Modified cyanobacteria, method for producing modified cyanobacteria, and method for producing proteins

Abstract

This modified cyanobacterium has expressed therein an organic matter channel protein (18) that improves protein permeability of an outer membrane (5).

Description

[Technical field]

[0001] The present disclosure relates to modified cyanobacteria having improved protein secretion productivity that extracellularly secretes a protein produced intracellularly, a method for producing the modified cyanobacteria, and a method for producing a protein. [Background technology]

[0002] A wide range of industrial fields, from the chemical industry to the agriculture, fisheries and livestock industries, require production methods that are not dependent on fossil fuels and have a low environmental impact. Production of substances using microorganisms can be carried out under normal temperature and pressure conditions, and with the recent development of genetic engineering technology, it has become possible to produce a wide range of chemical compounds. Therefore, it has attracted attention as a production system that meets the above requirements. Among these, photosynthetic microorganisms, such as cyanobacteria and algae, use light as an energy source to convert carbon dioxide (CO2) from the air into energy. carbon Because it can be used as a raw material, it is particularly expected to be a carbon-neutral, next-generation material production system.

[0003] For example, substances that have been reported to be produced using cyanobacteria include ethanol (Non-Patent Document 1), isobutanol (Non-Patent Document 2), alkanes (Patent Document 1), fatty acids (Patent Document 2), and proteins (Non-Patent Document 3).

[0004] Furthermore, for example, Non-Patent Document 4 discloses a technique for expressing a protein in cyanobacteria. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2015-109828 A [Patent Document 2] Patent No. 6341676 [Non-patent literature]

[0006] [Non-Patent Document 1] Jason Dexter and Pengcheng Fu, “Metabolic engineering of cyanobacteria for ethanol production”, Energy & Environmental Science, Royal Society of Chemistry, 2009, Vol.2, pp.857-864 [Non-Patent Document 2] Shota Atsumi et al., “Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde”, Nature Biotechnology, Nature Publishing Group, 2009, Vol.27, pp.1177-1180 [Non-Patent Document 3] Jie Zhou et al., “Discovery of a super-strong promoter enable efficient production of heterologous proteins in cyanobacteria”, Scientific Reports, Nature Research, 2014, Vol.4, Article No.4500 [Non-Patent Document 4] Andrew H. Ng et al., “Fine-Tuning of Photoautotrophic Synechocystis sp. Strain PCC 6803”, Applied and Environmental Microbiology, American Society for Microbiology, 2015, Vol.81, pp.6857-6863 Summary of the Invention [Problem to be solved by the invention]

[0007] However, with the above-mentioned conventional techniques, it is difficult to efficiently produce proteins using cyanobacteria.

[0008] Therefore, the present disclosure provides modified cyanobacteria with improved protein secretion productivity, a method for producing the modified cyanobacteria, and a method for producing a protein using the modified cyanobacteria. [Means for solving the problem]

[0009] In one embodiment of the present disclosure, a modified cyanobacterium expresses a channel protein that increases the protein permeability of the outer membrane. Effect of the Invention

[0010] The modified cyanobacterium and the method for producing the modified cyanobacterium of the present disclosure can provide a modified cyanobacterium with improved protein secretion productivity. Furthermore, the method for producing a protein of the present disclosure can efficiently produce a protein. [Brief description of the drawings]

[0011] [Figure 1] Figure 1 is a schematic diagram of the cell surface of a cyanobacterium. [Diagram 2] FIG. 2 is a graph showing the protein amount in the culture supernatant of the modified cyanobacteria of Example 1 and Comparative Example 1 (n=3, error bars=SD). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] (Findings on which this disclosure is based) Cyanobacteria (also called blue-green bacteria or blue-green algae) are a group of eubacteria that decompose water by photosynthesis to produce oxygen, and fix CO2 from the air using the energy obtained. Note that some species of cyanobacteria can also fix nitrogen (N2) from the air. Cyanobacteria are known to have characteristics such as rapid growth and high light utilization efficiency, and are easier to genetically manipulate than other algae species. Therefore, active research and development is being conducted on the use of cyanobacteria, among photosynthetic microorganisms. As mentioned above, as examples of substance production using cyanobacteria, the production of fuels such as ethanol (Non-Patent Document 1), isobutanol (Non-Patent Document 2), alkanes (Patent Document 1), and fatty acids (Patent Document 2) has been reported.

[0013] Research and development is also being conducted on the production of substances that serve as nutrient sources for living organisms using cyanobacteria. For example, since proteins can only be synthesized by living organisms, photosynthetic microorganisms that use light energy and atmospheric CO2 are thought to be useful as a species that can easily and efficiently produce proteins.

[0014] For example, Non-Patent Document 3 describes a method for producing a protein using the cyanobacterium Synechocystis sp. PCC 6803. This document discloses a promoter base sequence for activating the transcription of an ethylene synthase gene with high efficiency, and a ribosome binding sequence for enhancing translation.

[0015] In addition, Non-Patent Document 4 discloses a method for inserting a gene encoding a protein into the NS-pCC2 region on a plasmid carried by Synechocystis sp. PCC 6803 strain when expressing a recombinant protein using this strain as a host. It has been reported that the use of this method improves the expression efficiency of the recombinant protein by about 14 times compared to the case where the gene is inserted into chromosomal DNA (deoxyribonucleic acid).

[0016] However, in the above-mentioned conventional techniques, the cells of the PCC6803 strain must be disrupted in order to recover the protein produced in the cells of the PCC6803 strain. production In addition, since various substances exist within the bacterial cells, it may be necessary to remove these substances to produce the target protein. This results in a low protein recovery rate. In addition, a new bacterial strain must be prepared each time a protein is produced, which is time-consuming and increases production costs. Thus, in the above-mentioned conventional techniques, the efficiency of protein production using cyanobacteria is still low, and there is a need to develop techniques with higher production efficiency.

[0017] The present inventors have found that the protein permeability of the outer membrane is improved by expressing an outer membrane channel protein derived from plant chloroplasts in the outer membrane of a cyanobacterium, thereby increasing the amount of protein secreted per cyanobacterial culture solution or cell.

[0018] Therefore, according to the present disclosure, it is possible to provide a modified cyanobacterium with improved protein secretion productivity. Furthermore, by culturing the modified cyanobacterium of the present disclosure, proteins can be efficiently produced.

[0019] (Summary of the Disclosure) An outline of one aspect of the present disclosure is as follows.

[0020] In one embodiment of the present disclosure, a modified cyanobacterium expresses a channel protein that increases the protein permeability of the outer membrane.

[0021] This improves the protein permeability of the outer membrane, and therefore the modified cyanobacterium according to one embodiment of the present disclosure can provide a cyanobacterium with improved protein secretion productivity.

[0022] For example, in a modified cyanobacterium according to one embodiment of the present disclosure, the channel protein may be CppS having the amino acid sequence shown in SEQ ID NO: 1, CppF having the amino acid sequence shown in SEQ ID NO: 2, or a protein having an amino acid sequence that is 50% or more identical to either of these channel proteins.

[0023] As a result, the modified cyanobacterium expresses CppS (SEQ ID NO: 1) or CppF (SEQ ID NO: 2), which is a channel protein that improves the protein permeability of the outer membrane, or a protein having a function equivalent to either of these channel proteins. Therefore, the modified cyanobacterium according to one embodiment of the present disclosure improves the protein permeability of the outer membrane.

[0024] For example, in a modified cyanobacterium according to one embodiment of the present disclosure, a gene encoding the channel protein may be introduced.

[0025] This results in the expression of a channel protein that improves the protein permeability of the outer membrane. Therefore, according to the modified cyanobacterium according to one embodiment of the present disclosure, the protein permeability of the outer membrane is improved, and therefore it is possible to provide a cyanobacterium with improved protein secretion productivity.

[0026] For example, in the modified cyanobacterium according to an embodiment of the present disclosure, the gene may be a gene derived from chloroplasts. For example, in the modified cyanobacterium according to an embodiment of the present disclosure, the gene may be cppS having the nucleotide sequence shown in SEQ ID NO: 3, cppF having the nucleotide sequence shown in SEQ ID NO: 4, or a gene having a nucleotide sequence that is 50% or more identical to any of these genes.

[0027] As a result, a gene encoding any one of the channel proteins shown in SEQ ID NO: 3 and SEQ ID NO: 4 above, or a gene having 50% or more identity to the base sequence of any one of these genes, is introduced into the modified cyanobacterium. Therefore, a protein having a function of improving protein permeability in the outer membrane, or a protein having a function equivalent to that of the protein, is expressed in the modified cyanobacterium. Therefore, in the modified cyanobacterium according to one embodiment of the present disclosure, the protein permeability of the outer membrane is improved.

[0028] In addition, a method for producing modified cyanobacteria according to one embodiment of the present disclosure includes a step of expressing a channel protein that increases the protein permeability of the outer membrane.

[0029] This allows expression of a channel protein that improves the protein permeability of the outer membrane of the cyanobacterium. As a result, a protein produced within the bacterial cell is more likely to permeate through the channel protein and be secreted outside the outer membrane (i.e., outside the bacterial cell). Thus, according to the method for producing a modified cyanobacterium according to one embodiment of the present disclosure, a modified cyanobacterium having improved protein permeability of the outer membrane can be produced.

[0030] Furthermore, a method for producing a protein according to one aspect of the present disclosure includes culturing any of the modified cyanobacteria described above.

[0031] As a result, the protein produced within the bacterial body (also called cell) of the modified cyanobacterium is secreted outside the bacterial body (i.e., into the culture medium), and the protein secreted into the culture medium can be recovered. Therefore, there is no need to collect and disrupt the bacterial body to recover the protein. Furthermore, the bacterial body can be used repeatedly even after the protein is recovered. Furthermore, no special nutrients are required for culturing the modified cyanobacterium, and it can be easily cultured by simply providing light, water, air, and trace amounts of inorganic substances. Therefore, according to the method for producing a protein according to one aspect of the present disclosure, it is possible to efficiently produce a protein.

[0032] Hereinafter, the embodiment will be specifically described with reference to the drawings.

[0033] The embodiments described below are all comprehensive or specific examples. The numerical values, materials, steps, and order of steps shown in the following embodiments are merely examples and are not intended to limit the present disclosure. Furthermore, among the components in the following embodiments, those components that are not described in the independent claims that indicate the highest concept are described as optional components.

[0034] In addition, each drawing is not necessarily a strict illustration. In each drawing, substantially the same configuration is given the same reference numeral, and duplicated explanations may be omitted or simplified.

[0035] In the following description, numerical ranges do not only refer to strict ranges, but also include substantially equivalent ranges, such as measuring the amount (e.g., number or concentration) of a protein or a range thereof.

[0036] In addition, in this specification, the term "bacteria body" and "cell" both refer to an individual cyanobacterium.

[0037] (Embodiment) [1. Definition] 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 program available on the website of the NCBI (National Center for Biotechnology Information) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Information on genes and proteins of cyanobacteria and plants is published, for example, in the above-mentioned NCBI database and Cyanobase (http: / / genome.microbedb.jp / cyanobase / ). From these databases, the amino acid sequence of the target protein and the base sequence of the gene encoding the protein can be obtained.

[0038] Cyanobacteria, also called blue-green algae or blue-green bacteria, are a group of prokaryotes that capture light energy with chlorophyll, electrolyze water with the energy obtained, and perform photosynthesis while generating oxygen. Cyanobacteria are rich in diversity, for example, in terms of cell shape, there are unicellular species such as Synechocystis sp. PCC 6803 and filamentous species with multiple cells connected together such as Anabaena sp. PCC 7120. Regarding the growth environment, there are thermophilic species such as Thermosynechococcus elongatus, marine species such as Synechococcus elongatus, and freshwater species such as Synechocystis. In addition, there are many species with unique characteristics such as Microcystis aeruginosa, which has gas vesicles and produces toxins, and Gloeobacter violaceus, which does not have thylakoids and has a protein called phycobilisome, which is a light-gathering antenna, in the plasma membrane.

[0039] FIG. 1 is a schematic diagram of the cell surface of a cyanobacterium. As shown in FIG. 1, the cell surface of a cyanobacterium is composed of, from the inside, a plasma membrane (also called inner membrane 1), peptidoglycan 2, and an outer membrane 5, which is a lipid membrane forming the outermost layer of the cell. A sugar chain 3 composed of glucosamine and mannosamine is covalently bonded to the peptidoglycan 2, and pyruvic acid is bonded to these covalent sugar chains 3. In this specification, the peptidoglycan 2 and the covalent sugar chain 3 are collectively referred to as the cell wall 4. The sugar chain 3 of the cell wall 4 is bound to a binding domain 7 of an ion channel protein 6 described later, so that the outer membrane 5 covers the cell wall 4. The gap between the plasma membrane (i.e., inner membrane 1) and the outer membrane 5 is called the periplasm, and various enzymes involved in the decomposition or formation of a three-dimensional structure of proteins, the decomposition of lipids or nucleic acids, or the uptake of extracellular nutrients are present.

[0040] A channel protein is a membrane protein that forms a pathway (i.e., a channel) for selectively passing a specific substance from the inside to the outside of a lipid membrane (e.g., outer membrane 5) or from the outside to the inside. In the outer membrane of typical heterotrophic gram-negative bacteria such as Escherichia coli and Salmonella, there is a large amount of a channel protein called Porin that selectively passes relatively low molecular weight nutrients such as sugars and amino acids from the outside to the inside of the outer membrane and takes them into the cell (Non-Patent Document 5: Nikaido, 2003, Microbiol. Mol. Biol. Rev., 67(4):593-656). On the other hand, Porin does not exist in the outer membrane 5 of cyanobacteria, and instead, there is a large amount of an ion channel protein 6 that selectively passes only inorganic ions in the outer membrane 5. The ion channel protein 6 accounts for approximately 80% of the total protein in the outer membrane 5 (Non-Patent Document 6: Kowata et al., 2017, J. Bacteriol., 199(19):e00371-17). Therefore, in cyanobacteria, unless the properties of the outer membrane 5 are significantly modified using a technique such as gene introduction, it is difficult for a high molecular weight substance such as a protein to permeate the outer membrane 5 and diffuse outside the cell (i.e., outside the outer membrane 5).

[0041] The chloroplasts of plants originated from cyanobacteria that lived symbiotically within primitive eukaryotic cells about 1.5 to 2 billion years ago, and then evolved into chloroplasts (Non-Patent Document 7: Ponce-Toledo et al., 2017, Curr. Biol., 27(3):386-391). The chloroplasts of glaucophytes, which are unicellular algae considered to be the most primitive plants, have peptidoglycan and retain a surface structure very similar to that of cyanobacteria. On the other hand, peptidoglycan does not exist in the chloroplasts of seed plants, which are more evolved than unicellular algae. In addition, many of the outer membrane proteins of cyanobacteria have been lost from the outer membrane of chloroplasts during the evolutionary process, at the early stage of the birth of chloroplasts. Therefore, the outer membrane proteins of the chloroplasts of the glaucophytes described above are significantly different in composition from the outer membrane proteins of cyanobacteria. For example, the outer membrane 5 of cyanobacteria contains a large amount of ion channel proteins 6 that allow inorganic substances to pass through, such as Slr1841. The ion channel protein 6 accounts for about 80% of the total protein in the outer membrane 5. On the other hand, the outer membrane of the chloroplast of glaucophyte contains a large amount of channel proteins (hereinafter also referred to as organic channel protein 18) that allow organic matter to pass through, named CppS and CppF, in the outer membrane. The organic channel protein 18 accounts for more than 80% of the total protein in the outer membrane of the chloroplast of glaucophyte (Non-Patent Document 8: Kojima et al., 2016, J. Biol. Chem., 291:20198-20209). CppS and CppF are channel proteins that have a channel function that selectively allows relatively high molecular weight organic matter (e.g., biomolecules such as proteins) to pass through, and are thought to function as a substance transport pathway connecting the inside of the chloroplast in the plant cell to the cytoplasm of the plant cell. CppS and CppF are widely distributed in glaucophyte. On the other hand, in bacteria, similar proteins to CppS and CppF exist only in bacteria belonging to the Planctomycetes phylum. It should be noted that cyanobacteria do not possess CppS and CppF or proteins similar thereto (see Non-Patent Document 8).

[0042] In cyanobacteria, CO2 fixed by photosynthesis is converted into various amino acids through multi-step enzymatic reactions. These amino acids are used as raw materials to synthesize proteins 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 there. However, no cases of active extracellular secretion of proteins have been reported in cyanobacteria to date.

[0043] Cyanobacteria have high photosynthetic ability and do not necessarily need to take in organic matter from the outside as nutrients. Therefore, cyanobacteria have very few channels in the outer membrane 5 that allow organic matter to pass through, such as the organic matter channel protein 8 (e.g., Slr1270) in Figure 1. For example, in Synechocystis sp. PCC 6803, the organic matter channel protein 8 (e.g., Slr1270) that allows organic matter to pass through only about 4% of the total protein amount of the outer membrane 5. On the other hand, cyanobacteria have many ion channels in the outer membrane 5 that allow only inorganic ions to pass through, such as the ion channel protein 6 (e.g., Slr1841, etc.) in Figure 1, in order to take up inorganic ions necessary for growth into the cell with high efficiency. For example, in Synechocystis sp. PCC 6803, the ion channel protein 6 that allows inorganic ions to pass through accounts for about 80% of the total protein amount of the outer membrane 5.

[0044] As described above, in cyanobacteria, there are very few channels in the outer membrane 5 that allow the passage of organic matter such as proteins, and therefore it is thought to be difficult to actively secrete proteins produced within the cell.

[0045] [2. Modified cyanobacteria] Next, the modified cyanobacterium according to this embodiment will be described with reference to FIG.

[0046] In the modified cyanobacteria according to this embodiment, a channel protein (so-called organic channel protein 18) that improves the protein permeability of the outer membrane 5 is expressed. This improves the protein permeability of the outer membrane 5, thereby improving the secretion productivity of the protein produced within the bacterial cell and secreted outside the bacterial cell. In addition, since there is no need to disrupt the bacterial cell to recover the protein, the modified cyanobacteria can be used repeatedly to produce the protein even after the protein is recovered. In this specification, the production of a protein within the bacterial cell by the modified cyanobacteria is referred to as "production," and the secretion of the produced protein outside the bacterial cell is referred to as "secretory production."

[0047] The type of cyanobacteria (hereinafter referred to as parent cyanobacteria) before the protein permeability of the outer membrane 5 is improved, which is the parent microorganism of the modified cyanobacteria according to the present embodiment, is not particularly limited and may be any type of cyanobacteria. For example, the parent cyanobacteria may be S. yn The bacterium may be of the genus Echocystis, Synechococcus, Anabaena, or Thermosynechococcus, and in particular, may be Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, or Thermosynechococcus elongatus BP-1.

[0048] In this specification, expressing the organic matter channel protein 18 in the outer membrane 5 of a cyanobacterium means inserting a gene encoding the organic matter channel protein 18 into the chromosomal DNA or plasmid of a cyanobacterium, transporting the organic matter channel protein 18 synthesized through transcription and translation of the gene to the outer membrane 5, and expressing a channel function that selectively allows proteins to permeate in the outer membrane 5 of the cyanobacterium. The means for inserting and expressing the gene are not particularly limited as long as they are commonly used means, and are not limited by the type of promoter base sequence for transcription activation and ribosome binding sequence for translation, and signal sequence for transport to the outer membrane 5.

[0049] In this embodiment, the organic channel protein 18 expressed in the outer membrane 5 of the cyanobacterium may be an outer membrane channel protein derived from chloroplasts. The organic channel protein 18 may be, for example, CppS (SEQ ID NO: 1) or CppF (SEQ ID NO: 2) of the glaucoalga Cyanophora paradoxa (hereinafter also referred to as C. paradoxa). The organic channel protein 18 may also be a protein having an amino acid sequence that is 50% or more identical to that of CppS or CppF. Note that the protein having an amino acid sequence that is 50% or more identical to that of CppS or CppF is not limited to a protein derived from chloroplasts, and may be, for example, CppS derived from a microorganism such as a bacterium. S Or it may be a protein similar to CppF.

[0050] As a result, the modified cyanobacterium expresses CppS (SEQ ID NO: 1) or CppF (SEQ ID NO: 2), which is an organic channel protein 18 that improves the protein permeability of the outer membrane 5, or a protein having a function equivalent to either of these organic channel proteins 18. Therefore, the modified cyanobacterium according to this embodiment has improved protein permeability of the outer membrane 5.

[0051] In general, it is said that if the amino acid sequence of a protein is 30% or more identical, the homology of the three-dimensional structure of the protein is high, and therefore the protein is likely to have the same function as the protein. Therefore, the organic channel protein 18 may be, for example, a protein or polypeptide having an amino acid sequence having 40% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and even more preferably 90% or more identity with the amino acid sequence of any of the proteins shown in SEQ ID NO: 1 and SEQ ID NO: 2, and having the function of improving the permeability of the protein of the outer membrane 5.

[0052] In the present embodiment, a gene encoding the organic channel protein 18 may be introduced into the modified cyanobacterium. As a result, the modified cyanobacterium expresses the organic channel protein 18, which improves the protein permeability of the outer membrane 5.

[0053] The gene may be, for example, a gene derived from chloroplasts. The gene encoding the chloroplast-derived organic matter channel protein 18 may be, for example, cppS (SEQ ID NO: 3) or cppF (SEQ ID NO: 4) of the glaucophyte Cyanophora paradoxa. The organic matter channel protein 18 may be a gene having a base sequence identical to any of these genes by 50% or more. In this way, in the modified cyanobacterium, a gene encoding any of the organic matter channel proteins 18 shown in SEQ ID NO: 3 and SEQ ID NO: 4 or a gene having a base sequence identical to any of these genes by 50% or more is introduced. Therefore, a protein having a function of improving the protein permeability in the outer membrane 5 or a protein having a function equivalent to that of the protein is expressed. In this way, in the modified cyanobacterium, the protein permeability in the outer membrane 5 is improved.

[0054] The gene encoding the organic channel protein 18 is not limited to a gene derived from chloroplasts. The gene encoding the organic channel protein 18 may be, for example, a gene that has a base sequence with 40% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and still more preferably 90% or more identity with the base sequence of any of the above genes cppS (SEQ ID NO: 3) and cppF (SEQ ID NO: 4), and that encodes a protein or polypeptide that has a function of improving the protein permeability of the outer membrane 5.

[0055] [3. Method for producing modified cyanobacteria] Next, a method for producing the modified cyanobacterium according to the present embodiment will be described. The method for producing the modified cyanobacterium includes a step of expressing an organic channel protein 18 that improves the protein permeability of the outer membrane 5.

[0056] In this embodiment, the organic channel protein 18 that improves the protein permeability of the outer membrane 5 is, for example, a channel protein derived from chloroplasts, and specifically, may be CppS consisting of the amino acid sequence shown in SEQ ID NO: 1, or CppF consisting of the amino acid sequence shown in SEQ ID NO: 2. In addition, the organic channel protein 18 may be a protein whose amino acid sequence is 50% or more identical to any of these proteins.

[0057] In the step of expressing the organic channel protein 18, first, a gene encoding the organic channel protein 18 that improves the protein permeability of the outer membrane 5 is inserted into the chromosomal DNA or plasmid of the cyanobacterium. The organic channel protein 18 synthesized through transcription and translation of the gene is transported to the outer membrane 5 and expresses a channel function in the outer membrane 5 of the cyanobacterium. The means for inserting and expressing the gene are not particularly limited as long as they are commonly used means, and are not limited by the type of promoter base sequence for transcription activation and ribosome binding sequence for translation, and signal sequence for transport to the outer membrane 5.

[0058] By the above procedure, a channel protein that increases the protein permeability of the outer membrane 5 may be expressed to produce a modified cyanobacterium.

[0059] As a result, in the modified cyanobacteria produced by the production method according to the present embodiment, the organic channel protein 18 that improves protein permeability is expressed in the outer membrane 5, improving the protein permeability of the outer membrane 5. This makes it easier for proteins produced within the bacterial cell to permeate through the organic channel protein 18 and be secreted outside the outer membrane 5 (i.e., outside the bacterial cell). Therefore, the production method for modified cyanobacteria according to the present embodiment can provide modified cyanobacteria with improved protein secretion productivity.

[0060] In addition, in the modified cyanobacteria produced by the production method according to the present embodiment, the protein produced within the bacterial cells diffuses outside the bacterial cells as described above, so that it is not necessary to disrupt the bacterial cells in order to recover the protein. For example, the modified cyanobacteria can be cultured under appropriate conditions, and then the protein secreted into the culture solution can be recovered, so that the protein in the culture solution can be recovered while culturing the modified cyanobacteria. Therefore, by using the modified cyanobacteria obtained by the present production method, efficient microbiological protein production can be carried out. Therefore, according to the production method of the modified cyanobacteria according to the present embodiment, it is possible to provide a modified cyanobacterium with high utilization efficiency that can be used repeatedly even after the protein is recovered.

[0061] The modified cyanobacteria produced by the method for producing modified cyanobacteria according to the present embodiment secrete, outside the cells, mainly proteins originally present in the periplasm, such as peptidases or phosphatases. In the present embodiment, for example, it is possible to cause the modified cyanobacteria to produce a desired protein by modifying a gene encoding a protein originally produced within the cells of the cyanobacteria, such as a protein group present in the periplasm, and replacing it with a gene encoding another protein. Thus, according to the method for producing modified cyanobacteria according to the present embodiment, it is also possible to provide a modified cyanobacterium capable of producing a desired protein simply and efficiently.

[0062] [4. Protein manufacturing method] Next, a method for producing a protein according to the present embodiment will be described. The method for producing a protein according to the present embodiment includes a step of culturing the modified cyanobacterium.

[0063] Cyanobacteria can generally be cultured based on liquid culture using BG-11 medium (see Table 2) or a modified method thereof. Therefore, modified cyanobacteria may be cultured in a similar manner. The culture period of cyanobacteria for producing a protein may be any period that allows sufficient bacterial proliferation and accumulation of a high concentration of protein, and may be, for example, 1 to 3 days or 4 to 7 days. The culture method may be, for example, aeration agitation culture or shaking culture.

[0064] When cultured under the above conditions, the modified cyanobacteria produce proteins within the cells and secrete the proteins into the culture medium. When recovering the proteins secreted into the culture medium, the culture medium may be filtered or centrifuged to remove solid matter such as cells (so-called bacterial bodies) from the culture medium, and the culture supernatant may be recovered. According to the method for producing a protein of this embodiment, since the protein is secreted outside the cells of the modified cyanobacteria, there is no need to destroy the cells to recover the protein. Therefore, the modified cyanobacteria remaining after protein recovery can be repeatedly used to produce the protein.

[0065] The method for recovering the protein secreted into the culture solution is not limited to the above example, and the protein in the culture solution may be recovered while culturing the modified cyanobacterium. For example, a permeable membrane that allows the protein to pass through may be used to recover the protein that has permeated the permeable membrane. In this case, useful microorganisms such as lactic acid bacteria may be cultured using the protein that has permeated the permeable membrane as a nutrient source. In this way, since the protein in the culture solution can be recovered while culturing the modified cyanobacterium, a process for removing the cells of the modified cyanobacterium from the culture solution is not required. Therefore, the protein can be produced more simply and efficiently.

[0066] Furthermore, since the process of recovering the bacterial cells from the culture medium and the process of disrupting the bacterial cells are no longer necessary, damage and stress to the modified cyanobacterium can be reduced, making it difficult for the protein secretion productivity of the modified cyanobacterium to decrease, and allowing the modified cyanobacterium to be used for a longer period of time.

[0067] As described above, the method for producing proteins using modified cyanobacteria according to the present embodiment makes it possible to simply and efficiently obtain enzymes for producing food ingredient raw materials or compounds, diagnostic or therapeutic enzymes in the medical field, or feed enzymes in the agricultural, fishery and livestock fields. EXAMPLES

[0068] The modified cyanobacteria, the method for producing the modified cyanobacteria, and the method for producing the protein of the present disclosure will be specifically described in the following examples, but the present disclosure is in no way limited to the following examples.

[0069] In this example, as a method for improving the protein permeability of the outer membrane of a cyanobacterium, the chloroplast outer membrane channel protein CppS (SEQ ID NO: 1) possessed by the glaucophyte Cyanophora paradoxa was expressed in the outer membrane of the cyanobacterium. Then, the protein secretion productivity of the modified cyanobacterium in which the outer membrane channel protein CppS (SEQ ID NO: 1) was expressed was measured, and the secreted protein was identified. The cyanobacterial species used in this example was Synechocystis sp. PCC 6803 (hereinafter simply referred to as "cyanobacteria").

[0070] (1) Construction of cppS gene expression cassette A gene cassette containing the cppS gene, a promoter region (PL22) for controlling the expression of the cppS gene, an outer membrane transport signal sequence in cyanobacteria (slr0042-signal), and a kanamycin resistance marker gene (KmR) serving as a marker for gene introduction was constructed as follows.

[0071] First, the chromosomal DNA of a cyanobacterium was used as a template, and the primers slr0042-Fw (SEQ ID NO: 5) and slr0042-Rv (SEQ ID NO: 6) listed in Table 1 were used to amplify the gene by PCR (polymerase chain reaction), to obtain the slr0042 gene. Next, the chromosomal DNA of Synechocystis LY07 strain (Non-Patent Document 9: Yao et al., ACS Synth. Biol., 2016, 5(3):207-212) was used as a template, and the primers slr2030-Fw (SEQ ID NO: 7) and PL22-Rv (SEQ ID NO: 8) and KmR-Fw (SEQ ID NO: 9) and slr2031-Rv (SEQ ID NO: 10) listed in Table 1 were used to amplify the gene by PCR, to obtain PL22 and KmR. In the LY07 strain, these are inserted into the slr2030-slr2031 gene on the chromosome, so when amplified by PCR using the above four primers, the slr2030 gene fragment is amplified in a form linked to the 5' end of PL22, and the slr2031 gene fragment is amplified in a form linked to the 3' end of KmR. Next, the mixed solution of the slr0042 gene, PL22, and KmR obtained by the above procedure was used as a template, and amplified by PCR using the four primers (SEQ ID NOs: 7 to 10) listed in Table 1 to obtain a gene cassette (slr2030-2031::slr0042-KmR cassette) in which the slr2030 gene fragment, PL22, slr0042 gene, KmR, and slr2031 gene fragment were linked in order from the 5' end. Using the In-Fusion PCR cloning method®, the slr2030-2031::slr0042-KmR cassette was inserted into the pUC19 plasmid, resulting in the pUC19-slr0042 plasmid.

[0072] In parallel with the above procedure, total cDNA was prepared from the gray alga C. paradoxa NIES-547 using the SMART cDNA Library Synthesis Kit (Clontech). This cDNA was used as a template and amplified by PCR using the primers cppS-Fw (SEQ ID NO: 11) and cppS-Rv (SEQ ID NO: 12) listed in Table 1 to obtain the cppS gene. The cppS gene was inserted into the pUC19-slr0042 plasmid using the In-Fusion PCR Cloning Method (registered trademark) to obtain the pUC19-cppS plasmid. Note that, by this procedure, the cppS gene was inserted in a form linked to the 3'-end of the outer membrane transport signal sequence of the slr0042 gene, and the region other than the outer membrane transport signal sequence of the slr0042 gene was removed in a form replacing the coding region of cppS.

[0073] [Table 1]

[0074] (2) Construction of a promoter activity control cassette (tetR) for PL22 The promoter activity of PL22 is induced only in the presence of anhydrotetracycline (aTc) by the TetR repressor, so it is necessary to introduce the tetR gene into the engineered cyanobacterium in order to control the activity of PL22.

[0075] First, the chromosomal DNA of the LY07 strain was used as a template, and the tetR gene and the spectinomycin resistance marker gene (SpcR), which serves as a marker for gene introduction, were obtained by PCR amplification using a set of primers psbA1-Fw (SEQ ID NO: 13) and tetR-Rv (SEQ ID NO: 14) and a set of primers tetR-Fw (SEQ ID NO: 15) and psbA1-Rv (SEQ ID NO: 16) shown in Table 1. In the LY07 strain, these genes are inserted into the psbA1 gene on the chromosome, so when the above primers are used for PCR amplification, the upstream fragment of the psbA1 gene is amplified in a form linked to the 5' end of the tetR gene, and the downstream fragment of the psbA1 gene is amplified in a form linked to the 3' end of SpcR. Next, a mixture of the tetR gene and SpcR was used as a template, and amplification was performed by PCR using the primers psbA1-Fw (SEQ ID NO: 13) and psbA1-Rv (SEQ ID NO: 14) shown in Table 1 to obtain a gene cassette (psbA1::tetR cassette) in which the upstream fragment of the psbA1 gene, TetR, SpcR, and the downstream fragment of the psbA1 gene were linked in this order from the 5' end. The psbA1::tetR cassette was cloned using the In-Fusion PCR cloning method (registered trademark). To The fragment was inserted into the pUC19 plasmid to obtain the pUC19-tetR plasmid.

[0076] (3) Introduction of the cppS gene expression cassette and the tetR cassette 1 μg of the pUC19-cppS plasmid obtained by the above procedure was mixed with a cyanobacterial culture solution (cell concentration OD730 = about 0.5), and the plasmid was introduced into the cells by natural transformation. The transformed cells were selected by growing them on a BG-11 agar medium containing 30 μg / mL kanamycin. In the selected cells, homologous recombination occurs between the slr2030-2031 gene on the chromosome and the slr2030 gene fragment region and the slr2031 gene fragment region on the pUC19-cppS plasmid. This resulted in a Synechocystis cppS strain in which the cppS gene expression cassette was inserted into the slr2030-2031 gene region. The composition of the BG-11 medium used is as shown in Table 2.

[0077] [Table 2]

[0078] Next, 1 μg of the pUC19-tetR plasmid was mixed with Synechocystis cppS culture fluid (cell concentration OD730 = approximately 0.5) to introduce the plasmid into the cells by natural transformation. The transformed cells were selected by growing them on BG-11 agar medium containing 30 μg / mL kanamycin and 20 μg / mL spectinomycin, and the Synechocystis cppS tetR strain was obtained. As above, in this strain, the tetR cassette is inserted into the psbA1 gene on the chromosomal DNA.

[0079] (4) Protein secretion productivity test The Synechocystis cppS tetR strain obtained in (3) above was cultured under the conditions described in Example 1 and Comparative Example 1 below, and the amount of protein secreted outside the cells (hereinafter also referred to as the amount of secreted protein) was measured. Protein secretion productivity was evaluated based on the amount of protein in the culture medium. The protein secretion productivity refers to the ability to produce a protein by secreting a protein produced inside the cells outside the cells. Specific methods are described below.

[0080] (4-1) Cultivation of modified strains [Example 1] In Example 1, the Synechocystis cppS tetR strain was inoculated into a BG-11 medium containing 1 μg / mL aTc so that the initial cell concentration OD730 was 0.05, and the light intensity was 100 μmol / m 2 The mixture was cultured at 30°C for 5 days with shaking at 100 / s. The culture was carried out three times independently.

[0081] [Comparative Example 1] In Comparative Example 1, except that the aTc-free BG-11 medium was used, the Synechocystis cppS tetR strain was cultured under the same conditions as in Example 1. The culture was carried out three times independently.

[0082] (4-2) Quantification of secreted proteins The culture solution obtained in Example 1 in (4-1) above was centrifuged at room temperature at 2,500 g for 10 minutes to obtain a culture supernatant. The obtained culture supernatant was filtered using a membrane filter with a pore size of 0.22 μm to completely remove the cells of the Synechocystis cppS tetR strain. The total protein amount contained in the culture supernatant after filtration was quantified by the BCA (Bicinchoninic acid) method. This series of operations was performed for each of the three culture solutions independently cultured, and the average value and standard deviation of the protein amount secreted outside the cells of the Synechocystis cppS tetR strain of Example 1 were obtained. In addition, the protein amount of the three culture solutions of the Synechocystis cppS tetR strain cultured under the conditions of Comparative Example 1 was also quantified under the same conditions, and the average value and standard deviation of the protein amount in the three culture solutions were obtained.

[0083] The results are shown in Figure 2. Figure 2 shows the culture of the modified cyanobacteria of Example 1 and Comparative Example 1. Supernatant 1 is a graph showing the amount of protein in each well (n=3, error bars=SD).

[0084] As shown in FIG. 2, the Synechocystis cppS tetR strain cultured in the presence of aTc in Example 1 had an approximately 25-fold increase in the amount of protein (mg / L) secreted into the culture supernatant compared to the Synechocystis cppS tetR strain cultured in the absence of aTc in Comparative Example 1.

[0085] Although the data is omitted, the absorbance (730 nm) of the culture solution was measured and the amount of secreted protein per 1 g of dry cell weight (mg protein / g cell dry weight) was calculated. The amount of secreted protein per 1 g of dry cell weight (mg protein / g cell dry weight) of the Synechocystis cppS tetR strain cultured under the conditions of Example 1 was 100% higher than that of the Synechocystis cppS tetR strain cultured under the conditions of Comparative Example 1. S t This was approximately 36-fold improved compared to the etR strain.

[0086] (5) Identification of secreted proteins Next, the secretory proteins contained in the culture supernatant obtained in (4-2) above were identified by LC-MS / MS, as described below.

[0087] (5-1) Sample preparation Eight times the volume of cold acetone was added to the culture supernatant, and the mixture was left to stand at 20°C for 2 hours, followed by centrifugation at 20,000 g for 15 minutes to obtain a protein precipitate. 100 mM Tris pH 8.5, 0.5% sodium dodecanoate (SDoD) was added to the precipitate, and the protein was dissolved using a closed ultrasonic homogenizer. After adjusting the protein concentration to 1 μg / mL, dithiothreitol (DTT) was added to a final concentration of 10 mM and the mixture was left to stand at 50°C for 30 minutes. Next, iodoacetamide (IAA) was added to a final concentration of 30 mM and the mixture was left to stand at room temperature (shielded from light) for 30 minutes. To stop the IAA reaction, cysteine ​​was added to a final concentration of 60 mM and the mixture was left to stand at room temperature for 10 minutes. 400 ng of trypsin was added and the mixture was left to stand overnight at 37°C to fragment the protein into peptide fragments. After adding 5% TFA (Trifluoroacetic Acid), the mixture was centrifuged at 15,000 g for 10 minutes at room temperature to obtain the supernatant. This process removed SDoD. After desalting using a C18 spin column, the sample was dried using a centrifugal evaporator. Then, 3% acetonitrile and 0.1% formic acid were added, and the sample was dissolved using a closed ultrasonic disintegrator. The peptide concentration was adjusted to 200 ng / μL.

[0088] (5-2)LC-MS / MS analysis The sample obtained in (5-1) above was analyzed using an LC-MS / MS device (UltiMate 3000 RSLCnano LC System) under the following conditions.

[0089] Sample injection amount: 200 ng Column: CAPCELL CORE MP 75 μm × 250 mm Solvent: Solvent A is 0.1% formic acid in water, and Solvent B is 0.1% formic acid + 80% acetonitrile Gradient program: 8% B solvent 4 minutes after sample injection, 44% B solvent 27 minutes after, 80% B solvent 28 minutes after, measurement ends 34 minutes after

[0090] (5-3) Data analysis The obtained data was analyzed under the following conditions to identify proteins and peptides and calculate quantitative values.

[0091] Software: Scaffold DIA Database:UniProtKB / Swiss Prot database (Synechocystis sp. PCC 6803) Fragmentation:HCD Precursor Tolerance: 8 ppm Fragment Tolerance: 1.0 ppm Data Acquisition Type: Overlapping DIA Peptide Length:8-70 Peptide Charge:2-8 Max Missed Cleavages:1 Fixed Modification: Carbamidomethylation Peptide FDR: 1% or less

[0092] Among the identified proteins, the 10 proteins with the highest relative quantitative values ​​are shown in Table 3.

[0093] [Table 3]

[0094] All ten types of proteins were contained in the culture supernatants of Example 1 and Comparative Example 1. All of these proteins retained a periplasmic (referring to the gap between the outer membrane and the inner membrane) transport signal. From these results, it was confirmed that in the modified strain of Example 1, the expression of the cppS gene improved the protein permeability of the outer membrane, making it easier for proteins in the periplasm to permeate the channel protein CppS and be secreted outside the outer membrane (i.e., outside the bacterial cell). Therefore, it was shown that the modified cyanobacterium of this embodiment has significantly improved protein secretion productivity.

[0095] (6) Discussion In this example, it was confirmed that the modified cyanobacteria of the present disclosure secrete proteins present within the bacterial cell (here, within the periplasm) to the outside of the bacterial cell. In addition, by identifying the proteins secreted by the modified cyanobacteria, it is possible to replace the genes encoding these proteins with genes encoding other proteins by genetic modification. For example, by genetically modifying the modified cyanobacteria of the present disclosure so as to produce a desired protein instead of the protein identified above, it is possible to efficiently produce a desired protein using the modified cyanobacteria. In addition, since cyanobacteria have high photosynthetic ability, a required protein can be easily obtained when needed by culturing the cyanobacteria while providing light, water, air, and a trace amount of inorganic matter, so there is no need to use a complex device for producing a protein. In addition, a protein is likely to lose its function when processed into, for example, a supplement. Therefore, according to the modified cyanobacteria of the present disclosure, it is possible to provide a protein while maintaining its function. Due to the above advantages, the modified cyanobacteria of the present disclosure are expected to be applied in various fields.

[0096] The modified cyanobacteria, the method for producing the modified cyanobacteria, and the method for producing a protein according to the present disclosure have been described above based on the embodiments, but the present disclosure is not limited to these embodiments. As long as they do not deviate from the gist of the present disclosure, various modifications conceivable by those skilled in the art to the embodiments and other forms constructed by combining some of the components of the embodiments are also included in the scope of the present disclosure. [Industrial Applicability]

[0097] According to the modified cyanobacteria, the method for producing the modified cyanobacteria, and the method for producing a protein using the modified cyanobacteria of the present disclosure, the modified cyanobacteria can be cultured while being provided with water, light, air, and trace amounts of inorganic substances, thereby efficiently obtaining proteins. For example, enzymes for producing raw materials or compounds of food ingredients, diagnostic enzymes or therapeutic enzymes in the medical field, or enzymes for feed in the agricultural, fishery, and livestock fields can be obtained. [Explanation of symbols]

[0098] 1 Intima 2. Peptidoglycan 3 Glycosylation 4 cell wall 5 Adventitia 6. Ion Channel Proteins 7. Binding Domains 8, 18 Organic Channel Proteins

Claims

1. A channel protein that increases the protein permeability of the outer membrane is expressed in the outer membrane, The channel protein is CppS consisting of the amino acid sequence shown in SEQ ID NO: 1, or A protein having an amino acid sequence that is 90% or more identical to that of this channel protein. Modified cyanobacteria.

2. A gene encoding the channel protein is introduced. The modified cyanobacterium of claim 1.

3. The gene is a gene derived from chloroplast. The modified cyanobacterium of claim 2.

4. The gene is cppS consisting of the base sequence shown in SEQ ID NO: 3, or A gene whose base sequence is 90% or more identical to this gene. The modified cyanobacterium of claim 2 or 3.

5. expressing a channel protein in the outer membrane that increases the protein permeability of the outer membrane; The channel protein is CppS consisting of the amino acid sequence shown in SEQ ID NO: 1, or A protein having an amino acid sequence that is 90% or more identical to that of this channel protein. Methods for producing modified cyanobacteria.

6. Cultivating the modified cyanobacterium of any one of claims 1 to 4, Methods for producing proteins.

Images