickly frozen with liquid propane at -175°C and then fixed at -80°C for 2 days using an ethanol solution containing 2% glutaraldehyde and 1% tannic acid. The fixed cells were dehydrated with ethanol, the dehydrated cells were soaked in propylene oxide, and then sank in a resin (Quetol-651) solution. The resin was then allowed to stand at 60°C for 48 hours to solidify, thereby embedding the cells with the resin. The cells in the resin were thinly sliced ​​into 70nm thickness using an ultracut to prepare ultrathin sections. The ultrathin sections were stained with 2% uranyl acetate and 1% lead citrate solution to prepare transmission electron microscope samples of the slr1841 inhibitor strain of Example 1. In addition, the same operation was performed on the slr0688-suppressed strain of Example 2 and the control strain of Comparative Example 1, and samples for transmission electron microscopy were prepared.

[0197] (3-3) Observation using an electron microscope

[0198] The ultrathin sections obtained in (3-2) were observed using a transmission electron microscope (JEOL JEM-1400Plus) at an accelerating voltage of 100 kV. The observation results are shown in Figures 4 to 9 .

[0199] First, the slr1841-suppressed strain of Example 1 will be described. Figure 4 This is a TEM (Transmission Electron Microscope) image of the slr1841-suppressed strain of Example 1. Figure 5 yes Figure 4 A magnified image of the dashed area A. Figure 5 (a) is Figure 4 The enlarged TEM image of the dashed area A is shown in Figure 1. Figure 5 (b) depicts Figure 5 (a) is an enlarged TEM image of the sample.

[0200] like Figure 3 As shown, in the slr1841-suppressed strain of Example 1, the outer membrane was partially peeled off from the cell wall (ie, the outer membrane was partially peeled off), and the outer membrane was partially bent.

[0201] In order to confirm the state of the cell surface in more detail, the dotted area A was magnified and observed. Figure 5 (a) and Figure 5 As shown in (b), it is possible to confirm that the outer membrane is partially peeled off (the dot-dash areas a1 and a2 in the figure). In addition, a greatly bent portion of the outer membrane can be confirmed next to the dot-dash area a1. It is considered that this portion is a portion where the outer membrane is weakly bonded to the cell wall, and the culture fluid penetrates from the outer membrane into the periplasm, so the outer membrane expands outward and bends.

[0202] Next, the slr0688-suppressed strain of Example 2 will be described. Figure 6 This is a TEM image of the slr0688 inhibitor strain of Example 2. Figure 7 yes Figure 6 A magnified image of the dashed area B. Figure 7 (a) is Figure 6 The enlarged TEM image of the dashed area B is shown in Figure 1. Figure 7 (b) depicts Figure 7 (a) is an enlarged TEM image of the sample.

[0203] like Figure 6 As shown, in the slr0688-inhibited strain of Example 2, the outer membrane was partially peeled off from the cell wall, and the outer membrane was partially bent. In addition, in the slr0688-inhibited strain, it was confirmed that the outer membrane was partially separated from the cell wall.

[0204] In order to confirm the state of the cell surface in more detail, the dotted area B was magnified and observed. Figure 7 (a) and Figure 7As shown in (b), it is possible to confirm a portion where the outer membrane is greatly bent (the dotted line region b1 in the figure) and a portion where the outer membrane is partially peeled off (the dotted line regions b2 and b3 in the figure). In addition, it is possible to confirm a portion where the outer membrane is detached from the cell wall near each of the dotted line regions b1, b2 and b3.

[0205] Next, the control strain of Comparative Example 1 will be described. Figure 8 This is a TEM image of the control strain of Comparative Example 1. Fig. 9 yes Figure 8 A magnified image of the dashed area C. Fig. 9 (a) is Figure 8 The enlarged TEM image of the dashed area C, Fig. 9 (b) depicts Fig. 9 (a) is an enlarged TEM image of the sample.

[0206] like Figure 8 and Fig. 9 As shown, the cell surface layer of the control strain of Comparative Example 1 was flat, and the inner membrane, cell wall, outer membrane, and S layer were stacked in order. That is, in the control strain, there was no portion where the outer membrane was separated from the cell wall, no portion where the outer membrane was peeled off (i.e., peeled off) from the cell wall, and no portion where the outer membrane was bent as in Examples 1 and 2.

[0207] (4) Protein secretion productivity test

[0208] The slr1841 inhibitor strain of Example 1, the slr0688 inhibitor strain of Example 2, and the control strain of Comparative Example 1 were cultured separately, and the amount of protein secreted outside the cell (hereinafter, also referred to as the amount of secreted protein) was measured. The secretory productivity of the protein of each of the above-mentioned strains was evaluated by the amount of protein in the culture medium. In addition, the so-called secretory productivity of protein refers to the ability to produce protein by secreting the protein produced in the cell outside the cell. The specific method is described below.

[0209] (4-1) Cultivation of strains

[0210] The slr1841-suppressed strain of Example 1 was cultured in the same manner as in (3-1) above. The culture was performed independently three times. In addition, the strains of Example 2 and Comparative Example 1 were also cultured under the same conditions as the strain of Example 1.

[0211] (4-2) Quantification of proteins secreted outside cells

[0212] The culture solution obtained in (4-1) above was centrifuged at 2,500g for 10 minutes at room temperature to obtain a culture supernatant. The obtained culture supernatant was filtered using a membrane filter with a pore size of 0.22μm to completely remove the cells of the slr1841 inhibitor strain of Example 1. The amount of total protein contained in the filtered culture supernatant was quantified by the BCA (Bicinchoninic acid) method. This series of operations was performed on three culture solutions obtained by independent culture, and the average value and standard deviation of the amount of protein secreted outside the cells of the slr1841 inhibitor strain of Example 1 were calculated. In addition, for the strains of Example 2 and Comparative Example 1, the protein in three culture solutions was quantified under the same conditions, and the average value and standard deviation of the amount of protein in the three culture solutions were calculated.

[0213] The results are shown in Fig.10 . Fig.10 It is a bar graph showing the amount of protein in the culture supernatant of the modified cyanobacteria of Example 1, Example 2 and Comparative Example 1 (n=3, error bar=SD).

[0214] like Fig.10 As shown, the amount of protein secreted into the culture supernatant (mg / L) of both the slr1841-suppressed strain of Example 1 and the slr0688-suppressed strain of Example 2 was increased by about 25 times compared with the control strain of Comparative Example 1.

[0215] The data record is omitted, but the absorbance of the culture medium was measured (730nm) and the amount of secretory protein per 1g dry weight of the bacteria (mg protein / g cell dry weight) was calculated. The results showed that the amount of secretory protein per 1g dry weight of the bacteria (mg protein / g cell dry weight) of the slr1841 inhibition strain of Example 1 and the slr0688 inhibition strain of Example 2 was approximately 36 times higher than that of the control strain of Comparative Example 1.

[0216] In addition, if Fig.10As shown, compared with the slr1841 inhibition strain of Example 1 in which the expression of the gene (slr1841) encoding the SLH domain-retaining outer membrane protein was suppressed, the slr0688 inhibition strain of Example 2 in which the expression of the gene (slr0688) encoding the cell wall-pyruvate modification enzyme was suppressed, secreted a larger amount of protein into the culture supernatant. This is considered to be related to the fact that the number of covalently bonded sugar chains on the cell wall surface is greater than the number of SLH domain-retaining outer membrane proteins (Slr1841) in the outer membrane. In other words, it is considered that the slr0688 inhibition strain of Example 2 has a further reduced amount and strength of binding between the outer membrane and the cell wall compared to the slr1841 inhibition strain of Example 1, and thus the amount of secreted protein is increased compared to the slr1841 inhibition strain of Example 1.

[0217] Can confirm by above result, by suppressing the protein function of the combination of adventitia and cell wall, thereby the adventitia of cyanobacteria and the combination of cell wall are partially weakened, and adventitia partially breaks away from cell wall.Also can confirm a little less than the combination of adventitia and cell wall, thereby the protein produced in the cell of cyanobacteria becomes easy to leak into the extracellular.Therefore show, according to the transformation cyanobacteria that the present embodiment relates to and preparation method thereof, can obtain the transformation cyanobacteria that the secretion productivity of protein significantly improves.

[0218] (5) Identification of secreted proteins

[0219] Next, the secretory proteins contained in the culture supernatant obtained in (4-2) above were identified by LC-MS / MS. The method is described below.

[0220] (5-1) Sample preparation

[0221] Add 8 times the amount of cold acetone relative to the amount of the culture supernatant, let it stand at 20°C for 2 hours, and then centrifuge at 20,000g for 15 minutes to obtain a protein precipitate. Add 100mM Tris pH8.5 and 0.5% sodium dodecanoate (SDoD) to the precipitate, and dissolve the protein by a closed ultrasonic crusher. After adjusting the protein concentration to 1μg / mL, add dithiothreitol (DTT) at a final concentration of 10mM and let it stand at 50°C for 30 minutes. Next, add iodoacetamide (IAA) at a final concentration of 30mM and let it stand at room temperature (shaded) for 30 minutes. In order to stop the reaction of IAA, add cysteine ​​at a final concentration of 60mM and let it stand at room temperature for 10 minutes. Add 400ng of trypsin and let it stand at 37°C overnight to fragment the protein peptides. After adding 5% TFA (Trifluoroacetic Acid), centrifuge at 15,000g for 10 minutes at room temperature to obtain the supernatant. SDoD was removed by this operation. After desalting using a C18 spin column, the sample was dried using a centrifugal evaporator. Then, 3% acetonitrile and 0.1% formic acid were added and the sample was dissolved using a closed ultrasonic crusher. The peptide concentration was adjusted to 200 ng / μL.

[0222] (5-2) LC-MS / MS analysis

[0223] The sample obtained in the above (5-1) was analyzed under the following conditions using an LC-MS / MS apparatus (UltiMate 3000RSLCnano LCSystem).

[0224] Sample injection amount: 200ng

[0225] Column: CAPCELL CORE MP 75μm×250mm

[0226] Solvent: Solvent A is 0.1% formic acid aqueous solution, solvent B is 0.1% formic acid + 80% acetonitrile

[0227] Gradient program: 4 minutes after sample injection, 8% solvent B, 27 minutes after solvent B, 44% solvent B, 28 minutes after solvent B, 80% solvent B, 34 minutes after the end of the measurement

[0228] (5-3) Data Analysis

[0229] The obtained data were analyzed under the following conditions to identify proteins and peptides and calculate quantitative values.

[0230] Software: Scaffold DIA

[0231] Database: UniProtKB / Swiss Prot database (Synechocystis sp. PCC 6803)

[0232] Fragmentation: HCD

[0233] Precursor Tolerance: 8ppm

[0234] Fragment Tolerance: 10ppm

[0235] Data Acquisition Type: Overlapping DIA

[0236] Peptide Length: 8-70

[0237] Peptide Charge: 2-8

[0238] Max Missed Cleavages: 1

[0239] Fixed Modification: Carbamidomethylation

[0240] Peptide FDR: less than 1%

[0241] Table 4 shows the ten proteins in order from the one with the largest relative quantitative value among the identified proteins.

[0242]

[0243] All 10 proteins are contained in the culture supernatant of the slr1841 inhibitor strain of Example 1 and the slr0688 inhibitor strain of Example 2. In all these proteins, the periplasmic (referring to the gap between the outer membrane and the inner membrane) transfer signal is maintained. According to this result, it can be confirmed that in the modified strains of Example 1 and Example 2, the outer membrane is partially separated from the cell wall, so that the protein in the periplasm becomes easy to leak out of the outer membrane (that is, outside the bacterium). Therefore, the modified cyanobacteria involved in this embodiment show that the secretion productivity of the protein is greatly improved.

[0244] (6) Determination of outer membrane exfoliation of cyanobacteria

[0245] (6-1) Cultivation of cyanobacteria

[0246] As the wild strain of cyanobacterium Synechocystis sp. PCC 6803, the control strain of Comparative Example 1 was used, and as the outer membrane stripping type mutant strain of Synechocystis sp. PCC 6803, the slr0688 inhibitory strain of Example 2, which was confirmed to have outer membrane stripping by the secretion production test of the protein in the above (4), was used. Hereinafter, these cyanobacteria are referred to as cyanobacterial wild strains and outer membrane stripping type cyanobacteria, respectively. These two types of cyanobacteria were inoculated in 12 mL of BG-11 medium (see Table 2) containing 1 μg / mL of aTc in a 125 mL Erlenmeyer flask at an initial cell concentration of OD730 = 0.1, and incubated at a light intensity of 100 μmol / m 2 / s, 30°C, and shaking culture for 3 days.

[0247] After culturing for 3 days, the culture solution was centrifuged at 2,500 g for 10 minutes at room temperature, and the culture supernatant was filtered using Millex-GVSyringe Filter Unit, 0.22 μm (Millipore). The filtered supernatant was subjected to amino acid analysis using LC-MS / MS.

[0248] (6-2) Amino Acid Analysis by LC-MS / MS

[0249] Next, isoleucine, leucine, tyrosine, and phenylalanine contained in the culture supernatants of the two types of cyanobacteria obtained in (6-1) above were measured by LC-MS / MS. The method is described below.

[0250] (6-2-1) Sample preparation

[0251] 1 mL of the culture supernatant of the wild strain of cyanobacteria obtained in (6-1) above was freeze-dried and dissolved in 195 μL of APDS Tag (registered trademark) boric acid buffer. 5 μL of a solution obtained by dissolving 100 mg of APDS (3-aminopyridyl-N-hydroxysuccinimidyl carbamate) Tag reagent in 5 mL of acetonitrile was added thereto, mixed thoroughly, and heated at 55-60° C. for 5-15 minutes. In addition, the culture supernatant of the outer membrane peeling type cyanobacteria was treated in the same manner.

[0252] (6-2-2) LC-MS / MS analysis

[0253] The sample obtained in the above (6-2-1) was analyzed under the following conditions using an LC-MS / MS device (UltiMate 3000RSLCnano LCSystem).

[0254] Sample injection volume: 10μL

[0255] Column: Tosoh TSKgel ODS-100V 2.0mmI.D×15cm

[0256] Mobile phase: Solution A is APDS Tag (registered trademark) eluent for mobile phase, Solution B is 60% acetonitrile

[0257] Dissolution timetable (B solution %):

[0258] 0min, 6%; 0.05-1.70min, 8%; 1.71min, 12%; 4.95min, 30%; 5.95min, 60%; 6.70min, 95%; 6.71min, 6%; 12.0min, 6%

[0259] Flow rate: 0.3mL / min

[0260] Column temperature: 40°C

[0261] Ionization mode: ESI positive

[0262] (6-3) Results

[0263] Fig.11 The graph shows the results of amino acid analysis of culture supernatants of two types of cyanobacteria by LC-MS / MS. Fig.11 The TIC (total ion current chromatogram) of the culture supernatant of the outer membrane-shedding cyanobacteria and the culture supernatant of the wild strain of cyanobacteria is shown in FIG. A is the peak derived from tyrosine, B is the peak derived from isoleucine, C is the peak derived from leucine, and D is the peak derived from phenylalanine. Fig.11 As shown, since peaks A to D were hardly detected in the culture supernatant of wild-type cyanobacteria but were detected in the culture supernatant of membrane-shedding cyanobacteria, it was confirmed that the above four amino acids are characteristically secreted into the culture supernatant when the outer membrane of the cyanobacterium is shed from the cell wall.

[0264] In addition, the measurement results of the concentrations of the above four kinds of amino acids are shown in Table 5. As shown in Table 5, it was confirmed that in the culture supernatant of the outer membrane peeling type cyanobacteria, isoleucine, leucine, tyrosine and phenylalanine were present at a concentration of about 130nM or more and 320nM or less. On the other hand, it was confirmed that in the culture supernatant of the cyanobacterial wild strain, these four kinds of amino acids were not substantially present.

[0265] Therefore, if at least one amino acid selected from these 4 kinds of amino acids in the quantitative culture supernatant, it is possible to easily determine that the outer membrane is peeled off from the cell wall. By the result of Table 5, it can be believed that if the concentration of each amino acid exceeds 100nM, it is possible to determine that the outer membrane of cyanobacteria is peeled off from the cell wall. By using the determination method of the outer membrane peeling of cyanobacteria disclosed in the present invention, it is not necessary to gather the cell of cyanobacteria to confirm the state of the outer membrane with complicated techniques such as electron microscope observation, therefore the cell of the state of being suitable for material production can be selected quickly and easily and be applied to material production.

[0266]

[0267] (7) Investigation

[0268] In the present embodiment, it can be confirmed that the disclosed transformation cyanobacteria will secrete the protein present in the thalline (here in the periplasm) outside the thalline. The disclosed transformation cyanobacteria, for example, can carry out genetic modification in the mode of producing other proteins to replace the protein (i.e. the protein originally produced in the thalline) of the above-mentioned identification, thereby can efficiently produce desired protein. In addition, cyanobacteria are because the photosynthesis ability is high, thereby by giving light, water, air and a trace of inorganic matter and cultivating, thereby can easily obtain required protein when needed, thereby do not need to use complicated device for the production of protein. In addition, protein, for example, its function is easily lost when being processed into supplement etc. Therefore, according to the disclosed transformation cyanobacteria, protein can be provided under the state of having maintained the function of protein. By above advantages, the disclosed transformation cyanobacteria can be expected to be applied in various fields.

[0269] In addition, through "(3-3) Observation using an electron microscope", it was confirmed that in the modified strain in which the expression of the gene encoding the protein involved in the binding of the outer membrane to the cell wall was suppressed, the outer membrane partially peeled off due to the weakening of the binding of the outer membrane to the cell wall, and the outer membrane partially swelled. It is believed that this is because the weakening of the binding of the outer membrane to the cell wall makes it easier for the culture fluid to flow in from outside the cell due to osmotic pressure at the position where the outer membrane is partially separated from the cell wall, and the outer membrane partially swells due to the inflow of the culture fluid. It is also believed that due to the large difference between the protein concentration of the culture fluid and the protein concentration in the periplasm, the flow of the culture fluid from the outer membrane into the periplasm does not stop, and the outer membrane that cannot withstand the internal pressure ruptures and peels off (i.e., peels off).

[0270] In addition, Table 3 lists only 10 proteins in descending order of relative quantitative values ​​among all proteins identified in LC-MS / MS analysis. In addition, "(5-3) Data Analysis" states that all 10 proteins listed in Table 3 are contained in the culture supernatants of the slr1841-suppressed strain of Example 1 and the slr0688 strain of Example 2, but all other proteins identified in LC-MS / MS analysis are also contained in the culture supernatants of Examples 1 and 2. It was also confirmed that secreted proteins include proteins originally present in the periplasm and proteins transported from the cytoplasm to the periplasm and functioning in the periplasm.

[0271] In addition, the concentrations of 4 kinds of amino acids characteristically included in the culture supernatant of outer membrane stripping type cyanobacteria are recorded in Table 5 by LC-MS / MS analysis. These 4 kinds of amino acids are not basically present in the culture supernatant of wild strains of cyanobacteria. Therefore, it is confirmed that when the concentration of any one of these 4 kinds of amino acids in the culture supernatant exceeds a threshold value (e.g., 100 nM), it can be determined that the outer membrane of cyanobacteria is stripped from the cell wall.

[0272] (Other embodiments)

[0273] Although the determination method of the outer membrane peeling of cyanobacteria involved in the present disclosure, the determination device and program of the outer membrane peeling of cyanobacteria are described above based on the embodiments, the present disclosure is not limited to these embodiments. As long as it does not depart from the purport of the present disclosure, the various deformation modes that a person skilled in the art thinks of in the embodiments, and other modes constructed by combining some of the constituent elements in the embodiments are also within the scope of the present disclosure.

[0274] In the above-mentioned embodiment, for the total amount of the protein that participates in the combination of the outer membrane and the cell wall in the cyanobacteria is suppressed to more than 30% and less than 70% of the total amount of the protein in the parent strain, thereby weakening the combination of the outer membrane and the cell membrane and causing the protein produced in the thalline to leak out of the thalline, the example has been described, but the disclosure is not limited thereto. For example, the combination of the outer membrane and the cell wall can be weakened by applying an external force to the cyanobacteria, and the outer membrane can also be embrittled. In addition, the outer membrane can be embrittled by adding an enzyme or a medicament in the culture solution of the cyanobacteria.

[0275] Industrial Availability

[0276] According to the present disclosure, it is possible to easily determine whether the outer membrane of cyanobacteria is peeled off from the cell wall, thereby quickly grasping whether cyanobacteria is suitable for the state of material production and whether the state of material production is also maintained during the culture process. Therefore, according to the present disclosure, the cyanobacteria of the state of material production can be selected and used, thereby material productivity can be improved reliably.

[0277] Explanation of symbols

[0278] 1 Endometrium

[0279] 2 Peptidoglycan

[0280] 3 Sugar chains

[0281] 4 Cell Wall

[0282] 5 Outer membrane

[0283] 6 SLH domain-retaining outer membrane protein

[0284] 7 SLH domain

[0285] 8 Organic matter channel protein

[0286] 9 Cell wall-pyruvate modifying enzymes

Claims

1. The method for determining the outer membrane peeling of cyanobacteria includes: a step of measuring the concentration of at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine and phenylalanine in the culture supernatant of cyanobacteria; and A determination step of determining whether the outer membrane of the cyanobacterium has been peeled off from the cell wall based on the concentration of the at least one amino acid measured in the determination step.

2. The method for determining the outer membrane peeling of cyanobacteria according to claim 1, In the determination step, the concentrations of four amino acids, namely, isoleucine, leucine, tyrosine and phenylalanine, are determined. In the determination step, whether the outer membrane of the cyanobacterium is peeled off from the cell wall is determined based on the concentrations of the four amino acids.

3. The method for determining the outer membrane peeling of cyanobacteria according to claim 1, In the determination step, when at least one of the concentrations of the at least one amino acid is above a threshold value, it is determined that the outer membrane of the cyanobacterium is peeled off from the cell wall; and when all of the concentrations of the at least one amino acid are below the threshold value, it is determined that the outer membrane of the cyanobacterium is not peeled off from the cell wall.

4. The method for determining the outer membrane peeling of cyanobacteria according to claim 3, wherein the threshold value is 100 nM.

5. A device for determining the outer membrane peeling of cyanobacteria, comprising: a measuring unit for measuring the concentration of at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine, and phenylalanine in the culture supernatant of cyanobacteria; and A determination unit for determining whether the outer membrane of the cyanobacterium has been peeled off from the cell wall based on the concentration of the at least one amino acid measured by the determination unit.

6. A program for causing a computer to execute a method for determining whether an outer membrane of a cyanobacterium has been stripped from a cell wall based on the concentration of at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine and phenylalanine in a culture supernatant of the cyanobacterium.

7. A method for determining whether the outer membrane of cyanobacteria has been peeled off, comprising the step of determining whether the outer membrane of the cyanobacteria has been peeled off from the cell wall based on the concentration of amino acids in the culture supernatant of the cyanobacteria. The amino acid is at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine and phenylalanine.

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