JP2024010288A - Method for producing dried bacterial cell, and dried bacterial cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing dried bacterial cells that can preserve outer membrane-peeled, modified cyanobacteria in a dried state, while maintaining high survival rates.

SOLUTION: A method for producing dried bacterial cells includes preparing a suspension that contains outer membrane-peeled, modified cyanobacteria and sucrose or glucose (step S01) and removing moisture content from the prepared suspension (step S02).

SELECTED DRAWING: Figure 1

COPYRIGHT: (C)2024,JPO&INPIT

Description

The present disclosure relates to a method for producing dried bacterial cells and a method for producing dried bacterial cells.

Photosynthetic microorganisms such as cyanobacteria and algae can produce various substances by using light as an energy source and water and carbon dioxide (CO ₂ ) in the air at room temperature and pressure. It is attracting attention as a next-generation material production tool that is neutral and has a low environmental impact.

In utilizing such photosynthetic microorganisms, cell (hereinafter also referred to as microbial cells) preservation technology is one of the most important fundamental technologies. For example, cell preservation methods include subculture preservation, in which cells of photosynthetic microorganisms are periodically transferred to new media using a liquid or solid medium, and dry preservation, in which cells are preserved in a dry state. There are laws etc. For example, dry preservation methods include a cryopreservation method for cells at an ultralow temperature (for example, −80° C.), and an L-drying method in which a liquid phase containing bacterial cells is vacuum-dried. The dry preservation method does not require periodic subculturing of cells, nor does it require maintaining conditions necessary to support cell life, such as temperature, water, and air, significantly reducing labor and costs. It is possible to do so. Among these, the L-drying method allows vacuum-dried cells to be stored in ampoules at room temperature, and unlike the freeze-drying method, there is no need to install expensive equipment such as a cryogenic freezer, which significantly reduces costs. It is possible to do so.

However, depending on the species of photosynthetic microorganisms, there are microorganisms that are difficult to freeze-dry or vacuum-dry, so it is necessary to preserve cells using an appropriate drying preservation method depending on the species of photosynthetic microorganisms to be preserved. Furthermore, regarding the freeze-drying method, since freeze-drying resistance varies depending on the species of photosynthetic microorganisms, effective protective agents are being investigated.

For example, in the freeze-drying method of eukaryotic algae among photosynthetic microorganisms, skim milk and sucrose are effective protective agents (Non-patent Document 1), and milk solids are effective protective agents (non-patent document 1). Patent Document 2) has been reported. On the other hand, in the freeze-drying method for cyanobacteria, it has been reported that some cyanobacterial species exhibit freeze-drying resistance in the absence of a protective agent (Non-Patent Document 2); It has also been reported that the insects die (Non-Patent Document 3) and that sheep serum is an effective protective agent (Non-Patent Document 4).

Marian Sharp McGrath et al., "FREEZE‐DRYING OF ALGAE: CHLOROPHYTA AND CHRYSOPHYTA 1", Journal of Phycology, Phycological Society of America, December 1978, Vol.14, No.4, pp.521-525 Osmund Holm-Hansen, "Viability of lyophilized algae", Canadian Journal of Botany, Canadian Science Publishing, February 1964, Vol.42, No.2, pp.127-137 Alberto A. Esteves-Ferreira et al., "Comparative evaluation of different preservation methods for cyanobacterial strains", Journal of Applied Phycology, 2013, Vol.25, pp.919-929 LL Corbett and DL Parker, "Viability of lyophilized cyanobacteria (blue-green algae)", Applied and Environmental Microbiology, American Society for Microbiology, December 1976, Vo.32, No.6, pp.777-780

However, with the above-mentioned conventional techniques, it may be difficult to properly dry and preserve, for example, cells of modified photosynthetic microorganisms whose productivity has been improved through genetic manipulation. For example, the inventors of the present application have found that the desiccation resistance of a modified outer membrane-exfoliating type of cyanobacterium, in which the outer membrane of cyanobacteria (hereinafter also referred to as parent cyanobacteria) is removed from the cell wall through genetic manipulation, is significantly lower than that of the parent cyanobacteria. I discovered that.

Accordingly, the present disclosure provides a method for producing dried bacterial cells that allows outer membrane-exfoliating cyanobacteria to be stored in a dry state while maintaining a high survival rate, and the dried bacterial cells.

A method for producing dried bacterial cells according to one aspect of the present disclosure includes preparing a suspension containing membrane-exfoliating modified cyanobacteria and sucrose or glucose, and removing water from the prepared suspension. .

Further, a dried bacterial cell according to one aspect of the present disclosure is produced by the method for producing a dried bacterial cell.

Furthermore, the dried bacterial cells according to one aspect of the present disclosure include outer membrane-exfoliating modified cyanobacteria and sucrose or glucose.

According to the method for producing dried bacterial cells and the dried bacterial cells of the present disclosure, outer membrane-exfoliating modified cyanobacteria can be stored in a dry state while maintaining a high survival rate.

FIG. 1 is a flowchart showing an example of the flow of the method for producing dried bacterial cells according to the present embodiment. FIG. 2 is a diagram schematically showing the cell surface layer of cyanobacteria. Figure 3 is a graph showing the survival rate of the wild-type strain and the adventitial membrane-exfoliating strain when suspensions were prepared using water as a dispersion liquid (wild strain n=4; adventitial membrane-exfoliating strain n=3; error bars = SD). FIG. 4 is a graph (n=3, error bars=SD) showing the survival rate of freeze-dried bacterial cells of outer membrane detachment strains depending on the different protective agents contained in the suspension. FIG. 5 is a graph (n=3 to 4, error bars=SD) showing the relationship between the bacterial cell concentration of the suspension during the freeze-drying process and the growth of the freeze-dried bacterial cells. Figure 6 is a graph showing the relationship between the ratio of the number of sucrose molecules to the number of bacterial cells in the suspension during freeze-drying and the growth of freeze-dried bacterial cells (n=3, error bar = SD). . FIG. 7 is a bluff (average value of n=1 to 3) showing the relationship between the presence or absence of addition of sucrose to the suspension during freeze-drying treatment and the number of storage days for freeze-dried bacterial cells.

(Findings that formed the basis of this disclosure)
Photosynthetic microorganisms such as cyanobacteria and algae are attracting attention as tools for next-generation material production with low environmental impact. Substance production by photosynthetic microorganisms is performed in an environment of normal temperature and pressure using water and carbon dioxide (CO ₂ ) in the air with light as an energy source. Furthermore, with the recent development of genetic engineering technology, it has become possible to produce a wide range of chemical compounds using genetically modified photosynthetic microorganisms.Therefore, substance production using photosynthetic microorganisms is expected to be a next-generation technology that can achieve carbon neutrality. has been done.

As for the production of substances by photosynthetic microorganisms, for example, techniques have been disclosed to improve the productivity of substances using genetically modified strains in which genes involved in the metabolism or biosynthesis of substances of photosynthetic microorganisms are modified. With this technology, for example, sucrose (Non-Patent Document 5: 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), isobutanol (Non-Patent Document 6: 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), fatty acids (Non-Patent Document 7: 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), amino acids (Non-Patent Document 8: Arnav Deshpande et al., "Combining random mutagenesis and metabolic engineering for enhanced tryptophan production in Synechocystis sp. strain PCC 6803", Applied Environmental Microbiology, May 2020, Vol.86, No.9, e02816-19) and proteins (Non-Patent Document 9: James A. Gregory et al., "Alga-Produced Cholera Toxin-Pfs25 Fusion Proteins as Oral Vaccines" , Applied and Environmental Microbiology, American Society for Microbiology, June 2013, Vol.79, No.13, pp.3917-3925).

In utilizing such photosynthetic microorganisms, cell (hereinafter also referred to as microbial cells) preservation technology is one of the most important fundamental technologies. For example, cell preservation methods include subculture preservation, in which cells of photosynthetic microorganisms are periodically transferred to new media using a liquid or solid medium, and dry preservation, in which cells are preserved in a dry state. There are laws etc. For example, dry preservation methods include a cryopreservation method for cells at an ultralow temperature (for example, −80° C.), and an L-drying method in which a liquid phase containing bacterial cells is vacuum-dried. The dry preservation method does not require periodic subculturing of cells, nor does it require maintaining conditions necessary to support cell life, such as temperature, water, and air, significantly reducing labor and costs. It is possible to do so. Among these, the L-drying method allows vacuum-dried cells to be stored in ampoules at room temperature, and unlike the freeze-drying method, there is no need to install expensive equipment such as a cryogenic freezer, which significantly reduces costs. It is possible to do so.

However, depending on the species of photosynthetic microorganisms, there are microorganisms that are difficult to freeze-dry or vacuum-dry, so it is necessary to preserve cells using an appropriate drying preservation method depending on the species of photosynthetic microorganisms to be preserved. Furthermore, regarding the freeze-drying method, since freeze-drying resistance varies depending on the species of photosynthetic microorganisms, effective protective agents are being investigated.

For example, in the freeze-drying method of eukaryotic algae among photosynthetic microorganisms, skim milk and sucrose are effective protective agents (Non-patent Document 1), and milk solids are effective protective agents (non-patent document 1). Patent Document 2) has been reported. On the other hand, in the freeze-drying method for cyanobacteria, it has been reported that some cyanobacterial species exhibit freeze-drying resistance in the absence of a protective agent (Non-Patent Document 2); It has also been reported that the insects die (Non-Patent Document 3) and that sheep serum is an effective protective agent (Non-Patent Document 4).

However, with the above-mentioned conventional techniques, it may be difficult to properly dry and preserve cells of modified photosynthetic microorganisms whose productivity has been improved through genetic manipulation. For example, the present inventors genetically engineered cyanobacterium Synechocystis sp. In some cases, it has been found that the resistance to freeze-drying (so-called freeze-drying tolerance) is significantly lower than that of the parent cyanobacteria. The technology to dry and preserve membrane-exfoliated cyanobacterial strains while maintaining their high substance production capacity is extremely important industrially in order to stably produce next-generation substances with low environmental impact.

Therefore, the inventors of the present application have conducted extensive studies on a method for producing dried cells of modified cyanobacteria that can exfoliate their outer membranes. As a result, the inventors of the present application have found that by using sucrose or glucose as a protective agent, it is possible to preserve membrane-exfoliating modified cyanobacteria in good condition by a dry preservation method, and more specifically, to achieve high survival. We have discovered that modified cyanobacteria that can exfoliate their outer membrane can be stored in a dry state while maintaining the yield.

Therefore, according to the present disclosure, it is possible to provide a method for producing dried bacterial cells that allows outer membrane-exfoliating modified cyanobacteria to be stored in a dry state while maintaining a high survival rate, and the dried bacterial cells.

(Summary of this disclosure)
An overview of one aspect of the present disclosure is as follows.

A method for producing dried bacterial cells according to one aspect of the present disclosure includes preparing a suspension containing membrane-exfoliating modified cyanobacteria and sucrose or glucose, and removing water from the prepared suspension. .

Accordingly, in the method for producing dried bacterial cells, since sucrose or glucose acts as a protective agent for the bacterial cells, it is possible to preserve outer membrane-exfoliating modified cyanobacteria in a dry state while maintaining a high survival rate.

For example, in the method for producing dried bacterial cells according to one aspect of the present disclosure, further, in removing the water, the prepared suspension is frozen, and the water in the frozen suspension is removed under reduced pressure. It may also be sublimated.

As a result, the method for producing dried bacterial cells can preserve outer membrane-exfoliating modified cyanobacteria in a dry state while maintaining a high survival rate using the freeze-drying method.

For example, in the method for producing dried bacterial cells according to one aspect of the present disclosure, the bacterial cell concentration of the outer membrane-exfoliating modified cyanobacteria in the prepared suspension is 730 (OD (Optical Density)). .3 or more and 80 or less.

As a result, the method for producing dried bacterial cells has an appropriate ratio of the concentration of sucrose or glucose to the concentration of bacterial cells in the adjusted suspension, so that sucrose or glucose can effectively act as a protective agent for bacterial cells. can do. Therefore, the method for producing dried bacterial cells can preserve outer membrane-exfoliating modified cyanobacteria in a dry state while maintaining a high survival rate.

For example, in the method for producing dried bacterial cells according to one aspect of the present disclosure, the ratio of the number of sucrose or glucose molecules to the number of outer membrane-exfoliating modified cyanobacteria cells in the prepared suspension is: It may be ¹⁰⁹ or more.

The method for producing dried bacterial cells has an appropriate ratio of the number of molecules of sucrose or glucose to the number of bacterial cells of outer membrane-exfoliating modified cyanobacteria, so that sucrose or glucose effectively acts as a protective agent for bacterial cells. be able to. Therefore, the method for producing dried bacterial cells can preserve outer membrane-exfoliating modified cyanobacteria in a dry state while maintaining a high survival rate.

Furthermore, the dried bacterial cells according to one aspect of the present disclosure are produced by any of the methods for producing dried bacterial cells described above.

Thereby, the dried bacterial cells can maintain a high survival rate of the outer membrane-exfoliating modified cyanobacteria even in a dry state.

Furthermore, the dried bacterial cells according to one aspect of the present disclosure include outer membrane-exfoliating modified cyanobacteria and sucrose or glucose.

As a result, sucrose or glucose protects the outer membrane-exfoliating modified cyanobacteria from damage caused by drying, allowing the outer membrane-exfoliating modified cyanobacteria to maintain a high survival rate even in dry conditions. be able to.

For example, in the dried bacterial cells according to one aspect of the present disclosure, the ratio of the number of sucrose or glucose molecules to the number of bacterial cells of the outer membrane-exfoliating modified cyanobacteria may be 10 ⁹ or more.

As a result, in dried bacterial cells, the number of molecules of sucrose or glucose is in an appropriate ratio to the number of bacterial cells of outer membrane-exfoliating modified cyanobacteria, so that sucrose or glucose effectively acts as a protective agent for bacterial cells. be able to. Therefore, dry bacterial cells allow outer membrane-exfoliating modified cyanobacteria to maintain a high survival rate even in a dry state.

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

Note that the embodiments described below are all inclusive or specific examples. The numerical values, materials, steps, order of steps, etc. shown in the following embodiments are merely examples, and do not limit the present disclosure. Further, among the constituent elements in the following embodiments, constituent elements that are not described in the independent claims indicating the most significant concept will be described as arbitrary constituent elements.

Further, each figure is not necessarily strictly illustrated. In each figure, substantially the same configurations are denoted by the same reference numerals, and overlapping explanations may be omitted or simplified.

Further, in the following, a numerical range does not represent only a strict meaning, but includes a substantially equivalent range, for example, measuring the amount (for example, number or concentration, etc.) of a protein or its range.

Furthermore, in this specification, both a bacterial body and a cell represent one individual cyanobacterium.

(Embodiment)
[1. Definition]
In this specification, the identity of base sequences and amino acid sequences is calculated by the BLAST (Basic Local Alignment Search Tool) algorithm. Specifically, it was calculated by performing pairwise analysis using the BLAST program available on the website of NCBI (National Center for Biotechnology Information) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Ru. Information regarding cyanobacterial genes and proteins encoded by the genes is published, for example, in the NCBI database mentioned above and Cyanobase (http://genome.microbedb.jp/cyanobase/). From these databases, the amino acid sequences of proteins of interest and the base sequences of genes encoding those proteins can be obtained.

[2. Method for producing dried bacterial cells]
Next, a method for producing dried bacterial cells according to the present embodiment will be explained. FIG. 1 is a flowchart showing an example of the flow of the method for producing dried bacterial cells according to the present embodiment.

The method for producing dried bacterial cells includes preparing a suspension containing outer membrane exfoliating modified cyanobacteria (hereinafter also referred to as modified cyanobacteria) and sucrose or glucose (step S01); Water is removed from the suspension (step S02).

In step S01, first, modified cyanobacteria of outer membrane exfoliating type are prepared. The modified cyanobacteria and the method for producing the modified cyanobacteria will be described later. Here, preparing the outer membrane-exfoliating modified cyanobacteria may mean restoring the cells from dried cells, freeze-dried cells, or frozen stock of the modified cyanobacteria, and Alternatively, a modified cyanobacterium may be produced by genetically modifying the cyanobacteria. The method for producing the modified cyanobacteria will be described later.

Next, the prepared modified cyanobacteria are cultured in a liquid medium, and bacterial cells in the late logarithmic growth phase or early stationary phase are collected from the culture solution. The method for recovering bacterial cells from the culture solution after completion of culture is not particularly limited, and examples thereof include decanting, centrifugation, filtration, and the like. Next, the collected bacterial cells are suspended in a solution of a protective agent (also referred to as a protective substance) to prepare a bacterial suspension. The protective agent (protective substance) is, for example, a sugar such as sucrose or glucose, and is added to a solvent (also referred to as a dispersion liquid) in which the bacterial cells are suspended. These saccharides stabilize the bacterial cells by preventing damage to the bacterial cells due to drying or freezing during the drying or freeze-drying process in step S02. Specifically, these saccharides have properties similar to water molecules, and stabilize the higher-order structure of cellular components in place of water during the freezing or drying process.

In addition, the bacterial cell concentration of the outer membrane-exfoliating modified cyanobacteria in the suspension prepared in step S01 is such that, for example, the absorbance at 730 nm (OD (Optical Density) 730 value) is 7.3 or more and 80 or less. . Among them, the bacterial cell concentration (OD730 value) may be 60 or less. As a result, the method for producing dried bacterial cells has an appropriate ratio of the concentration of sucrose or glucose to the concentration of bacterial cells in the adjusted suspension, so that sucrose or glucose can effectively act as a protective agent for bacterial cells. can do. Therefore, the method for producing dried bacterial cells can preserve outer membrane-exfoliating modified cyanobacteria in a dry state while maintaining a high survival rate.

Further, for example, the ratio of the number of molecules of sucrose or glucose to the number of cells (also referred to as the number of cells) of the outer membrane-exfoliating modified cyanobacteria in the suspension prepared in step S01 is 10 ⁹ or more. Good too. As a result, the method for producing dried bacterial cells has an appropriate ratio of the number of molecules of sucrose or glucose to the number of bacterial cells of modified outer membrane-exfoliating cyanobacteria, so that sucrose or glucose is effective as a protective agent for bacterial cells. can act on Therefore, the method for producing dried bacterial cells can preserve outer membrane-exfoliating modified cyanobacteria in a dry state while maintaining a high survival rate.

In step S02, water is removed from the bacterial suspension prepared in step S01. Methods for removing water from the suspension are not particularly limited, and examples include vacuum drying, freeze vacuum drying (also referred to as freeze drying), reduced pressure spray drying, spray drying, pulse combustion drying, etc. can be mentioned. Examples of the vacuum drying method include the L-drying method. The L-drying method is a method in which water is directly removed from a suspension by reducing the pressure of the suspension and evaporating the water. In addition, in the freeze-vacuum drying method, the suspension is frozen by immersing it in a low-temperature bath (for example, -30°C or less) of liquid nitrogen, dry ice, or alcohol, and then reducing the pressure to remove moisture. (i.e. let it dry). At this time, in the freeze-vacuum drying method, drying is accelerated by capturing water sublimated under reduced pressure in a cold trap. The suspension may be obtained by suspending outer membrane exfoliating modified cyanobacteria collected from the culture solution in a medium containing sucrose or glucose as a protective agent (also referred to as a protective medium), or by suspending it in a medium containing sucrose or glucose as a protective agent (also referred to as a protective medium). It may also be obtained by scraping the bacterial cells cultured in a medium or slant medium with a platinum loop and suspending them in a solution of a protective agent or a protective medium.

[3. Dried bacterial cells]
Next, the dried bacterial cells according to this embodiment will be explained. Dried bacterial cells are produced by any of the methods for producing dried bacterial cells described above. Thereby, the dried bacterial cells can maintain a high survival rate of the outer membrane-exfoliating modified cyanobacteria even in a dry state. The dried bacterial cells contain, for example, outer membrane-exfoliating modified cyanobacteria and sucrose or glucose. As described above, sucrose or glucose is a protective agent for bacterial cells during the drying or freeze-drying process in step S02. Therefore, since sucrose or glucose protects the outer membrane-exfoliating modified cyanobacteria from damage caused by drying, the outer membrane-exfoliating modified cyanobacteria can maintain a high survival rate even in dry conditions. Can be done. For example, in the case of dried bacterial cells, the ratio of the number of sucrose or glucose molecules to the number of bacterial cells of the modified outer membrane-exfoliating cyanobacteria may be 10 ⁹ or more. As a result, in dried bacterial cells, the number of molecules of sucrose or glucose is in an appropriate ratio to the number of bacterial cells of outer membrane-exfoliating modified cyanobacteria, so that sucrose or glucose effectively acts as a protective agent for bacterial cells. be able to. Therefore, dry bacterial cells allow outer membrane-exfoliating modified cyanobacteria to maintain a high survival rate even in a dry state.

[4. cyanobacteria]
Next, cyanobacteria (parent cyanobacteria) will be explained. Cyanobacteria, also called blue-green algae or cyanobacteria, are a group of prokaryotes that perform photosynthesis while collecting light energy with chlorophyll and electrolyzing water to generate oxygen. Cyanobacteria are highly diverse, and include, for example, unicellular species such as Synechocystis sp. PCC 6803 and filamentous multicellular species such as Anabaena sp. PCC 7120. Regarding the growing environment, there are thermophilic species such as Thermosynechococcus elongatus, marine species such as Synechococcus elongatus, and freshwater species such as Synechocystis. In addition, species such as Microcystis aeruginosa, which has gas vesicles and produces toxins, and Gloeobacter violaceus, which does not have thylakoid but has a protein called phycobilisome, which is a light-harvesting antenna on its plasma membrane, have unique characteristics. There are many species that can be mentioned.

FIG. 2 is a diagram schematically showing the cell surface layer of cyanobacteria. As shown in Figure 2, the cell surface layer of cyanobacteria is composed of, in order from the inside, a plasma membrane (also called inner membrane 1), peptidoglycan 2, and outer membrane 5, which is a lipid membrane that forms the outermost layer of the cell. Ru. Sugar chains 3 composed of glucosamine, mannosamine, etc. are covalently bonded to peptidoglycan 2, and pyruvate is bonded to these covalently bonded sugar chains 3 (Non-Patent Document 10: Jurgens and Weckesser, 1986, J. Bacteriol., 168:568-573). In this specification, the peptidoglycan 2 and the covalent sugar chain 3 are collectively referred to as a cell wall 4. In addition, the gap between the plasma membrane (that is, the inner membrane 1) and the outer membrane 5 is called the periplasm, and is used for protein decomposition or three-dimensional structure formation, lipid or nucleic acid decomposition, or the uptake of extracellular nutrients. There are various enzymes involved.

The SLH domain-retaining outer membrane protein (for example, Slr1841 in the figure) consists of a C-terminal region embedded in the lipid membrane (also referred to as outer membrane 5) and an N-terminal SLH domain 7 that protrudes from the lipid membrane. , is widely distributed in cyanobacteria and bacteria belonging to the Negativicutes class, which is a group of Gram-negative bacteria (Non-Patent Document 11: Kojima et al., 2016, Biosci. Biotech. Biochem., 10:1954-1959). The region embedded in the lipid membrane (i.e. outer membrane 5) forms a channel to allow the passage of hydrophilic substances through the outer membrane, while the SLH domain 7 has the function of binding to the cell wall 4 (non-transparent membrane 5). Patent Document 12: Kowata et al., 2017, J. Bacteriol., 199:e00371-17). In order for SLH domain 7 to bind to cell wall 4, covalent sugar chain 3 in peptidoglycan 2 needs to be modified with pyruvate (Non-Patent Document 13: Kojima et al., 2016, J. Biol Chem., 291:20198-20209). Examples of genes encoding SLH domain-retaining outer membrane protein 6 include slr1841 or slr1908 held by Synechocystis sp. PCC 6803, or oprB held by Anabaena sp. 90.

The enzyme that catalyzes the pyruvate modification reaction of the covalent sugar chain 3 in peptidoglycan 2 (hereinafter referred to as cell wall-pyruvate modification enzyme 9) was identified in the Gram-positive bacterium Bacillus anthracis and named CsaB. (Non-Patent Document 14: Mesnage et al., 2000, EMBO J., 19:4473-4484). Among cyanobacteria whose genome sequences have been published, many species possess genes encoding homologous proteins with amino acid sequence identity of 30% or more with CsaB. Examples include slr0688 held by Synechocystis sp. PCC 6803 or synpcc7502_03092 held by Synechococcus sp. 7502.

In cyanobacteria, _CO2 fixed through photosynthesis is converted into various amino acids through multi-step enzymatic reactions. Using these as raw materials, proteins are synthesized within the cytoplasm of cyanobacteria. Some of these proteins function within the cytoplasm, while others are transported from the cytoplasm to the periplasm and function within the periplasm. However, no case of active secretion of proteins outside the cell has been reported in cyanobacteria to date.

Cyanobacteria have a high photosynthetic ability, so they do not necessarily need to take in organic matter from the outside as nutrients. Therefore, cyanobacteria have very few channel proteins in their outer membrane 5 that allow organic substances to pass therethrough, such as the organic substance channel protein 8 (eg, Slr1270) in FIG. For example, in Synechocystis sp. PCC 6803, the organic substance channel protein 8 that allows organic substances to pass therethrough is present in only about 4% of the total protein content of the outer membrane 5. On the other hand, in order for cyanobacteria to take in inorganic ions necessary for growth into cells with high efficiency, only inorganic ions can be permeated by cyanobacteria, such as SLH domain-retaining outer membrane protein 6 (e.g., Slr1841) shown in Figure 2. The outer membrane 5 contains many ion channel proteins that cause For example, in Synechocystis sp. PCC 6803, ion channel proteins that permeate inorganic ions account for about 80% of the total protein content of the outer membrane 5.

In this way, cyanobacteria have very few channels in the outer membrane 5 that allow organic substances such as proteins to pass through, so it is thought that it is difficult to actively secrete proteins produced within the bacterium to the outside of the bacterium. There is. Furthermore, although the structure of the cell wall and cell membrane of cyanobacteria influences protein permeability, it is not easy to artificially modify the cell membrane and cell wall structure to improve protein secretion production ability. For example, Non-Patent Document 10 and Non-Patent Document 11 state that when the slr1841 gene or slr0688 gene, which is involved in adhesion between the outer membrane and the cell wall and contributes to the structural stability of the cell surface, is deleted, the proliferation ability of the cell is reduced. It is stated that it will be lost.

[5. Modified cyanobacteria]
Next, the cyanobacteria (hereinafter referred to as modified cyanobacteria) according to the present embodiment will be described with reference to FIG. 2. Further, below, proteins will be explained as an example of substances produced using modified cyanobacteria.

In the modified cyanobacteria according to the present embodiment, the function of a protein involved in binding between the outer membrane 5 and the cell wall 4 (so-called binding-related protein) is suppressed or lost in the cyanobacterium. More specifically, for example, the modified cyanobacteria is such that the total amount of proteins involved in binding between the outer membrane 5 and the cell wall 4 (i.e., binding-related proteins) is lower than that in the parent strain (i.e., parent cyanobacteria). The total amount of protein is suppressed to 30% or more and 70% or less. Here, for example, "the total amount of binding-related proteins is suppressed to 30% of the total amount of the protein in the parent strain" means that 70% of the total amount of the protein in the parent strain has been lost and 30% remains. It means the state of being. As a result, in the modified cyanobacteria, the bond between the outer membrane 5 and the cell wall 4 (for example, the amount of bond and the binding force) is partially reduced, so that the outer membrane 5 is easily partially detached from the cell wall 4. Therefore, the modified cyanobacteria has improved protein secretion productivity, which secretes proteins produced within the bacterial cells to the outside of the bacterial cells. Furthermore, since it is not necessary to crush the bacterial cells to recover proteins, the modified cyanobacteria can be repeatedly used to produce proteins even after the proteins are recovered. In this specification, the production of proteins by modified cyanobacteria within the bacterial cells is referred to as production, and the secretion of the produced proteins to the outside of the bacterial cells is referred to as secretory production.

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. In the present embodiment, in the modified cyanobacteria, the function of at least one protein, for example, the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9, is suppressed. For example, in the modified cyanobacteria, (i) at least one function of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 may be suppressed, and (ii) the SLH domain-retaining type that binds to the cell wall 4 may be suppressed. At least one of the expression of the outer membrane protein 6 and the expression of an enzyme that catalyzes the pyruvate modification reaction of sugar chains bound to the surface of the cell wall 4 (ie, the cell wall-pyruvate modifying enzyme 9) may be suppressed. This reduces the bond (that is, the bond amount and binding force) between the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 and the covalent sugar chain 3 on the surface of the cell wall 4. Therefore, the outer membrane 5 easily detaches from the cell wall 4 in areas where these bonds are weakened. By partially detaching the outer membrane 5 from the cell wall 4, proteins present in the cells of the modified cyanobacteria, particularly in the periplasm, tend to leak out of the cells (outside the outer membrane 5).

As mentioned above, cyanobacteria have a high photosynthetic ability, so they do not necessarily need to take in organic matter from the outside as nutrients. Therefore, cyanobacteria only need light, air, water, and a small amount of inorganic substances to cultivate cyanobacteria, and cyanobacteria take inorganic substances into cells through ion channels in the outer membrane 5 and produce proteins within the cells. In particular, various proteins are present in the periplasm, which is the space between the outer membrane 5 and the cell wall 4. In the modified cyanobacterium according to the present embodiment, the functions of proteins involved in binding between the outer membrane 5 and the cell wall 4 are suppressed. As a result, the outer membrane 5 is easily partially peeled off from the cell wall 4. Then, proteins within the periplasm leak into the culture solution from the peeled portion of the outer membrane 5. As a result, the modified cyanobacteria improves the productivity of protein secretion, which secretes proteins produced within the bacterial cells to the outside of the bacterial cells.

Hereinafter, the outer membrane 5 is modified to partially detach from the cell wall 4 by suppressing the function of at least one binding-related protein of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9. The following cyanobacteria will be explained in more detail.

The cyanobacterium that is the parent microorganism of the modified cyanobacterium according to the present embodiment, which is the parent microorganism of the modified cyanobacteria (hereinafter referred to as The type of the "parent strain" or "parent cyanobacteria") is not particularly limited, and may be any type of cyanobacteria. For example, the parent cyanobacteria may be of the genus Synechocystis, Synechococcus, Anabaena, or Thermosynechococcus, among which Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, or Thermosynechococcus elongatus BP-1. Good too. The parent strain may be a wild type of cyanobacteria, as long as it has not yet suppressed the total amount of binding-related proteins to 30% or more and 70% or less, or it may be a modified strain that is equivalent to the wild type. binding-related proteins.

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., the cell wall-pyruvate modification enzyme 9) in these parent cyanobacteria, and the genes encoding their binding-related proteins. The base sequence and the position of the gene on the chromosomal DNA or plasmid can be confirmed in the NCBI database and Cyanobase mentioned above.

Note that the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvic acid modifying enzyme 9, whose functions are suppressed in the modified cyanobacteria according to the present embodiment, can be used in any parent cyanobacteria as long as they are possessed by the parent cyanobacteria. They are not limited by the location of the genes encoding them (for example, on chromosomal DNA or on plasmids).

For example, the SLH domain-retaining outer membrane protein 6 may be Slr1841, Slr1908, or Slr0042 when the parent cyanobacterium belongs to the genus Synechocystis, or may be NIES970_09470 when the parent cyanobacterium belongs to the genus Synechococcus. Often, when the parent cyanobacteria is of the genus Anabaena, it may be Anacy_5815 or Anacy_3458, etc. When the parent cyanobacteria is of the genus Microcystis, it may be A0A0F6U6F8_MICAE, etc. When the parent cyanobacteria is of the genus Cyanothece, it may be A0A3B8XX12_9CYAN, etc. When the parent cyanobacterium belongs to the genus Leptolyngbya, it may be A0A1Q8ZE23_9CYAN, etc. When the parent cyanobacterium belongs to the genus Calothrix, it may be A0A1Z4R6U0_9CYAN, and when the parent cyanobacterium belongs to the genus Nostoc, it may be A0A1C0VG86_9NOSO, etc. When the parent cyanobacterium belongs to the genus Crocosphaera, it may be B1WRN6_CROS5, and when the parent cyanobacterium belongs to the genus Pleurocapsa, it may be K9TAE4_9CYAN.

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

As a result, in the modified cyanobacteria, for example, (i) any of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOs: 1 to 3 above or any of these SLH domain-retaining outer membrane proteins 6 and amino acids; The function of the protein whose sequence is 50% or more identical may be suppressed, and (ii) any of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOs: 1 to 3 above or any of these SLHs. Expression of a protein whose amino acid sequence is 50% or more identical to domain-retaining outer membrane protein 6 may be suppressed. 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 a function equivalent to the SLH domain-retaining outer membrane protein 6 is suppressed, or (ii) ) The expression level of the SLH domain-retaining outer membrane protein 6 or a protein having the same function as the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 is reduced. Therefore, in the modified cyanobacteria according to the present embodiment, the binding domain (for example, SLH domain 7) for binding the outer membrane 5 to the cell wall 4 has a reduced binding amount and binding force with the cell wall 4, so The membrane 5 is easily partially detached from the cell wall 4.

Generally, it is said that if the amino acid sequences of a protein are 30% or more identical, the protein has a high degree of homology in its three-dimensional structure and is therefore likely to have the same function as the protein. Therefore, the SLH domain-retaining outer membrane protein 6 whose function is suppressed is, for example, one of the amino acid sequences of the SLH domain-retaining outer membrane protein 6 shown in SEQ ID NOs: 1 to 3 above, and 40% or more of the amino acid sequence, Preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, and the cell wall 4 is shared. It may be a protein or a polypeptide that has the function of binding to a bound sugar chain 3.

Furthermore, for example, the cell wall-pyruvate modifying enzyme 9 may be Slr0688, etc. when the parent cyanobacterium belongs to the genus Synechocystis, and may be Syn7502_03092 or Synpcc7942_1529, etc. when the parent cyanobacterium belongs to the genus Synechococcus. If the cyanobacterium belongs to the genus Anabaena, it may be ANA_C20348 or Anacy_1623, and if the parent cyanobacterium belongs to the genus Microcystis, it may be CsaB (NCBI access ID: TRU80220), or if the parent cyanobacterium belongs to the genus Cyanothece. If the parent cyanobacterium belongs to the genus Spirulina, it may be CsaB (NCBI access ID: WP_026079530.1), etc. If the parent cyanobacterium belongs to the genus Calothrix, it may be CsaB (NCBI access ID: WP_096658142.1), etc., and if the parent cyanobacterium belongs to the genus Nostoc, it may be CsaB (NCBI access ID: WP_099068528.1), etc. , if the parent cyanobacterium belongs to the genus Crocosphaera, it may be CsaB (NCBI access ID: WP_012361697.1), and if the parent cyanobacterium belongs to the genus Pleurocapsa, it may be CsaB (NCBI access ID: WP_036798735), etc. Good too.

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

As a result, in the modified cyanobacteria, for example, (i) any of the cell wall-pyruvate modifying enzymes 9 shown in SEQ ID NOs: 4 to 6 above or any of these cell wall-pyruvate modifying enzymes 9 and the amino acid sequence The function of a protein that is 50% or more identical is suppressed, or (ii) any of the cell wall-pyruvic acid modifying enzymes 9 shown in SEQ ID NOs: 4 to 6 above or any of these cell wall-pyruvic acid Expression of a protein whose amino acid sequence is 50% or more identical to modified enzyme 9 is suppressed. Therefore, in the modified cyanobacteria, (i) the function of the cell wall-pyruvate modifying enzyme 9 or a protein having an equivalent function to the enzyme is suppressed, or (ii) the function of the cell wall-pyruvate modifying enzyme 9 or the enzyme is suppressed. The expression level of proteins with equivalent functions decreases. This makes it difficult for the covalent sugar chains 3 on the surface of the cell wall 4 to be modified with pyruvate, so that the sugar chains 3 on the cell wall 4 interact with the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5. The amount of binding and the binding strength are reduced. Therefore, in the modified cyanobacteria according to the present embodiment, the covalently bonded sugar chains 3 on the surface of the cell wall 4 are difficult to be modified with pyruvate, so the binding force between the cell wall 4 and the outer membrane 5 is weakened, and the outer membrane 5 becomes easily partially detached from the cell wall 4.

Moreover, as mentioned above, if the amino acid sequences of a protein are 30% or more identical, it is said that the protein is likely to have the same function as the protein. Therefore, the cell wall-pyruvate modifying enzyme 9 whose function is suppressed is, for example, an amino acid sequence of any one of the cell wall-pyruvate modifying enzymes 9 shown in SEQ ID NOs: 4 to 6 above, and 40% or more, preferably It consists of an amino acid sequence having an identity of 50% or more, more preferably 60% or more, still more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, and It may be a protein or polypeptide that has the function of catalyzing a reaction that modifies covalent sugar chain 3 with pyruvate.

In this specification, suppressing the function of the SLH domain-retaining outer membrane protein 6 refers to suppressing the ability of the protein to bind to the cell wall 4, or suppressing or losing the transport of the protein to the outer membrane 5. or suppress the ability of the protein to become embedded in the outer membrane 5 and function.

Note that suppressing the function of the cell wall-pyruvic acid modifying enzyme 9 means suppressing the function of the protein to modify the covalent sugar chain 3 of the cell wall 4 with pyruvate.

The means for suppressing the functions of these proteins is not particularly limited as long as it is a means normally used for suppressing the functions of proteins. The means includes, 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, The method may include inhibiting the translation of transcription products of these genes, or administering an inhibitor that specifically inhibits these proteins.

In the present embodiment, the modified cyanobacteria may have a gene that expresses a protein involved in bonding the outer membrane 5 and the cell wall 4 deleted or inactivated. As a result, in the modified cyanobacteria, the expression of the protein involved in the bond between the cell wall 4 and the outer membrane 5 is suppressed, or the function of the protein is suppressed, so that the bond between the cell wall 4 and the outer membrane 5 is suppressed. (so-called bond amount and bond strength) are partially reduced. As a result, in the modified cyanobacteria, the outer membrane 5 tends to partially detach from the cell wall 4, so that proteins produced within the bacterial body tend to leak out of the outer membrane 5, that is, to the outside of the bacterial body. Therefore, the modified cyanobacteria according to the present embodiment has improved protein secretion productivity for secreting proteins produced within the bacterial cells to the outside of the bacterial cells. Furthermore, since it is not necessary to crush the bacterial cells to recover proteins, the modified cyanobacteria can be repeatedly used to produce proteins even after the proteins are recovered.

The gene that expresses the protein involved in the binding between the outer membrane 5 and the cell wall 4 is, for example, at least one of the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9. There may be. In the modified cyanobacteria, at least one of the genes encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9 has been deleted or inactivated. Therefore, in the modified cyanobacteria, for example, (i) the expression of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 is suppressed, or (ii) the SLH domain-retaining outer membrane protein At least one function of cell wall-pyruvate modifying enzyme 6 and cell wall-pyruvate modifying enzyme 9 is suppressed. Therefore, the bond between the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 and the covalent sugar chain 3 on the surface of the cell wall 4 (that is, the bond amount and binding force) is reduced. This makes it easier for the outer membrane 5 to detach from the cell wall 4 at the portion where the bond between the outer membrane 5 and the cell wall 4 is weakened. Therefore, according to the modified cyanobacteria according to the present embodiment, the binding between the outer membrane 5 and the cell wall 4 is reduced, making it easier for the outer membrane 5 to be partially detached from the cell wall 4. This makes it easier for protein to leak out of the bacterial body.

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

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

More specifically, the genes encoding SLH domain-retaining outer membrane protein 6 include slr1841 (SEQ ID NO: 7) of Synechocystis sp. PCC 6803, nies970_09470 (SEQ ID NO: 8) of Synechococcus sp. NIES-970, and Anabaena cylindrica PCC. 7122 anacy_3458 (SEQ ID NO: 9), or a gene whose base sequence is 50% or more identical to these genes.

As a result, in the modified cyanobacteria, the gene encoding any of the SLH domain-retaining outer membrane protein 6 shown in SEQ ID NOs: 7 to 9 above, or the base sequence of any of these genes, is 50% or more identical. Genes are deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the above-mentioned SLH domain-retaining outer membrane protein 6 or a protein having a function equivalent to any of these proteins is suppressed, or (ii) the above-mentioned The function of any of the SLH domain-retaining outer membrane proteins 6 or a protein having a function equivalent to any of these proteins is suppressed. Therefore, in the modified cyanobacteria according to the present embodiment, the binding amount and binding force of the binding domain (for example, SLH domain 7) for binding the outer membrane 5 to the cell wall 4 with the cell wall 4 are reduced, 5 becomes easily partially detached from the cell wall 4.

As mentioned above, if the amino acid sequences of a protein are 30% or more identical, it is said that the protein is likely to have the same function as the protein. Therefore, if the base sequences of genes encoding proteins are 30% or more identical, it is considered that there is a high possibility that a protein having the same function as the protein will be expressed. Therefore, as a gene encoding the SLH domain-retaining outer membrane protein 6 whose function is suppressed, for example, any of the genes encoding the SLH domain-retaining outer membrane protein 6 shown in SEQ ID NOs: 7 to 9 above can be used. From a base sequence having an identity of 40% or more, preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, even more preferably 80% or more, still more preferably 90% or more with the base sequence. It may also be a gene that encodes a protein or polypeptide that has the function of binding to the covalent sugar chain 3 of the cell wall 4.

Furthermore, for example, the gene encoding cell wall-pyruvate modifying enzyme 9 may be slr0688, etc. when the parent cyanobacterium belongs to the genus Synechocystis, and may be syn7502_03092 or synpcc7942_1529, etc. when the parent cyanobacterium belongs to the genus Synechococcus. If the parent cyanobacterium belongs to the genus Anabaena, it may be ana_C20348 or anacy_1623, and if the parent cyanobacterium belongs to the genus Microcystis, it may be csaB (NCBI access ID: TRU80220), etc. If the parent cyanobacterium belongs to the genus Cynahothece, it may be csaB (NCBI access ID: WP_107667006.1), etc., and if the parent cyanobacterium belongs to the genus Spirulina, it may be csaB (NCBI access ID: WP_026079530.1), etc. , if the parent cyanobacteria belongs to the genus Calothrix, it may be csaB (NCBI access ID: WP_096658142.1), etc., and if the parent cyanobacteria belongs to the genus Nostoc, it may be csaB (NCBI access ID: WP_099068528.1), etc. If the parent cyanobacterium belongs to the genus Crocosphaera, it may be csaB (NCBI access ID: WP_012361697.1), and if the parent cyanobacterium belongs to the genus Pleurocapsa, it may be csaB (NCBI access ID: WP_036798735). etc. may be used. The base sequences of these genes can be obtained from the NCBI database or Cyanobase mentioned above.

More specifically, the gene encoding cell wall-pyruvate modification enzyme 9 is slr0688 (SEQ ID NO: 10) of Synechocystis sp. PCC 6803, synpcc7942_1529 (SEQ ID NO: 11) of Synechococcus sp. PCC 7942, or Anabaena cylindrica PCC. 7122 anacy_1623 (SEQ ID NO: 12). Alternatively, the gene may have a base sequence that is 50% or more identical to these genes.

As a result, in the modified cyanobacteria, the base sequence of the gene encoding any of the cell wall-pyruvic acid modifying enzymes 9 shown in SEQ ID NOs: 10 to 12 above or the gene encoding any of these enzymes is 50% or more. Genes that are identical are deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the above cell wall-pyruvate modifying enzymes 9 or a protein having a function equivalent to any of these enzymes is suppressed, or (ii) the above-mentioned The function of any cell wall-pyruvate modifying enzyme 9 or a protein having a function equivalent to any of these enzymes is suppressed. This makes it difficult for the covalent sugar chains 3 on the surface of the cell wall 4 to be modified with pyruvate, so that the sugar chains 3 on the cell wall 4 interact with the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5. The amount of binding and the binding strength are reduced. Therefore, in the modified cyanobacteria according to the present embodiment, the amount of modification of sugar chains 3 with pyruvate for bonding the cell wall 4 to the outer membrane 5 is reduced, so that the binding strength between the cell wall 4 and the outer membrane 5 is reduced. is weakened, and the outer membrane 5 becomes easily partially detached from the cell wall 4.

As mentioned above, if the base sequences of genes encoding proteins are 30% or more identical, it is considered that there is a high possibility that a protein having the same function as the protein will be expressed. Therefore, as a gene encoding cell wall-pyruvate modifying enzyme 9 whose function is suppressed, for example, any of the base sequences of the genes encoding cell wall-pyruvate modifying enzyme 9 shown in SEQ ID NOs: 10 to 12 above. and a base sequence having an identity of 40% or more, preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, even more preferably 80% or more, still more preferably 90% or more, In addition, it may be a gene encoding a protein or polypeptide that has the function of catalyzing a reaction that modifies the covalent sugar chain 3 of the peptidoglycan 2 of the cell wall 4 with pyruvate.

[6. Method for producing modified cyanobacteria]
Next, a method for producing modified cyanobacteria according to this embodiment will be explained. The method for producing a modified cyanobacterium includes the step of suppressing the function of a protein involved in bonding the outer membrane 5 and the cell wall 4 in the cyanobacterium.

In this embodiment, the protein involved in 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.

Means for suppressing protein function include, but are not particularly limited to, deletion or inactivation of the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9. It may be possible to inhibit the transcription of these genes, inhibit the translation of the transcripts of these genes, or administer an inhibitor that specifically inhibits these proteins.

Means for deleting or inactivating the above gene include, for example, introducing a mutation into one or more bases on the base sequence of the gene, replacing the base sequence with another base sequence, or replacing the base sequence with another base sequence. It may be insertion, or deletion of part or all of the base sequence of the gene concerned.

Means for inhibiting the transcription of the above genes include, for example, introducing mutations into the promoter region of the gene, inactivating the promoter by substituting or inserting other base sequences, or CRISPR interference (non-transfer). Patent Document 15: Yao et al., ACS Synth. Biol., 2016, 5:207-212). Specific methods for the above-mentioned mutagenesis or base sequence substitution or insertion may be, for example, ultraviolet irradiation, site-specific mutagenesis, or homologous recombination.

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

Modified cyanobacteria may be produced by using any of the above methods to suppress the function of a protein involved in bonding the outer membrane 5 and cell wall 4 in cyanobacteria.

As a result, in the modified cyanobacteria produced by this production method, the bond between the cell wall 4 and the outer membrane 5 (that is, the amount of bond and binding force) is partially reduced, so that the outer membrane 5 is partially separated from the cell wall 4. It becomes easier to detach. Therefore, in the modified cyanobacteria, proteins produced within the bacterial cells tend to leak out of the outer membrane 5 (that is, out of the bacterial cells). Therefore, according to the method for producing modified cyanobacteria according to the present embodiment, modified cyanobacteria with improved protein secretion productivity can be provided.

Furthermore, in the modified cyanobacteria produced by this production method, proteins produced within the bacterial cells leak out of the bacterial cells, so there is no need to crush the bacterial cells for protein recovery. For example, it is sufficient to culture the modified cyanobacteria under appropriate conditions and then collect the proteins secreted into the culture solution, so it is also possible to collect the proteins in the culture solution while culturing the modified cyanobacteria. Therefore, by using the modified cyanobacteria obtained by this production method, efficient microbial protein production can be carried out. Therefore, according to the method for producing a modified cyanobacterium according to the present embodiment, it is possible to provide a modified cyanobacterium with high utilization efficiency that can be used repeatedly even after protein recovery.

Note that the modified cyanobacteria produced by this production method secretes, to the outside of the cell, a group of proteins, such as peptidases or phosphatases, which are primarily present in the periplasm. In this embodiment, for example, by modifying a gene encoding a protein originally produced within cyanobacterial cells, such as a group of proteins present in the periplasm, and replacing it with a gene encoding another protein, It is also possible to cause modified cyanobacteria to produce desired proteins. Therefore, according to the method for producing a modified cyanobacterium according to the present embodiment, it is also possible to provide a modified cyanobacterium that can easily and efficiently produce a desired protein.

The present invention will be specifically explained with reference to the following examples, but the present invention is not limited to the following examples.

(1) Preparation of cyanobacteria to be used in the production of dried bacterial cells First, cyanobacteria to be used in the production of dried bacterial cells were prepared. As an outer membrane-exfoliating modified cyanobacterium (hereinafter also referred to as an outer membrane-exfoliating strain), a modified cyanobacterium in which the expression of the slr0688 gene encoding a cell wall-pyruvate modifying enzyme was suppressed was obtained by the following procedure. The cyanobacterial species used in this example is Synechocystis sp. PCC 6803 (hereinafter simply referred to as "cyanobacteria").

(1-1) Creation of adventitial membrane detachment strain CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference method was used as a gene expression suppression method. In this method, the expression of the slr0688 gene is suppressed by introducing the gene encoding the dCas9 protein (hereinafter referred to as the dCas9 gene) and the slr0688_sgRNA (single-guide Ribonucleic Acid) gene into the chromosomal DNA of cyanobacteria. Can be done. Furthermore, by controlling the transcriptional activity of slr0688_sgRNA, the degree of suppression of the slr0688 gene can be controlled.

The mechanism of gene expression suppression by this method is as follows.

First, a Cas9 protein lacking nuclease activity (dCas9) and sgRNA (slr0688_sgRNA) that binds complementary to the base sequence of the slr0688 gene form a complex.

Next, this complex recognizes the slr0688 gene on the cyanobacterial chromosomal DNA and specifically binds to the slr0688 gene. This binding causes steric hindrance, which inhibits transcription of the slr0688 gene. As a result, the expression of the cyanobacterial slr0688 gene is suppressed. Furthermore, by controlling the transcriptional activity of slr0688_sgRNA, the degree of suppression of the slr0688 gene can be controlled.

Hereinafter, a method for introducing each of the above two genes into the chromosomal DNA of cyanobacteria will be specifically explained.

(1-1-1) Introduction of dCas9 gene
Using the chromosomal DNA of Synechocystis strain LY07 (hereinafter also referred to as LY07 strain) (see Non-Patent Document 15) as a template, the dCas9 gene, an operator gene for controlling the expression of the dCas9 gene, and spectinomycin, which serves as a marker for gene introduction. The resistance marker gene was amplified by the PCR (polymerase chain reaction) method using the primers psbA1-Fw (SEQ ID NO: 13) and psbA1-Rv (SEQ ID NO: 14) listed in Table 1. In the LY07 strain, the three genes mentioned above are inserted into the psbA1 gene on the chromosomal DNA in a linked state, so they can be amplified as one DNA fragment by PCR. Here, the obtained DNA fragment is referred to as "psbA1::dCas9 cassette." The psbA1::dCas9 cassette was inserted into the pUC19 plasmid using the In-Fusion PCR cloning method (registered trademark) to obtain the pUC19-dCas9 plasmid.

1 μg of the obtained pUC19-dCas9 plasmid was mixed with a cyanobacterial culture solution (bacteria cell concentration OD ₇₃₀ = approximately 0.5), and the pUC19-dCas9 plasmid was introduced into cyanobacterial cells by natural transformation. Transformed cells were selected by growing on BG-11 agar medium containing 20 μg/mL spectinomycin. In the selected cells, homologous recombination occurs between the psbA1 gene on the chromosomal DNA and the psbA1 upstream fragment region and psbA1 downstream fragment region on the pUC19-dCas9 plasmid. As a result, a Synechocystis dCas9 strain in which the dCas9 cassette was inserted into the psbA1 gene region was obtained. The composition of the BG-11 medium used is shown in Table 2.

(1-1-2) Introduction of slr1841_sgRNA gene
In CRISPR interference, sgRNA specifically binds to a target gene by introducing a sequence of approximately 20 bases that is complementary to the target sequence into a region called a protospacer on the sgRNA gene. The protospacer sequences used in this example are shown in Table 3.

In Synechocystis LY04, LY05, or LY07 strains, the sgRNA gene (excluding the protospacer region) and the kanamycin resistance marker gene are inserted into the slr2030-slr2031 genes on the chromosomal DNA (non-patent literature). 15). Therefore, by adding a protospacer sequence (SEQ ID NO: 19) complementary to the slr0688 gene (SEQ ID NO: 4) to the primers used when amplifying the sgRNA gene by PCR method, an sgRNA (slr0688_sgRNA) that specifically recognizes slr0688 (slr0688_sgRNA ) can be easily obtained. Furthermore, by controlling the transcriptional activity of slr0688_sgRNA, the degree of suppression of the slr0688 gene can be controlled.

First, using the chromosomal DNA of the LY07 strain as a template, a set of primers slr2030-Fw (SEQ ID NO: 15) and sgRNA_slr0688-Rv (SEQ ID NO: 17) listed in Table 1, as well as sgRNA_slr0688-Fw (SEQ ID NO: 18) and slr2031- Two DNA fragments were amplified by PCR using a set of Rv (SEQ ID NO: 16).

Next, by using the mixed solution of the above DNA fragments as a template and using the primers slr2030-Fw (SEQ ID NO: 15) and slr2031-Rv (SEQ ID NO: 16) listed in Table 1 by PCR method, ( A DNA fragment (slr2030-2031::slr0688_sgRNA) was obtained in which i) slr2030 gene fragment, (ii) slr0688_sgRNA, (iii) kanamycin resistance marker gene, and (iv) slr2031 gene fragment were linked in this order. Using the In-Fusion PCR cloning method (registered trademark), slr2030-2031::slr0688_sgRNA was inserted into the pUC19 plasmid to obtain the pUC19-slr0688_sgRNA plasmid.

The pUC19-slr0688_sgRNA plasmid was introduced into the Synechocystis dCas9 strain in the same manner as in (1-1-1) above, and the transformed cells were selected on a BG-11 agar medium containing 30 μg/mL kanamycin. As a result, a transformant Synechocystis dCas9 slr0688_sgRNA strain (so-called outer membrane detachment strain) in which slr0688_sgRNA was inserted into the slr2030-slr2031 gene on the chromosomal DNA was obtained.

(1-1-3) Suppression of slr0688 gene The promoter sequences of the dCas9 gene and slr0688_sgRNA gene are designed to induce expression in the presence of anhydrotetracycline (aTc). In this example, the expression of the slr0688 gene was suppressed by adding aTc at a final concentration of 1 μg/mL to the medium.

As described above, the amount of the protein involved in binding the outer membrane and cell wall in cyanobacteria can be adjusted to the amount of the protein in the parent strain (Synechocystis dCas9 strain, wild strain described below) without impairing cell growth ability. We obtained a modified cyanobacterium Synechocystis dCas9 slr0688_sgRNA strain (so-called outer membrane detachment strain) that was suppressed to about 50% compared to the above. Here, the protein involved in binding the outer membrane to the cell wall is Slr0688.

(1-2) Preparation of dCas9 gene-introduced strain A Synechocystis dCas9 strain in which the dCas9 cassette was inserted into the psbA1 gene region was obtained by the same procedure as in (1-1-1) above. Hereinafter, the Synechocystis dCas9 strain will be referred to as the wild strain.

(2) Cultivation of cyanobacteria The outer membrane detachment strain produced in (1-1) above and the wild strain produced in (1-2) above were cultured under the following conditions.

First, the outer membrane detachment strain and the wild type strain were each inoculated into a BG-11 medium (see Table 2) containing 1 μg/mL aTC so that the initial bacterial cell concentration was OD ₇₃₀ = 0.1. 12 mL of this culture solution was dispensed into a 125 mL Erlenmeyer flask, and cultured with shaking under conditions of light irradiation at a light intensity of 100 μmol/m ² /s and 30° C. for 5 days.

(3) Production of dried bacterial cells (3-1) Evaluation of freeze-drying tolerance of cyanobacteria Subsequently, freeze-drying tolerance was evaluated for each of the outer membrane detachment strain and the wild strain.

(3-1-1) About the wild strain For the wild strain, the culture solution obtained in (2) above on the 5th day of culture is centrifuged at 2,500 g for 10 minutes at room temperature to remove bacterial cells from the culture solution. (also called cells) were collected.

The collected cells were suspended in water to a cell concentration OD ₇₃₀ = 7.3 to prepare a suspension, and then the suspension was frozen with liquid nitrogen. The frozen suspension was then freeze-dried overnight at a trap temperature of -80°C and a pressure of 10 Pa or less to remove water from the suspension.

Immediately after freeze-drying, the cells were inoculated into 5 mL of BG-11 medium in a test tube so that the cell concentration was OD ₇₃₀ = 0.1, and the cells were incubated at a light intensity of 100 μmol/m ² /s and at 30°C for 8 days. After shaking culture, the bacterial cell concentration OD ₇₃₀ value (A in the following formula (1)) of the culture solution was measured. The measurement results are shown in Table 4.

In addition, among the collected cells, the cells that were not subjected to freeze-drying were inoculated to a cell concentration of OD ₇₃₀ = 0.1, and cultured with shaking for 8 days under the same light and temperature conditions as the freeze-dried cells. After that, the bacterial cell concentration OD ₇₃₀ value (B in formula (1) below) of the culture solution was measured.

The survival rate C (%) of dried bacterial cells was calculated using the following formula (1). The calculation results are shown in Table 4. Table 4 shows the average value ± standard deviation (SD) of the measured value and survival rate when the bacterial cell concentration OD ₇₃₀ value of the culture solution of the wild strain was measured four times.

(3-1-2) Regarding the outer membrane detachment strain For the outer membrane detachment strain, the bacterial cells recovered from the culture solution were mixed with water, BG-11 medium (see Table 2), and sucrose (Suc) as a protective agent. The same procedure as above (3-1-1) was carried out except that the cells were suspended in BG-11 medium containing 100 mM sucrose added to BG-11 medium. Table 4 shows the measurement results of the bacterial cell concentration OD ₇₃₀ (A in the following formula (1)) of the culture solution on the 8th day of culturing the freeze-dried bacterial cells and the calculation results of the survival rate (%). Table 4 shows the average value ± standard deviation (SD) of the bacterial cell concentration OD ₇₃₀ value of the culture solution of the outer membrane detachment strain measured 3 to 4 times and the survival rate.

In addition, Figure 3 is a graph showing the survival rate of wild-type strains and adventitial membrane-exfoliating strains when suspensions are prepared using water as a dispersion liquid (wild strain n=4; adventitial membrane-exfoliating strains n=3; error bar = SD).

As shown in Figure 3 and Table 4, the wild strain has a survival rate of over 90% even when a suspension of bacterial cells is prepared using water as a dispersion liquid and subjected to freeze-drying. showed that. On the other hand, for the outer membrane detachment strain, when a suspension of bacterial cells was prepared using water as a dispersion liquid and subjected to lyophilization, the lyophilized bacterial cells showed a survival rate of only about 30%. In other words, it was shown that approximately 70% of the outer membrane detachment strain was killed by freeze-drying treatment.

In addition, as shown in Table 4, the outer membrane detachment strain only showed a survival rate of about 70% even when a suspension of bacterial cells was prepared using BG-11 medium as a dispersion liquid and subjected to freeze-drying. There wasn't. From this result, it is considered that in the outer membrane detachment strain, the outer membrane detaches from the cell wall, thereby impairing the freeze-drying resistance inherent in cyanobacterial cells.

However, when a suspension of bacterial cells was prepared using BG-11 medium supplemented with sucrose as a dispersion liquid and subjected to freeze-drying, the freeze-dried bacterial cells showed a survival rate of over 90%. This survival rate was comparable to that of the wild type. Therefore, sucrose was confirmed to be an effective protective agent for freeze-drying outer membrane detachment strains.

From the above, when a suspension containing an outer membrane-exfoliating strain and sucrose is subjected to freeze-drying, modified outer membrane-exfoliating cyanobacteria (outer membrane-exfoliating strain) can be stored in a dry state while maintaining a high survival rate. It was shown that a method for producing dried bacterial cells and that dried bacterial cells can be provided.

(3-2) Examination of a protective agent that is effective for freeze-drying a strain that has peeled off the outer membrane. In (3-1-2) above, it was shown that sucrose is an effective protective agent for freeze-drying a strain that has peeled off the outer membrane. However, we investigated whether there are any protective agents other than sucrose that would have the same effect. As a dispersion liquid, (a) BG-11 medium, (b) culture supernatant of a culture solution obtained by culturing adventitia detachment strain in BG-11 medium, and (c) 100mM trehalose added to BG-11 medium as a protective agent. BG-11 medium containing trehalose, (d) BG-11 medium containing 100mM glucose added to BG-11 medium with glucose as a protectant, (e) BG-100mM sucrose added to BG-11 medium with sucrose as a protectant. 11 medium, (f) BG-11 medium containing 10% (v/v) methanol with methanol added to BG-11 medium as a protective agent, and (g) sheep serum added to BG-11 medium as a protective agent. The same procedure as above (3-1-2) was carried out except that BG-11 medium containing 10% (v/v) sheep serum was used. The results are shown in Figure 4. FIG. 4 is a graph (n=3, error bars=SD) showing the survival rate of freeze-dried bacterial cells of outer membrane detachment strains depending on the different protective agents contained in the suspension.

As shown in Figure 4, when (a) BG-11 medium was used as the dispersion liquid, the freeze-dried cells of the outer membrane detachment strain showed a survival rate of 65 ± 8% (approximately 70%). . In addition, when (b) culture supernatant is used as a dispersion liquid, the lyophilized cells of the outer membrane detachment strain have a survival rate of 68 ± 17 (approximately 70%), and c) BG-11 medium containing 100 mM trehalose. When using BG-11, the lyophilized cells of the outer membrane detachment strain had a survival rate of 62 ± 11% (approximately 70%), and these two showed only the same survival rate as (a) BG-11. Ta. Therefore, it was confirmed that neither the intracellular metabolites of the outer membrane detachment strain nor trehalose contained in the culture supernatant are effective protective agents for freeze-drying the outer membrane detachment strain.

Furthermore, when (f) 10% (v/v) methanol-containing BG-11 medium was used as the dispersion liquid, the lyophilized cells of the outer membrane detachment strain had a survival rate of 38 ± 15% (approximately 40%). (g) When using BG-11 medium containing 10% (v/v) sheep serum, the lyophilized cells of the outer membrane detachment strain had a survival rate of 22 ± 17% (approximately 20%), It was confirmed that these two media (a) significantly lowered the freeze-drying resistance of the outer membrane detachment strain than the BG-11 medium. Therefore, it was confirmed that methanol and sheep serum are not effective protective agents for freeze-drying the outer membrane detachment strain.

On the other hand, when (d) 100mM glucose-containing BG-11 medium was used as a dispersant, the freeze-dried cells of the outer membrane detachment strain showed a survival rate of 99±16% (approximately 90%), and (e ) It was confirmed that the survival rate (95 ± 12 (approximately 90%)) was equivalent to that when using BG-11 medium containing 100 mM sucrose. Therefore, glucose was confirmed to be an effective protective agent for freeze-drying outer membrane detachment strains.

(4) Examination of the appropriate bacterial cell concentration for freeze-drying the outer membrane-exfoliating strain Next, we examined the appropriate bacterial cell concentration for freeze-drying the outer membrane-exfoliating strain. In the same manner as in (3-1-1) above, collect cells from the culture solution of the outer membrane detachment strain on the 5th day of culture, and adjust the collected cells to a cell concentration OD ₇₃₀ = 3.7 to 234. A suspension was prepared by suspending the cells in BG11 medium containing 100mM sucrose. After freezing this suspension with liquid nitrogen, the frozen suspension was freeze-dried under the same conditions as in (3-1-1) above to remove water from the suspension. Immediately after freeze-drying, the cells were inoculated into 5 mL of BG-11 medium in a test tube so that the cell concentration was OD ₇₃₀ = 0.1, and the cells were incubated at a light intensity of 100 μmol/m ² /s and at 30°C for 8 days. After shaking culture, the bacterial cell concentration (OD ₇₃₀ value) of the culture solution was measured. FIG. 5 is a graph (n-3 to 4, error bars = SD) showing the relationship between the bacterial cell concentration of the suspension during the freeze-drying process and the growth of the freeze-dried bacterial cells.

As shown in Figure 5, the freeze-dried cells of the outer membrane detachment strain have a cell concentration (OD ₇₃₀ value) at the time of freeze-drying (in other words, the cell concentration (OD ₇₃₀ value) of the suspension subjected to freeze-drying). When the value)) was 60, the absorbance (OD ₇₃₀ value) of the culture medium was the highest. However, if the bacterial cell concentration (OD ₇₃₀ value) of the suspension subjected to freeze-drying is 120 or higher, the absorbance (OD ₇₃₀ value) of the culture solution is lower than 0.6, which indicates poor growth. Ta. In addition, in this case, the absorbance (OD ₇₃₀ value) of the culture solution is 0.2 or more lower than when the bacterial cell concentration (OD ₇₃₀ value) is 60 or less, so the growth of the freeze-dried bacterial cells is significantly reduced. It was confirmed that Generally, if the bacterial cell concentration (OD ₇₃₀ value) of the culture solution is 0.8 or more on the 8th day, it can be said that the growth is good. OD ₇₃₀ value) may be 3.7 or more and 90 or less, more preferably 7.3 or more and 80 or less, still more preferably 7.3 or more and 60 or less.

In addition, the optimal bacterial cell concentration (OD ₇₃₀ value) for freeze-drying is that if the bacterial cell concentration (OD ₇₃₀ value) in the culture solution is 0.9 or higher on the 8th day, growth is quite good. In this example, the bacterial cell concentration of the outer membrane-exfoliating strain in the prepared suspension was OD ₇₃₀ of 7.3 or more and 80 or less, and more preferably, the bacterial cell concentration was optimal for freeze-drying the outer membrane-exfoliating strain. The concentration (OD ₇₃₀ value) was shown to be 7.3 or more and 60 or less. Note that the optimal bacterial cell concentration for freeze-drying may be determined as appropriate by conducting preliminary tests and the like.

(5) Examination of the ratio of the number of sucrose molecules/the number of bacterial cells appropriate for freeze-drying the outer membrane-exfoliating strain Next, we examined the ratio of the number of sucrose molecules/the number of bacterial cells appropriate for the freeze-drying of the outer membrane-exfoliating strain. In the same manner as in (3-1-1) above, cells were collected from the culture solution of the outer membrane detachment strain on the 5th day of culture, and the collected cells were adjusted to a cell concentration OD ₇₃₀ = 7.3. A suspension was prepared by suspending it in BG-11 medium containing 1.6mM to 400mM sucrose. After freezing this suspension with liquid nitrogen, the frozen suspension was freeze-dried under the same conditions as in (3-1-1) above to remove water from the suspension. Immediately after freeze-drying, the cells were inoculated into 5 mL of BG-11 medium in a test tube at a cell concentration of OD ₇₃₀ = 0.1, and incubated at 30℃ for 8 days at a light intensity of 100 μmol/m ² /s. After shaking culture, the bacterial cell concentration (OD ₇₃₀ value) of the culture solution was measured. Figure 6 is a graph (n-3, error bar = SD) showing the relationship between the ratio of the number of sucrose molecules to the number of bacterial cells in the suspension during freeze-drying and the growth of freeze-dried bacterial cells. .

As shown in Figure 6, when the absorbance (OD ₇₃₀ value) of the culture solution of freeze-dried cells of outer membrane detachment strain is 0.8 or higher, growth can be considered to be good. It was confirmed that when the ratio of the number of sucrose molecules to the number of bacterial cells is 10 ⁹ or more, sucrose effectively acts as a protective agent for bacterial cells.

(6) Storage test of freeze-dried bacterial cells at room temperature Next, we investigated whether the freeze-dried bacterial cells of the outer membrane detachment strain could be stored at room temperature for a long period of time. In the same manner as in (3-1-1) above, cells were collected from the culture solution of the outer membrane detachment strain on the 5th day of culture, and the collected cells were added to BG so that the cell concentration was OD ₇₃₀ = 7.3. -11 medium to prepare a suspension without the addition of a protective agent (in this case, sucrose). Similarly, the bacterial cells of the outer membrane detachment strain recovered from the culture solution were suspended in BG-11 medium containing 100 mM sucrose so that the bacterial cell concentration OD ₇₃₀ = 7.3, and the suspension was added with a protective agent (sucrose). A liquid was prepared. After freezing each of these suspensions with liquid nitrogen, each frozen suspension was freeze-dried under the same conditions as in (3-1-1) above to remove water from each suspension. did. After freeze-drying, the cells were stored under vacuum at room temperature, protected from light, and after 0, 55, and 119 days, the cells were placed in a test tube at a cell concentration of OD ₇₃₀ = 0.1. The cells were inoculated into 5 mL of BG-11 medium and cultured with shaking at a light intensity of 100 μmol/m ² /s at 30°C for 8 days, and then the bacterial cell concentration (OD ₇₃₀ value) of the culture solution was measured. FIG. 7 is a graph (average value of n=1 to 3) showing the relationship between the presence or absence of addition of sucrose to the suspension during freeze-drying and the number of days the freeze-dried cells were stored. In Figure 7, the graph indicated by a black triangle indicates the growth of freeze-dried bacterial cells that were freeze-dried by adding sucrose to the suspension, and the graph indicated by a black circle indicates the growth of freeze-dried cells that were freeze-dried by adding sucrose to the suspension. The figure shows the growth of freeze-dried bacterial cells that were freeze-dried without drying.

As shown in Figure 7, the growth of the freeze-dried cells of the outer membrane detachment strain decreased significantly with each storage day, regardless of whether sucrose was added to the suspension. However, by adding sucrose to the suspension as a protective agent, the decline in growth was significantly suppressed compared to when no sucrose was added. From this, it was confirmed that when sucrose is added to the suspension as a protective agent, it is possible to store the open-separated strain in a dry state for a long period of time while maintaining a high survival rate. Therefore, it was shown that sucrose acts as an effective protective agent even when the freeze-dried cells of the outer membrane-exfoliating strain are stored for a long period of time.

(Other embodiments)
Above, the method for producing dried bacterial cells of outer membrane exfoliating modified cyanobacteria and the dried bacterial cells according to the present disclosure have been described based on the embodiments, but the present disclosure is limited to these embodiments. It's not a thing. Unless departing from the spirit of the present disclosure, various modifications to the embodiments that can be thought of by those skilled in the art and other forms constructed by combining some of the constituent elements of the embodiments are also within the scope of the present disclosure. included.

In the above embodiment, in the outer membrane-exfoliating modified cyanobacteria, the total amount of proteins involved in binding between the outer membrane and the cell wall in cyanobacteria is suppressed to 30% or more and 70% or less of the total amount of the proteins in the parent strain. Although an example has been described in which the bond between the outer membrane and the cell membrane is weakened by causing proteins produced within the bacterial cell to leak out of the bacterial cell, the present disclosure is not limited thereto. For example, by applying an external force to cyanobacteria, the bond between the outer membrane and the cell wall may be weakened, or the outer membrane may be weakened. Alternatively, the outer membrane may be weakened by adding an enzyme or a drug to the culture solution of cyanobacteria.

According to the present disclosure, there is provided a method for producing dried bacterial cells capable of preserving membrane-exfoliating modified cyanobacteria in a dry state while maintaining a high survival rate, and dried bacterial cells. Therefore, the modified cyanobacteria with outer membrane exfoliation can be used in good condition.

1 Inner membrane 2 Peptidoglycan 3 Sugar chain 4 Cell wall 5 Outer membrane 6 SLH domain-retaining outer membrane protein 7 SLH domain 8 Organic channel protein 9 Cell wall-pyruvate modifying enzyme

Claims (7)

preparing a suspension containing outer membrane-exfoliating modified cyanobacteria and a saccharide that is at least one of sucrose and glucose;
removing water from the prepared suspension;
Method for producing dried bacterial cells. Furthermore, in the removal of water,
freezing the prepared suspension;
subliming the water in the frozen suspension under reduced pressure;
The method for producing dried bacterial cells according to claim 1. The bacterial cell concentration of the outer membrane-exfoliating modified cyanobacteria in the prepared suspension is 7.3 or more and 80 or less in terms of OD (Optical Density) 730 value.
The method for producing dried bacterial cells according to claim 1. The ratio of the number of molecules of the saccharide to the number of cells of the outer membrane-exfoliating modified cyanobacteria in the prepared suspension is 10 ⁹ or more.
The method for producing dried bacterial cells according to claim 1. Produced by the method for producing dried bacterial cells according to any one of claims 1 to 4,
Dried bacterial cells. comprising a modified cyanobacterium that exfoliates its outer membrane and a sugar that is at least one of sucrose and glucose;
Dried bacterial cells. The ratio of the number of molecules of the saccharide to the number of bacterial cells of the outer membrane-exfoliating modified cyanobacteria is 10 ⁹ or more.
The dried bacterial cells according to claim 6.

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