JP2022134907A - Methods for producing biofilms, biofilms and methods for producing organic materials - Google Patents

Abstract

To provide methods for producing biofilms advantageous for producing organic materials using cyanobacteria.SOLUTION: In a method for producing a biofilm 20, a culture fluid 15 containing a modified cyanobacterium 10 is stored such that a gas-liquid interface 17 to be present between the culture fluid 15 and the air. In addition, the modified cyanobacterium 10 aggregates along the gas-liquid interface 17 as a sheet. In the modified cyanobacterium 10, the total amount of a protein participating the connection between outer membrane 5 and cell wall 4 is suppressed to from 30% to 70% of the total amount of that protein in a parent strain.SELECTED DRAWING: Figure 15

Description

There is an application for the application of Article 30, Paragraph 2 of the Patent Act. Published in publication Publication date: March 5, 2nd year of Reiwa, Publication: 2020 Annual Meeting of the Japan Society for Bioscience, Biotechnology and Agrochemistry 2. Announced via telecommunications line Posting date: March 25, 2020 Posting address: https://www. biorxiv. org/content/10.1101/2020.03.24.006684v1. full

The present disclosure relates to methods of producing biofilms, biofilms, and methods of producing organic matter.

Substance production methods that do not depend on fossil fuels and have a low environmental load are required in a wide range of industrial fields from the chemical industry to the agriculture, aquaculture and livestock industries. Substance production using microorganisms can be performed in an environment of normal temperature and normal pressure, and with the recent development of genetic manipulation technology, it has become possible to produce a wide range of chemical compounds. attention as a system. Among them, photosynthetic microorganisms such as cyanobacteria and algae can use light as an energy source and carbon dioxide (CO ₂ ) in the air as a raw material. .

For example, substances produced using cyanobacteria include ethanol (see Non-Patent Document 1), isobutanol (see Non-Patent Document 2), alkanes (see Patent Document 2), fatty acids (see Patent Document 1), and Proteins (see Non-Patent Document 3) and the like have been reported.

Further, for example, Non-Patent Document 4 discloses a technique for expressing proteins in cyanobacteria.

Japanese Patent No. 6341676 JP 2015-109828 A

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 Shota Atsumi et al., “Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde”, Nature Biotechnology, Nature Publishing Group, 2009, Vol.27, No.12, pp.1177-1180 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 Andrew H. Ng et al., ``Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803'', Applied and Environmental Microbiology, American Society for Microbiology, 2015, Vol.81, pp. .6857-6863 Hikaru Kohata, Studies on molecular basis of cyanobacterial outer membrane function and its evolutionary relationship with primitive chloroplasts, Doctoral thesis,[Online], 2018.03.27,Internet:<URL: http://hdl.handle.net/10097/00122689> Seiji Kojima, Elucidation and application of bacterial-derived membrane stabilization and material permeation mechanisms that function in the chloroplast surface membrane, KAKENHI, [Online], 2018.04.23, Internet:<URL: https://kaken.nii .ac.jp/grant/KAKENHI-PROJECT-18H02117> Jurgens and Weckesser, 1986, J. Bacteriol., 168:568-573 Kojima et al., 2016, Biosci. Biotech. Biochem., 10:1954-1959 Kowata et al., 2017, J. Bacteriol., 199:e00371-17 Kojima et al., 2016, J. Biol. Chem., 291:20198-20209 Mesnage et al., 2000, EMBO J., 19:4473-4484 Yao et al., ACS Synth. Biol., 2016, 5:207-212

However, it is difficult to efficiently produce organic substances such as proteins using cyanobacteria with the above conventional techniques.

Therefore, the present disclosure provides a biofilm production method that is advantageous for the production of organic matter using cyanobacteria.

A method for producing a biofilm according to one aspect of the present disclosure includes:
A culture medium containing a modified cyanobacterium in which the total amount of proteins involved in binding between the outer membrane and the cell wall in cyanobacteria is suppressed to 30% to 70% of the total amount of the proteins in the parent strain is placed between the culture medium and air. storing such that there is a gas-liquid interface of
aggregating the modified cyanobacteria into a planar shape along the air-liquid interface.

According to the biofilm production method of the present disclosure, a biofilm can be produced that is advantageous for the production of organic matter using cyanobacteria.

FIG. 1 is a diagram schematically showing the cell surface layer of cyanobacteria. FIG. 2 is a transmission electron microscope observation image of an ultra-thin section of modified cyanobacteria according to an example of the embodiment. FIG. 3 is a diagram including an enlarged image of the dashed line area A in FIG. 2 and a copy thereof. FIG. 4 is a transmission electron microscope image of an ultra-thin section of modified cyanobacteria according to another example of the embodiment. FIG. 5 is a diagram including an enlarged image of the dashed line area B in FIG. 4 and a replica thereof. FIG. 6 is a transmission electron microscope image of an ultra-thin section of modified cyanobacteria according to Reference Example. FIG. 7 is a diagram including an enlarged image of the dashed line area C in FIG. 6 and a copy thereof. FIG. 8 is a graph showing the amount of protein in culture media of modified cyanobacteria of samples 1, 2, and 3; FIG. 9 is an electropherogram showing the amounts of proteins involved in the binding of the outer membrane and the cell wall in the modified cyanobacteria of samples 1, 2, 3, 4, and 5; 10A is a transmission electron micrograph of an ultra-thin section of modified cyanobacteria of sample 4. FIG. FIG. 10B is an enlarged image of the portion enclosed by the dashed line in FIG. 10A. 11A is a transmission electron micrograph of an ultra-thin section of modified cyanobacteria of sample 5. FIG. FIG. 11B is an enlarged image of the portion enclosed by the dashed line in FIG. 11A. FIG. 12 is a graph showing the amount of protein in cultures of modified cyanobacteria of samples 1, 2, 3, 4, and 5; 13 is a graph showing amounts of pyruvic acid covalently bound to cell wall-bound sugar chains of modified cyanobacteria of samples 2 and 3. FIG. FIG. 14 is a diagram showing an example of a biofilm production method. FIG. 15 is a diagram showing another example of a biofilm production method. 16A is a photograph showing the state of the culture solution in Example 1. FIG. 16B is a photograph showing the state of the culture solution in Comparative Example 1. FIG. 17 is a graph showing measurement results of absorption spectra of suspensions of Reference Examples 4 and 5. FIG. 18 is a photograph of suspensions of Reference Examples 4 and 5. FIG. 19 is a cross-sectional view of a culture vessel used in Reference Example 6. FIG. FIG. 20 is a graph showing the relationship between the amount of secreted protein, the floating cell concentration, and the number of culture days in Reference Example 6.

(Findings on which this disclosure is based)
Cyanobacteria, also called cyanobacteria or cyanobacteria, are a group of eubacteria that split water through photosynthesis to produce oxygen and use the energy obtained to fix CO ₂ in the air. Depending on the species, cyanobacteria can also fix nitrogen (N ₂ ) in the air. In addition, cyanobacteria are known to grow quickly and use light efficiently as a characteristic of cyanobacteria. Active research and development is taking place. As described above, examples of substance production using cyanobacteria include ethanol (see Non-Patent Document 1), isobutanol (see Non-Patent Document 2), alkanes (see Patent Document 2), and fatty acids (Patent Document 1). See) and other fuel production has been reported.

Research and development related to the production of substances that serve as nutrients for organisms using cyanobacteria is also being conducted. For example, since proteins can only be synthesized by living organisms, photosynthetic microorganisms that utilize light energy and CO ₂ in the atmosphere are useful as one biological species that can be used to produce proteins easily and efficiently. It is considered.

For example, Non-Patent Document 3 describes a protein production method using the cyanobacterial Synechocystis sp. PCC6803 strain. This document discloses a promoter nucleotide sequence for activating transcription of the ethylene synthase gene with high efficiency and a ribosome binding sequence for enhancing translation.

Non-Patent Document 4 discloses a method of inserting a gene encoding a protein into the NS-pCC2 region of a plasmid carried by this strain when expressing a recombinant protein using the Synechocystis sp. PCC6803 strain as a host. . It has been reported that the expression efficiency of the recombinant protein is improved by about 14 times compared with the case where the gene is inserted onto chromosomal DNA (deoxyribonucleic acid) by using the technique.

However, in the conventional technology described above, it is necessary to disrupt the cells of the PCC6803 strain in order to recover the proteins produced in the cells of the PCC6803 strain. Therefore, it takes time and effort to recover the protein. In addition, since various substances are present in the cells, it may be necessary to remove these substances and purify the target protein. Therefore, protein recovery is low. Moreover, since it is necessary to prepare a new strain each time protein is produced, it takes time and effort, and the production cost also increases. As described above, in the above-described prior art, the efficiency of protein production using cyanobacteria is still at a low level, and the development of techniques with higher production efficiency is desired.

The structure of the cell wall and cell membrane of cyanobacteria affects protein permeability, but it is not easy to artificially modify the cell membrane and cell wall structure to improve the protein secretion production capacity. For example, Non-Patent Documents 5 and 6 disclose that deletion of 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 layer, results in loss of cell proliferation ability. It is stated that

Accordingly, the present inventors diligently studied methods for optimally modifying the structure of cell membranes and cell walls to enhance the ability to produce organic substances such as proteins while maintaining the growth ability of cyanobacterial cells. As a result, by suppressing the total amount of proteins involved in the binding between the outer membrane and the cell wall of cyanobacteria to 30% to 70% of the total amount of the proteins in the parent strain, the organic substances produced in the cells are released outside the cells. It was found that it is easily secreted. By using such modified cyanobacteria, it is possible to efficiently recover organic substances such as extracellularly secreted proteins without disrupting the cells of the cyanobacteria. In addition, since the cyanobacteria can be used continuously even after organic substances such as proteins are recovered, the production efficiency of organic substances tends to be high.

In addition, the present inventors believe that if biofilms containing such modified cyanobacteria can be formed, it will be easier to produce organic matter than when using a culture medium in which the modified cyanobacteria are uniformly dispersed. I wondered. Therefore, we have made intensive investigations into methods capable of forming biofilms containing modified cyanobacteria, and have finally found the method.

Therefore, according to the present disclosure, there is provided a biofilm production method that is advantageous for producing organic matter using cyanobacteria. The present disclosure also provides such biofilms. Furthermore, the present disclosure provides methods for producing organic matter using such biofilms.

(Summary of this disclosure)
A summary of one aspect of the disclosure follows.

In the method for producing a biofilm according to the first aspect of the present disclosure, the total amount of proteins involved in binding between the outer membrane and the cell wall in cyanobacteria is suppressed from 30% to 70% of the total amount of the proteins in the parent strain. Storing a culture solution containing cyanobacteria such that an air-liquid interface exists between the culture solution and air; and aggregating the modified cyanobacteria into a planar shape along the air-liquid interface. include.

As a result, in the modified cyanobacteria, the binding between the cell wall and the outer membrane is partially reduced, and the outer membrane is more likely to partially detach from the cell wall, without impairing the ability of the cells to proliferate. Binding means binding amount and binding strength. Therefore, organic substances such as proteins produced in the cells tend to leak out of the outer membrane, that is, out of the cells. Therefore, according to the modified cyanobacteria according to the first aspect, it is possible to provide cyanobacteria with improved secretion productivity of organic substances such as proteins. In addition, according to the modified cyanobacteria according to the first aspect, it is not necessary to crush the cells to recover organic matter such as proteins. can be made If the total amount of proteins involved in the binding of the outer membrane to the cell wall is suppressed to less than 30% of the total amount of these proteins in the parent strain, the cell's ability to proliferate will be impaired. In addition, when the total amount of proteins involved in binding between the outer membrane and the cell wall exceeds 70% of the total amount of the proteins in the parent strain, the proteins produced in the cells cannot leak out of the cells.

Additionally, according to the first aspect, biofilms comprising modified cyanobacteria can be produced. Such biofilms are more likely to produce organic matter than when using a culture solution in which modified cyanobacteria are uniformly dispersed. When producing an organic matter using a culture medium in which modified cyanobacteria are uniformly dispersed, agitation and aeration are required, and cell density monitoring in the culture medium is also necessary. In addition, a separate container in contact with the container containing the culture solution is required in order to recover the organic matter from the culture solution, and the cleaning of the container is complicated. Therefore, it is difficult to scale up the method of producing an organic substance using a culture solution in which modified cyanobacteria are uniformly dispersed. On the other hand, when producing an organic substance using a biofilm produced by the method for producing a biofilm according to the first aspect, the above disadvantages of producing an organic substance using a culture solution in which modified cyanobacteria are uniformly dispersed is less likely to occur. Therefore, the biofilm produced by the method for producing a biofilm according to the first aspect is easy to scale up, which is advantageous for producing organic substances using cyanobacteria.

In the second aspect of the present disclosure, in the method for producing a biofilm according to the first aspect, when the modified cyanobacteria are planarly aggregated along the air-liquid interface, the culture solution is irradiated with light. may Thereby, a biofilm containing the modified cyanobacteria can be produced while culturing the modified cyanobacteria.

In the third aspect of the present disclosure, in the method for producing a biofilm according to the first aspect or the second aspect, when the modified cyanobacteria are planarly aggregated along the air-liquid interface, the modified The cyanobacteria may be statically cultured. This makes it easier for the modified cyanobacteria to aggregate in a plane along the gas-liquid interface.

In the fourth aspect of the present disclosure, in the biofilm production method according to any one aspect of the first to third aspects, the pH of the culture solution is, for example, 6.5 or more and 8.4 or less. good. This makes it easier for the modified cyanobacteria to aggregate in a plane along the gas-liquid interface.

In the fifth aspect of the present disclosure, in the method for producing a biofilm according to any one of the first to fourth aspects, the protein is a Surface Layer Homology (SLH) domain-retaining outer membrane protein, and a cell wall - at least one pyruvate modifying enzyme. A cell wall-pyruvate-modifying enzyme is an enzyme that catalyzes a reaction that modifies sugar chains bound to the cell wall surface with pyruvate. As a result, in the modified cyanobacteria, for example, at least one function of an SLH domain-retaining outer membrane protein that binds to the cell wall and an enzyme that catalyzes a reaction that catalyzes pyruvic acid modification of sugar chains bound to the surface of the cell wall is suppressed. Alternatively, expression of at least one of an SLH domain-retaining outer membrane protein and a cell wall-pyruvate modifying enzyme is suppressed. Therefore, the binding between the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane and the covalent sugar chain on the surface of the cell wall is reduced. Binding means binding amount and binding strength. This makes it easier for the outer membrane to detach from the cell wall at the portion where the bond between the outer membrane and the cell wall is weakened. Therefore, organic substances such as proteins produced within the cells are likely to leak out of the cells.

The biofilm according to the sixth aspect of the present disclosure is a modified cyanobacterium in which the total amount of proteins involved in binding the outer membrane and the cell wall in cyanobacteria is suppressed to 30% to 70% of the total amount of the proteins in the parent strain. include.

In a seventh aspect of the present disclosure, the biofilm according to the sixth aspect may be arranged along an air-liquid interface between the culture medium and the air.

In the method for producing an organic substance according to the eighth aspect of the present disclosure, the total amount of proteins involved in binding between the outer membrane and the cell wall in cyanobacteria is suppressed from 30% to 70% of the total amount of the proteins in the parent strain. Producing organic matter in said modified cyanobacteria in said modified cyanobacteria of a biofilm containing bacteria and secreting said organic matter to the outside of said modified cyanobacteria.

In the ninth aspect of the present disclosure, the biofilm may be arranged along an air-liquid interface between the medium and the air.

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

It should be noted that the embodiments described below are all comprehensive or specific examples. Numerical values, materials, steps, order of steps, and the like shown in the following embodiments are examples and are not intended to limit the present disclosure. In addition, among the constituent elements in the following embodiments, constituent elements that are not described in independent claims representing the highest concept will be described as arbitrary constituent elements.

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

In addition, hereinafter, the numerical range does not represent only a strict meaning, but a substantially equivalent range, such as those specified by measuring the amount or range such as the number and concentration of proteins include.

Also, in this specification, both fungal bodies and cells represent a single cyanobacterial individual.

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

Cyanobacteria, also called cyanobacteria or cyanobacteria, are a group of prokaryotic organisms that perform photosynthesis while generating oxygen by electrolyzing water with energy obtained by capturing light energy with chlorophyll. Cyanobacteria are rich in diversity, and in terms of cell shape, for example, there are unicellular species such as Synechocystis sp. PCC 6803 and filamentous species such as Anabaena sp. As for habitat, there are thermophilic species such as Thermosynechococcus elongatus, marine species such as Synechococcus elongatus, and freshwater species such as Synechocystis. Other species, such as Microcystis aeruginosa, which have gas vesicles and produce toxins, and Gloeobacter violaceus, which lacks thylakoids but have proteins called phycobilisomes, which are light-harvesting antennas in the plasma membrane, have unique characteristics. Many species are also included.

FIG. 1 is a diagram schematically showing the cell surface layer of cyanobacteria. As shown in FIG. 1, the cell surface layer of cyanobacteria is composed of plasma membrane 1, peptidoglycan 2, and outer membrane 5 in order from the inside. The plasma membrane 1 is also called the inner membrane 1 . Outer membrane 5 is a lipid membrane that forms the outermost layer of the cell. Peptidoglycan 2 is covalently bound to sugar chain 3 composed of glucosamine, mannosamine, and the like. Pyruvate is bound to these covalent sugar chains 3 (see Non-Patent Document 7). In the present specification, the cell wall 4 including the peptidoglycan 2 and the covalent sugar chain 3 is referred to. The space between the plasma membrane 1, that is, the inner membrane 1, and the outer membrane 5 is called the periplasm. Various enzymes involved in events such as protein degradation or conformation formation, lipid or nucleic acid degradation, or extracellular nutrient uptake are present in this space.

SLH domain-retaining outer membrane proteins such as Slr1841 shown in FIG. 1 consist of a C-terminal region embedded in the lipid membrane, which is the outer membrane 5, and an N-terminal SLH domain 7 protruding from the lipid membrane. This SLH domain-retaining outer membrane protein is widely distributed in bacteria belonging to the Negativicutes class, which is a group of cyanobacteria and Gram-negative bacteria (see Non-Patent Document 8). The region embedded in the lipid membrane, which is the outer membrane 5, forms a channel to allow hydrophilic substances to permeate the outer membrane. On the other hand, SLH domain 7 has a function of binding to cell wall 4 in this region (see Non-Patent Document 9). In order for SLH domain 7 to bind to cell wall 4, covalent sugar chain 3 in peptidoglycan 2 must be modified with pyruvate (see Non-Patent Document 10). Examples of genes encoding SLH domain-retaining outer membrane protein 6 include slr1841 or slr1908 retained by Synechocystis sp. PCC 6803, and oprB retained by Anabaena sp.

An enzyme that catalyzes the pyruvate modification reaction of the covalent sugar chain 3 in peptidoglycan 2 is hereinafter referred to as cell wall-pyruvate modification enzyme 9 . A cell wall-pyruvate modifying enzyme 9 was identified in the Gram-positive bacterium Bacillus anthracis and named CsaB (see Non-Patent Document 11). Among cyanobacteria whose genome nucleotide sequences have been published, many species possess genes encoding homologous proteins having an amino acid sequence identity of 30% or more with CsaB. Examples of such genes include slr0688 retained by Synechocystis sp. PCC 6803 and synpcc7502_03092 retained by Synechococcus sp. 7502.

In cyanobacteria, CO ₂ fixed by photosynthesis is converted into various amino acids through multistep enzymatic reactions. Using these amino acids as raw materials, proteins are synthesized in 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 cyanobacteria that actively secrete proteins outside the cell have been reported so far.

Since cyanobacteria have high photosynthetic ability, they do not necessarily need to take in organic matter from the outside as nutrients. Therefore, cyanobacteria have very few channel proteins permeable to organic matter in the outer membrane 5, such as the organic matter channel protein 8 such as Slr1270 shown in FIG. For example, in Synechocystis sp. PCC 6803, organic matter channel protein 8, which allows organic matter to permeate, is present in only about 4% of the total protein content of outer membrane 5. On the other hand, cyanobacteria allow only inorganic ions to permeate, like SLH domain-retaining outer membrane protein 6 such as Slr1841 shown in Fig. 1, in order to take in inorganic ions necessary for growth into cells with high efficiency. The outer membrane 5 has many ion channel proteins. For example, in Synechocystis sp. PCC 6803, ion channel proteins permeable to inorganic ions account for approximately 80% of the total protein content of outer membrane 5 .

In this way, cyanobacteria have very few channels through which organic substances such as proteins permeate in the outer membrane 5, so it is considered difficult to positively secrete organic substances produced inside the cells to the outside of the cells. In addition, although the structure of the cell wall and cell membrane of cyanobacteria affects protein permeability, it is not easy to artificially modify the cell membrane and cell wall structure to improve the protein secretion production capacity. For example, in Non-Patent Documents 5 and 6, deletion of 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 layer, increases the cell proliferation ability. stated to be lost.

[2. modified cyanobacteria]
The modified cyanobacteria according to this embodiment will be described with reference to FIG.

In the modified cyanobacteria according to the present embodiment, the total amount of proteins involved in binding between the outer membrane 5 and the cell wall 4 in cyanobacteria is suppressed to 30 to 70% of the total amount of the proteins in the parent strain. This protein is hereinafter also referred to as binding-associated protein. Here, for example, "the total amount of the binding-related protein is suppressed to 30% of the total amount of the protein in the parent strain" means that the protein corresponding to 30% to 70% of the total amount of the protein in the parent strain is lost. , means a state in which protein corresponding to 30% to 70% of the total amount remains. As a result, in the modified cyanobacteria, the binding between the outer membrane 5 and the cell wall 4 is partially reduced, so that the outer membrane 5 becomes easier to partially detach from the cell wall 4 . The level of binding can be specified, for example, by the amount and strength of binding. In the modified cyanobacteria, the productivity of secretion of organic substances such as proteins produced in the cells is improved. In addition, since it is not necessary to crush the cells to recover the organic matter, the modified cyanobacteria can be repeatedly used to produce the organic matter even after the organic matter such as proteins has been recovered. In the present specification, production by a modified cyanobacterium to produce a protein inside the cell is referred to as production, and secretion of an organic substance such as the produced protein to the outside of the cell is referred to as secretory production.

The protein involved in binding between the outer membrane 5 and the cell wall 4 may be at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9, for example. In the present embodiment, in the modified cyanobacterium, for example, the function of at least one of SLH domain-retaining outer membrane protein 6 and cell wall-pyruvate modifying enzyme 9 is suppressed. For example, in modified cyanobacteria, at least one function of SLH domain-retaining outer membrane protein 6 and cell wall-pyruvate modifying enzyme 9 may be suppressed. Alternatively, at least one of the expression of the SLH domain-retaining outer membrane protein 6 that binds to the cell wall 4 and the expression of the cell wall-pyruvate modifying enzyme 9 may be suppressed. As a result, 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 amount and strength of the bond is reduced. Therefore, the outer membrane 5 is easily detached from the cell wall 4 at the portion where these bonds are weakened. Partial detachment of the outer membrane 5 from the cell wall 4 facilitates the leakage of organic matter such as proteins present in the cell of the modified cyanobacterium, particularly in the periplasm, to the outside of the outer membrane 5 outside the cell.

As described above, cyanobacteria have high photosynthetic ability, so they do not necessarily need to take in organic matter from the outside as nutrients. Therefore, cyanobacteria need only be cultured with light, air, water, and a small amount of inorganic substances. produce. In particular, organic substances such as various proteins are present in the periplasm of 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 outer membrane 5 and cell wall 4 are suppressed. As a result, the outer membrane 5 becomes easier to partially peel off from the cell wall 4 . Organic matter such as proteins in the periplasm leaks into the culture solution from the exfoliated portion of the outer membrane 5 . As a result, the modified cyanobacteria have a high protein secretion productivity that secretes intracellularly produced proteins to the outside of the cells.

Subsequently, the outer membrane 5 is partially detached 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. cyanobacteria will be described more specifically.

The type of the parent strain, which is the parent microorganism of the modified cyanobacterium according to the present embodiment, is not limited to a specific type, and may be any type of cyanobacterium. The parental strain is a cyanobacterium before at least one of expression of SLH domain-retaining outer membrane protein 6 and expression of cell wall-pyruvate modifying enzyme 9 is repressed. Parental strains are also referred to herein as parental cyanobacteria. The parent cyanobacterium may be of the genera Syenechocystis, Synechococcus, Anabaena, or Thermosynechococcus. Among these, the parent cyanobacterium may be Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, or Thermosynechococcus elongatus BP-1. The parental strain can be wild or modified, as long as it is a cyanobacterium prior to suppressing the total amount of binding-associated proteins from 30% to 70%, and has binding associations equivalent to those in the wild. It may have protein.

The amino acid sequences of SLH domain-retaining outer membrane protein 6 and cell wall-pyruvate modifying enzyme 9 in these parent cyanobacteria can be found in the NCBI database and Cyanobase mentioned above. In addition, the nucleotide sequences of genes encoding these binding-related proteins and the locations of the genes on chromosomal DNA or plasmids can also be confirmed with the above-mentioned NCBI database and Cyanobase.

The SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 whose functions are suppressed in the modified cyanobacteria may be of any parent cyanobacterium as long as they are possessed by the parent cyanobacterium. good too. SLH domain-retaining outer membrane protein 6 and cell wall-pyruvate modifying enzyme 9, whose functions are suppressed in modified cyanobacteria, are not restricted by the location of genes encoding them, for example, on chromosomal DNA or plasmids. .

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

More specifically, the SLH domain-retaining outer membrane protein 6 is, for example, Synechocystis sp. PCC 6803 Slr1841 (SEQ ID NO: 1), Synechococcus sp. Anacy_3458 (SEQ ID NO: 3), etc. may be used. Moreover, the SLH domain-retaining outer membrane protein 6 may be a protein having 50% or more of the same amino acid sequence as these SLH domain-retaining outer membrane proteins.

In the modified cyanobacteria, for example, any SLH domain-retaining outer membrane protein 6 shown in SEQ ID NOs: 1 to 3 above or any of these SLH domain-retaining outer membrane proteins 6 has an amino acid sequence identical to 50% or more. The function of the protein may be suppressed. Alternatively, in modified cyanobacteria, the amino acid sequence is 50% or more identical to 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. The expression of the protein may be suppressed. Therefore, in the modified cyanobacteria, the function of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 or a protein having a function equivalent to that of the SLH domain-retaining outer membrane protein 6 is suppressed. Alternatively, the expression level of the SLH domain-retaining outer membrane protein 6 or a protein having a function equivalent to that of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 is decreased. Therefore, in the modified cyanobacterium according to the present embodiment, the binding amount and binding strength of the binding domain for binding the outer membrane 5 to the cell wall 4, such as the SLH domain 7, with the cell wall 4 are reduced. tends to partially detach from the cell wall 4.

In general, if the amino acid sequences of different kinds of proteins are 30% or more identical, it is said that there is a high degree of homology in the three-dimensional structures of these proteins, and that these proteins are highly likely to have the same functions as each other. It is Therefore, the SLH domain-retaining outer membrane protein 6 whose function is suppressed has, for example, 40% or more identity with any of the amino acid sequences of the SLH domain-retaining outer membrane proteins shown in SEQ ID NOS: 1 to 3 above. has an amino acid sequence with In addition, the SLH domain-retaining outer membrane protein 6 can be a protein or polypeptide that has a function of binding to the sugar chain 3 on the cell wall 4 . The identity of the amino acid sequence of SLH domain-retaining outer membrane protein 6 with any of the amino acid sequences of SLH domain-retaining outer membrane proteins shown in SEQ ID NOs: 1 to 3 above is desirably 50% or more. The identity is more preferably 60% or higher, still more preferably 70% or higher, particularly preferably 80% or higher, particularly preferably 90% or higher. Amino acid sequence identity can be evaluated by the ratio of the number of identical amino acid sequences to the total number of amino acid sequences.

Further, for example, when the parent cyanobacterium belongs to the genus Synenchocystis, the cell wall-pyruvate modifying enzyme 9 may be Slr0688 or the like. When the parent cyanobacterium is of the genus Synechococcus, the cell wall-pyruvate modifying enzyme 9 may be Syn7502_03092, Synpcc7942_1529, or the like. When the parent cyanobacterium is of the genus Anabaena, the cell wall-pyruvate modifying enzyme 9 may be ANA_C20348, Anacy_1623, or the like. When the parent cyanobacterium is of the genus Microcystis, the cell wall-pyruvate modifying enzyme 9 may be CsaB (NCBI Accession ID: TRU80220) or the like. When the parent cyanobacterium is of the genus Cyanothese, the cell wall-pyruvate modifying enzyme 9 may be CsaB (NCBI access ID: WP_107667006.1) or the like. When the parent cyanobacterium is of the genus Spirulina, the cell wall-pyruvate modifying enzyme 9 may be CsaB (NCBI access ID: WP_026079530.1) or the like. When the parent cyanobacterium is of the genus Calothrix, the cell wall-pyruvate modifying enzyme 9 may be CsaB (NCBI Access ID: WP_096658142.1) or the like. When the parent cyanobacterium is of the genus Nostoc, the cell wall-pyruvate modifying enzyme 9 may be CsaB (NCBI access ID: WP_099068528.1) or the like. When the parent cyanobacterium is of the genus Crocosphaera, the cell wall-pyruvate modifying enzyme 9 may be CsaB (NCBI access ID: WP_012361697.1) or the like. When the parent cyanobacterium is of the genus Pleurocapsa, the cell wall-pyruvate modifying enzyme 9 may be CsaB (NCBI access ID: WP_036798735) or the like.

More specifically, the cell wall-pyruvate modifying enzyme 9 is Slr0688 (SEQ ID NO: 4) of Synechocystis sp. PCC 6803, Synpcc7942_1529 (SEQ ID NO: 5) of Synechococcus sp. PCC 7942, or Anacy_1623 of Anabaena cylindrica PCC 7122 ( Sequence number 6) etc. may be sufficient. Moreover, the cell wall-pyruvate modifying enzyme 9 may be a protein whose amino acid sequence is 50% or more identical to that of these cell wall-pyruvate modifying enzymes.

In modified cyanobacteria, for example, a protein whose amino acid sequence is 50% or more identical to any of the cell wall-pyruvate modifying enzymes shown in SEQ ID NOS: 4 to 6 or any of these cell wall-pyruvate modifying enzymes 9 function can be inhibited. Alternatively, in the modified cyanobacteria, any of the cell wall-pyruvate modifying enzymes shown in SEQ ID NOs: 4 to 6 above, or any of these cell wall-pyruvate modifying enzymes and proteins whose amino acid sequences are 50% or more identical Expression can be suppressed. Therefore, in modified cyanobacteria, the function of cell wall-pyruvate modifying enzyme 9 or a protein having a function equivalent to this enzyme can be suppressed. Alternatively, in the modified cyanobacteria, the expression level of cell wall-pyruvate modifying enzyme 9 or a protein having a function equivalent to this enzyme can be reduced. As a result, the covalent sugar chain 3 on the surface of the cell wall 4 is less likely to be modified with pyruvic acid, so that the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 and the sugar chain 3 on the cell wall 4 are The binding amount and binding strength are reduced. Therefore, in the modified cyanobacterium according to the present embodiment, the covalent sugar chains 3 on the surface of the cell wall 4 are less likely to be modified with pyruvic acid. 5 becomes easier to partially detach from the cell wall 4.

In addition, as described above, it is said that if different proteins have 30% or more of the same amino acid sequence, the proteins are highly likely to have equivalent functions. Therefore, the cell wall-pyruvate modifying enzyme 9 whose function is suppressed is, for example, an amino acid having 40% or more identity with any of the amino acid sequences of the cell wall-pyruvate modifying enzymes shown in SEQ ID NOs: 4 to 6 above. Contains arrays. In addition, the cell wall-pyruvate modifying enzyme 9 is a protein or polypeptide that has the function of catalyzing the reaction of modifying the covalent sugar chain 3 of the peptidoglycan 2 on the cell wall 4 with pyruvate. The identity between the amino acid sequence of cell wall-pyruvate modifying enzyme 9 and the amino acid sequence of any of the cell wall-pyruvate modifying enzymes shown in SEQ ID NOS: 4 to 6 above is desirably 50% or more. The identity is more preferably 60% or higher, still more preferably 70% or higher, particularly preferably 80% or higher, particularly preferably 90% or higher.

Suppression of the function of the SLH domain-retaining outer membrane protein 6 includes suppression of the binding ability of the protein to the cell wall 4, suppression of transport of the protein to the outer membrane 5, or embedding of the protein in the outer membrane 5. include inhibiting the ability to function as

Suppression of the function of the cell wall-pyruvate modifying enzyme 9 means suppression of the function of the protein to modify the covalently bound sugar chain 3 of the cell wall 4 with pyruvate.

Means for inhibiting the functions of these proteins are not limited to specific means as long as the protein functions can be inhibited. The means is, for example, to delete or inactivate the gene encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9 . The means may inhibit the transcription of these genes, may inhibit the translation of transcripts of these genes, or may be inhibitors that specifically inhibit these proteins. may be administered, or by other means.

In the present embodiment, the modified cyanobacterium may have deleted or inactivated genes that express proteins involved in binding between the outer membrane 5 and the cell wall 4 . As a result, in the modified cyanobacteria, the expression of proteins involved in binding between the cell wall 4 and the outer membrane 5 is suppressed, or the functions of the proteins are suppressed. As a result, the bond between the cell wall 4 and the outer membrane 5, that is, the amount and strength of bond, is partially reduced. As a result, in the modified cyanobacteria, the outer membrane 5 tends to partially detach from the cell wall 4, so that organic substances such as proteins produced in the cells tend to leak out of the outer membrane 5, that is, outside the cells. . Therefore, the modified cyanobacterium according to the present embodiment improves the secretory productivity of organic substances produced in the cells to the outside of the cells. In addition, since it is not necessary to crush the cells to recover organic matter such as proteins, the modified cyanobacteria can be repeatedly used to produce organic matter even after the organic matter has been recovered.

The gene that expresses the protein involved in the binding of 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, for example, at least one of the gene encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9 is deleted or inactivated. Therefore, in modified cyanobacteria, for example, expression of at least one of SLH domain-retaining outer membrane protein 6 and cell wall-pyruvate modifying enzyme 9 is suppressed. Alternatively, in modified cyanobacteria, at least one function of SLH domain-retaining outer membrane protein 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 covalently bound sugar chain 3 on the surface of the cell wall 4, that is, the amount and strength of binding decreases. 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 cyanobacterium according to the present embodiment, since the bond between the outer membrane 5 and the cell wall 4 is reduced, the outer membrane 5 becomes easier to partially detach from the cell wall 4. proteins easily leak out of the cells.

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

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

More specifically, the gene encoding SLH domain-retaining outer membrane protein 6 is Synechocystis sp. PCC 6803 slr1841 (SEQ ID NO: 7), Synechococcus sp. 7122 anacy_3458 (SEQ ID NO: 9), or genes having 50% or more identical amino acid sequence with these genes.

Thus, in modified cyanobacteria, for example, genes encoding any of the SLH domain-retaining outer membrane proteins shown in SEQ ID NOs: 7 to 9 above, or nucleotide sequences that are 50% or more identical to the nucleotide sequences of any of these genes. A gene is deleted or inactivated. Therefore, in modified cyanobacteria, the expression of any of the above SLH domain-retaining outer membrane proteins or proteins having functions equivalent to any of these proteins is suppressed. Alternatively, the functions of any of the above SLH domain-retaining outer membrane proteins or proteins having functions equivalent to any of these proteins are suppressed. Therefore, in the modified cyanobacterium according to the present embodiment, the binding amount and binding strength of the binding domain for binding the outer membrane 5 to the cell wall 4, such as the SLH domain 7, to the cell wall 4 are reduced. 5 becomes easier to partially detach from the cell wall 4.

As described above, it is said that if the amino acid sequence of a protein is 30% or more identical, it is highly likely that the protein has a function equivalent to that of the protein. Therefore, if the base sequences of genes encoding proteins are 30% or more identical, it is highly likely that proteins having functions equivalent to those of the proteins will be expressed. Therefore, the gene encoding the SLH domain-retaining outer membrane protein 6 whose function is suppressed is, for example, any of the nucleotide sequences of the genes encoding the SLH domain-retaining outer membrane proteins shown in SEQ ID NOs: 7 to 9 above. and base sequences having 40% or more identity. In addition, the gene encodes a protein or polypeptide that has the function of binding to the covalent sugar chain 3 on the cell wall 4 . The nucleotide sequence of the gene encoding the SLH domain-retaining outer membrane protein 6 whose function is suppressed and the nucleotide sequence of any of the genes encoding the SLH domain-retaining outer membrane proteins shown in SEQ ID NOs: 7 to 9 above The identity of is desirably 50% or more. The identity is more preferably 60% or higher, still more preferably 70% or higher, particularly preferably 80% or higher, particularly preferably 90% or higher.

For example, when the parent cyanobacterium belongs to the genus Synchocystis, the gene encoding cell wall-pyruvate modifying enzyme 9 may be slr0688 or the like. When the parent cyanobacterium belongs to the genus Synechococcus, the gene encoding cell wall-pyruvate modifying enzyme 9 may be syn7502_03092, synpcc7942_1529, or the like. When the parent cyanobacterium is of the genus Anabaena, the gene encoding cell wall-pyruvate modifying enzyme 9 may be ana_C20348, anacy_1623, or the like. When the parent cyanobacterium belongs to the genus Microcystis, the gene encoding cell wall-pyruvate modifying enzyme 9 may be csaB (NCBI Accession ID: TRU80220) or the like. When the parent cyanobacterium belongs to the genus Cynahothese, the gene encoding cell wall-pyruvate modifying enzyme 9 may be csaB (NCBI Accession ID: WP_107667006.1) or the like. When the parent cyanobacterium is of the genus Spirulina, the gene encoding cell wall-pyruvate modifying enzyme 9 may be csaB (NCBI Accession ID: WP_026079530.1) or the like. When the parent cyanobacterium belongs to the genus Calothrix, the gene encoding cell wall-pyruvate modifying enzyme 9 may be csaB (NCBI accession ID: WP_096658142.1) or the like. When the parent cyanobacterium belongs to the genus Nostoc, the gene encoding cell wall-pyruvate modifying enzyme 9 may be csaB (NCBI access ID: WP_099068528.1) or the like. When the parent cyanobacterium is the genus Crocosphaera, the gene encoding cell wall-pyruvate modifying enzyme 9 may be csaB (NCBI Accession ID: WP_012361697.1) or the like. When the parent cyanobacterium is of the genus Pleurocapsa, the gene encoding cell wall-pyruvate modifying enzyme 9 may be csaB (NCBI Accession ID: WP_036798735) or the like. The nucleotide sequences of these genes can be obtained from the NCBI database or Cyanobase mentioned above.

More specifically, the gene encoding cell wall-pyruvate modifying enzyme 9 is Synechocystis sp. PCC 6803 slr0688 (SEQ ID NO: 10), Synechococcus sp. PCC 7942 synpcc7942_1529 (SEQ ID NO: 11), or Anabaena cylindrica PCC 7122 anacy_1623 (SEQ ID NO: 12). Also, the gene encoding the cell wall-pyruvate modifying enzyme 9 may be a gene having 50% or more of the same base sequence as these genes.

In the modified cyanobacteria, for example, any gene encoding cell wall-pyruvate modifying enzyme 9 shown in SEQ ID NOs: 10 to 12 above is deleted or inactivated. Alternatively, in modified cyanobacteria, for example, genes whose base sequences are 50% or more identical to genes encoding any of these enzymes are deleted or inactivated. Therefore, in the modified cyanobacteria, expression of any of the cell wall-pyruvate modifying enzymes 9 or proteins having functions equivalent to any of these enzymes is suppressed. Alternatively, the function of any of the above cell wall-pyruvate modifying enzymes 9 or proteins having functions equivalent to any of these enzymes is inhibited. As a result, the covalent sugar chain 3 on the surface of the cell wall 4 is less likely to be modified with pyruvic acid, so that the sugar chain 3 on the cell wall 4 becomes the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5. The amount of binding and binding strength associated with binding are reduced. Therefore, in the modified cyanobacterium according to the present embodiment, the amount of pyruvic acid modification of the sugar chain 3 for binding the cell wall 4 to the outer membrane 5 is reduced. is weakened, and the outer membrane 5 tends to partially detach from the cell wall 4 .

If the base sequences of genes encoding different proteins are 30% or more identical, it is highly likely that proteins having equivalent functions will be expressed. The gene encoding the cell wall-pyruvate modifying enzyme 9 whose function is suppressed is, for example, any of the nucleotide sequences of the genes encoding the cell wall-pyruvate modifying enzymes shown in SEQ ID NOS: 10 to 12 above and 40% or more contains a base sequence having the identity of In addition, the gene encodes a protein or polypeptide that has the function of catalyzing the reaction of modifying the covalent sugar chain 3 of the peptidoglycan 2 on the cell wall 4 with pyruvate. The identity of the nucleotide sequence of the gene encoding the cell wall-pyruvate modifying enzyme 9 with any of the nucleotide sequences of the genes encoding the cell wall-pyruvate modifying enzymes shown in SEQ ID NOS: 10 to 12 above is preferably 50% or more. The identity is more preferably 60% or higher, still more preferably 70% or higher, particularly preferably 80% or higher, particularly preferably 90% or higher.

[3. Method for Producing Modified Cyanobacteria]
A method for producing a modified cyanobacterium according to this embodiment will be described. A method for producing a modified cyanobacterium includes, for example, a step of suppressing the function of a protein involved in binding between the outer membrane 5 and the cell wall 4 in cyanobacteria.

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

Means for inhibiting protein function are not limited to specific means. For example, the means include deleting or inactivating the gene encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9 . The means may involve inhibiting transcription of these genes, inhibiting translation of transcripts of these genes, or administering inhibitors that specifically inhibit these proteins.

A means of deleting or inactivating the above gene is, for example, introduction of mutation to one or more bases on the base sequence of the gene. The means may be replacement of the base sequence with another base sequence, insertion of another base sequence, deletion of part or all of the base sequence of the gene, or the like.

A means of inhibiting the transcription of the above gene is, for example, mutation introduction to the promoter region of the gene. The means may be inactivation of the promoter by substitution with or insertion of another base sequence, CRISPR interference method (see Non-Patent Document 12), or the like. Specific techniques for the introduction of mutation or substitution or insertion of base sequences may be, for example, UV irradiation, site-directed mutagenesis, homologous recombination, or the like.

Means for inhibiting translation of the transcription product of the gene may be, for example, ribonucleic acid (RNA) interference method or the like.

A modified cyanobacterium may be produced by suppressing the function of a protein involved in binding between the outer membrane 5 and the cell wall 4 in cyanobacteria by using any one of the above means.

As a result, in the modified cyanobacterium produced by the production method according to the present embodiment, the binding between the cell wall 4 and the outer membrane 5, that is, the amount of binding and the binding strength are partially reduced. It becomes easier to partially detach from 4. Therefore, in the modified cyanobacteria, organic substances such as proteins produced within the cells tend to leak out of the outer membrane 5, that is, out of the cells. Therefore, according to the method for producing a modified cyanobacterium according to the present embodiment, it is possible to provide a modified cyanobacterium with improved secretion productivity of organic matter.

In addition, in the modified cyanobacteria produced by the production method according to the present embodiment, organic substances such as proteins produced in the cells leak out of the cells, so it is necessary to crush the cells in order to recover the organic substances. There is no For example, modified cyanobacteria may be cultured under appropriate conditions, and then the organic matter secreted into the culture medium may be collected. Therefore, it is possible to collect the organic matter in the culture medium while culturing the modified cyanobacteria. Therefore, by using the modified cyanobacteria obtained by this production method, organic matter production such as microbiological protein production can be carried out with high efficiency. 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 organic substances such as proteins are recovered.

The modified cyanobacteria produced by the method for producing modified cyanobacteria according to the present embodiment extracellularly secretes, for example, peptidases, phosphatases, and other organic substances originally present mainly in the periplasm. In the present embodiment, for example, genes encoding proteins originally produced in cyanobacterial cells, such as proteins present in the periplasm, may be modified and replaced with genes encoding other proteins. . This also allows the modified cyanobacterium to produce the desired protein. Therefore, according to the method for producing a modified cyanobacterium according to the present embodiment, it is possible to provide a modified cyanobacterium capable of easily and efficiently producing an organic substance such as a desired protein.

[4. Production Example of Modified Cyanobacteria]
Examples of production of modified cyanobacteria are shown below. Sample 1 is obtained by suppressing the expression of the slr1841 gene, which encodes an SLH domain-retaining outer membrane protein, as a method for partially detaching the outer membrane of cyanobacteria from the cell wall. In addition, sample 2 is obtained by adopting expression suppression of the slr0688 gene, which encodes a cell wall-pyruvate modifying enzyme, as a method for partially detaching the outer membrane of cyanobacteria from the cell wall. The cyanobacterial species used to prepare Samples 1 and 2 was Synechocystis sp. PCC 6803, and this species is sometimes referred to simply as "cyanobacteria" in this section.

(Sample 1)
In the modified cyanobacteria of sample 1, the expression of the slr1841 gene, which encodes an SLH domain-retaining outer membrane protein, is suppressed.

(1) Construction of Cyanobacterial Modified Strain with Suppressed Expression of slr1841 Gene Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) interference method was used as a method for suppressing gene expression. In this method, the expression of the slr1841 gene can be suppressed by introducing the dCas9 gene, which is a gene encoding the dCas9 protein, and the slr1841_sgRNA (single-guide Ribonucleic Acid) gene into the chromosomal DNA of cyanobacteria. In addition, the degree of suppression of the slr1841 gene can be controlled by controlling the transcriptional activity of slr1841_sgRNA.

The mechanism of gene expression suppression by this method is as follows. First, Cas9 protein lacking nuclease activity (dCas9) forms a complex with sgRNA (slr1841_sgRNA) that complementarily binds to the base sequence of slr1841 gene. This complex then recognizes the slr1841 gene on the cyanobacterial chromosomal DNA and binds specifically to the slr1841 gene. The steric hindrance of this binding inhibits transcription of the slr1841 gene. As a result, the expression of the cyanobacterial slr1841 gene is suppressed. In addition, the degree of suppression of the slr1841 gene can be controlled by controlling the transcriptional activity of slr1841_sgRNA.

A method for introducing each of the above two genes into the chromosomal DNA of cyanobacteria will be specifically described below.

(1-1) Introduction of dCas9 gene
Using the chromosomal DNA of the Synechocystis LY07 strain (hereinafter also referred to as LY07 strain) (see Non-Patent Document 12) as a template, the dCas9 gene, an operator gene for regulating the expression of the dCas9 gene, and spectinomycin as a marker for gene introduction. Amplify the resistance marker gene. This amplification is performed by the polymerase chain reaction (PCR) method using primers psbA1-Fw (SEQ ID NO: 13) and psbA1-Rv (SEQ ID NO: 14) shown in Table 1, for example. In the LY07 strain, the above three genes are inserted into the psbA1 gene on the chromosomal DNA in a ligated state, so that they can be amplified as one DNA fragment by PCR. Here, the resulting DNA fragment is referred to as "psbA1::dCas9 cassette". The psbA1::dCas9 cassette is inserted into the pUC19 plasmid using the In-Fusion PCR Cloning Method®, resulting in the pUC19-dCas9 plasmid.

1 μg of the obtained pUC19-dCas9 plasmid was mixed with a cyanobacterial culture medium having a cell concentration OD ₇₃₀ of about 0.5, and the pUC19-dCas9 plasmid was introduced into cyanobacterial cells by spontaneous 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. This yields a Synechocystis dCas9 strain with a dCas9 cassette inserted into the psbA1 gene region. The composition of the BG-11 medium is as shown in Table 2, for example.

(1-2) Introduction of slr1841_sgRNA gene
In the CRISPR interference method, sgRNA specifically binds to a target gene by introducing a sequence of about 20 bases complementary to the target sequence into a region called protospacer on the sgRNA gene. Table 3 shows the protospacer sequences used in sample 1.

In the Synechocystis LY04, LY05, or LY07 strain, the sgRNA gene excluding the protospacer region and the kanamycin resistance marker gene are linked and inserted into the slr2030-slr2031 gene on the chromosomal DNA. Therefore, slr1841_sgRNA can be easily obtained by adding a protospacer sequence (SEQ ID NO: 21) complementary to the slr1841 gene (SEQ ID NO: 7) to the primers used for amplifying the sgRNA gene by PCR. In addition, the degree of suppression of the slr1841 gene can be controlled by controlling the transcriptional activity of slr1841_sgRNA.

Using the chromosomal DNA of the LY07 strain as a template, a set of primers slr2030-Fw (SEQ ID NO: 15) and sgRNA_slr1841-Rv (SEQ ID NO: 16) described in Table 1, and sgRNA_slr1841-Fw (SEQ ID NO: 17) and slr2031-Rv ( Two DNA fragments are amplified by PCR using the set of SEQ ID NO: 18).

Next, using the mixed solution of the above DNA fragments as a template, the primers slr2030-Fw (SEQ ID NO: 15) and slr2031-Rv (SEQ ID NO: 18) listed in Table 1 are used for amplification by PCR. As a result, a DNA fragment (slr2030-2031::slr1841_sgRNA) is obtained in which (i) the slr2030 gene fragment, (ii) slr1841_sgRNA, (iii) the kanamycin resistance marker gene, and (iv) the slr2031 gene fragment are linked in this order. Using the In-Fusion PCR Cloning Method®, slr2030-2031::slr1841_sgRNA is inserted into the pUC19 plasmid, resulting in the pUC19-slr1841_sgRNA plasmid.

The pUC19-slr1841_sgRNA plasmid was introduced into the Synechocystis dCas9 strain in the same manner as in (1-1) above, and the transformed cells were selected on BG-11 agar medium containing 30 μg/mL kanamycin. As a result, a transformant Synechocystis dCas9 slr1841_sgRNA strain in which slr1841_sgRNA is inserted into the slr2030-slr2031 gene on the chromosomal DNA is obtained. Hereinafter, this strain is also referred to as slr1841-suppressed strain.

(1-3) Suppression of slr1841 gene The promoter sequences of the dCas9 gene and slr1841_sgRNA gene are designed to induce expression in the presence of anhydrotetracycline (aTc). In sample 1, the expression of the slr1841 gene can be suppressed by adding aTc at a final concentration of 1 μg/mL to the medium.

As a result, the total amount of proteins involved in the binding of the outer membrane and the cell wall in cyanobacteria was suppressed to about 30% compared to the amount of the proteins in the parent strain, without impairing the ability of the cells to proliferate. A cyanobacterium is obtained. Here, the Synechocystis dCas9 strain of sample 3 described later corresponds to the parent strain, and the proteins involved in binding between the outer membrane and the cell wall are slr1841, slr1908, and slr0042. The results of measuring the amount of proteins involved in binding between the outer membrane and the cell wall will be described later in (6-1).

(Sample 2)
In sample 2, a modified cyanobacterium in which the expression of the slr0688 gene encoding a cell wall-pyruvate modifying enzyme is suppressed is obtained by the following procedure.

(2) Construction of a cyanobacterial modified strain in which the expression of the slr0688 gene is suppressed sgRNA containing a protospacer sequence (SEQ ID NO: 22) complementary to the slr0688 gene (SEQ ID NO: 4) by the same procedure as in (1-2) above The gene is introduced into the Synechocystis dCas9 strain to obtain the Synechocystis dCas9 slr0688_sgRNA strain. The set of primers slr2030-Fw (SEQ ID NO: 15) and sgRNA_slr0688-Rv (SEQ ID NO: 19) and the set of sgRNA_slr0688-Fw (SEQ ID NO: 20) and slr2031-Rv (SEQ ID NO: 18) described in Table 1 were used. In-Fusion PCR was performed on a DNA fragment (slr2030-2031::slr0688_sgRNA) in which (i) the slr2030 gene fragment, (ii) slr0688_sgRNA, (iii) the kanamycin resistance marker gene, and (iv) the slr2031 gene fragment were linked in order. It can be performed under the same conditions as in (1-2) above, except that it is inserted into the pUC19 plasmid using the cloning method (registered trademark) to obtain the pUC19-slr0688_sgRNA plasmid. In addition, the degree of suppression of the slr0688 gene can be controlled by controlling the transcriptional activity of slr0688_sgRNA.

Furthermore, the expression of the slr0688 gene can be suppressed by the same procedure as (1-3) above.

As described above, the amount of a protein involved in the binding of the outer membrane and the cell wall in cyanobacteria was suppressed to about 50% of the amount of the protein in the parent strain without impairing the ability of the cells to proliferate. A sample 2 modified cyanobacterium is obtained. Here, the Synechocystis dCas9 strain of Sample 3, which will be described later, corresponds to the parent strain, and the protein involved in binding between the outer membrane and the cell wall is slr0688. The results of measurement of the amount of pyruvic acid, which is related to the amount of proteins involved in binding between the outer membrane and the cell wall, will be described later in (6-4).

(Sample 3)
As sample 3, a Synechocystis dCas9 strain is obtained by the same procedure as in sample 1 (1-1). This strain is also called the control strain.

For the strains obtained from Sample 1, Sample 2, and Sample 3, the details of the observation of the state of the cell surface layer and the protein secretion productivity test will be described.

(3) Observation of the state of the cell surface layer of the strain An ultra-thin section containing each of the modified cyanobacteria of sample 1, the modified cyanobacteria of sample 2, and the modified cyanobacteria of sample 3 is prepared, and the ultra-thin section is By observing with an electron microscope, the state of the cell surface layer, in other words, the structure of the outer membrane can be confirmed. An ultra-thin section can be produced, for example, as follows.

(3-1) Cultivation of strain The slr1841-suppressing strain of sample 1 was inoculated into BG-11 medium containing 1 μg/mL ^aTc so that the initial cell concentration OD ₇₃₀ was 0.05. Shaking culture is performed for 5 days under the conditions of light irradiation at a light intensity of s ^-1 and 30°C. The slr0688-suppressed strain of sample 2 and the control strain of sample 3 can also be cultured under the same conditions as sample 1.

(3-2) Preparation of ultra-thin sections of the strain The culture solution obtained in (3-1) above is centrifuged at room temperature with a centrifugal force of 2,500 g for 10 minutes to extract the slr1841-suppressed strain of sample 1. Cells can be harvested. Next, the cells are rapidly frozen in liquid propane at −175° C. and then fixed at −80° C. for 2 days with an ethanol solution containing 2% by mass of glutaraldehyde and 1% by mass of tannic acid. The fixed cells are dehydrated with ethanol, the dehydrated cells are permeated with propylene oxide, and then submerged in a solution of resin Quetol-651. After that, the resin is allowed to stand at 60° C. for 48 hours to cure and embed the cells in the resin. Cells in resin are sliced to a thickness of 70 nm using an ultramicrotome Ultracut to obtain ultrathin sections. This ultra-thin section can be stained with a lead solution containing 2% by weight uranium acetate and 1% by weight citric acid to prepare a sample for transmission electron microscopy of the slr1841 suppressor strain of Sample 1. The slr0688-suppressed strain of sample 2 and the control strain of sample 3 are also subjected to the same operation to prepare samples for transmission electron microscopy.

(3-3) Observation with an electron microscope Using a transmission electron microscope JEM-1400 Plus manufactured by JEOL Ltd., under the conditions of an acceleration voltage of 100 kV, observe the ultrathin section obtained in (3-2) above. It can be carried out. The observation results are shown in FIGS. 2 to 7. FIG.

2 is a transmission electron microscope (TEM) image of the slr1841-suppressed strain of sample 1. FIG. FIG. 3 is a diagram including an enlarged image of the dashed line area A in FIG. 2 and a copy thereof. FIG. 3(a) is an enlarged image of the dashed line area A in FIG. 2, and FIG. 3(b) is a copy of the enlarged image of FIG. 3(a).

As shown in FIG. 2, in the slr1841-suppressed strain of sample 1, the outer membrane is partially detached from the cell wall, that is, the outer membrane is partially sloughed off and the outer membrane is partially flexed.

In order to confirm the state of the cell surface layer in more detail, when the broken line area A is enlarged and observed, as shown in areas a1 and a2 surrounded by dashed lines in FIGS. Partially peeled off parts are confirmed. Also, a portion where the adventitia is greatly bent can be confirmed near the region a1. This part is a part where the bond between the outer membrane and the cell wall is weakened, and it is thought that the culture medium permeates from the outer membrane into the inside of the periplasm, causing the outer membrane to expand and bend outward. be done.

4 is a TEM image of the slr0688-suppressed strain of sample 2. FIG. FIG. 5 is a diagram including an enlarged image of the dashed line area B in FIG. 4 and a replica thereof. FIG. 5(a) is an enlarged image of the dashed line area B in FIG. 4, and FIG. 5(b) is a copy of the enlarged image of FIG. 5(a).

As shown in Figure 4, in the sample 2 slr0688-suppressed strain, the outer membrane is partially detached from the cell wall and the outer membrane is partially flexed. In addition, in the slr0688-suppressed strain, it can be confirmed that the outer membrane is partially detached from the cell wall.

In order to confirm the state of the cell surface layer in more detail, when the broken line area B is magnified and observed, there is a portion where the membrane is greatly bent, such as the area b1 surrounded by the dashed line in FIGS. It is confirmed. In addition, portions where the adventitia has partially peeled off are confirmed, such as regions b2 and b3 surrounded by dashed lines in FIGS. 5(a) and 5(b). In addition, portions where the outer membrane is detached from the cell wall can be confirmed in the vicinity of each of the regions b1, b2, and b3.

6 is a TEM image of the control strain of sample 3. FIG. FIG. 7 is an enlarged image of the dashed line area C in FIG. 6 and its copy. FIG. 7(a) is an enlarged image of the dashed line area C in FIG. 6, and FIG. 7(b) is a copy of the enlarged image of FIG. 7(a).

As shown in FIG. 6, the cell surface layer of the control strain of sample 3 is well-ordered, and maintains a state in which the inner membrane, cell wall, outer membrane, and S layer are layered in this order. In the control strain, as in samples 1 and 2, the part where the outer membrane detached from the cell wall, the part where the outer membrane detached from the cell wall, that is, the part where the outer membrane fell off, and the part where the outer membrane flexed Not confirmed.

(4) Protein secretion productivity test The slr1841-suppressed strain of sample 1, the slr0688-suppressed strain of sample 2, and the control strain of sample 3 are cultured, respectively, and the extracellular secreted protein amount can be measured. This amount of protein is hereinafter also referred to as the amount of secreted protein. The protein secretion productivity of each of the above strains can be evaluated from the amount of protein in the culture medium. The protein secretion productivity refers to the ability to produce a protein by secreting the protein produced in the cell to the outside of the cell.

(4-1) Culture of strain The slr1841-suppressed strain of sample 1 is cultured in the same manner as in (3-1) above. Cultures are performed three times independently. The strains of samples 2 and 3 are also cultured under the same conditions as the strain of sample 1.

(4-2) Quantification of Extracellularly Secreted Protein The culture medium obtained in (4-1) above is centrifuged at room temperature with a centrifugal force of 2,500 g for 10 minutes to obtain a culture supernatant. The resulting culture supernatant is filtered using a membrane filter with a pore size of 0.22 μm to completely remove the cells of the slr1841-suppressing strain of sample 1. The total amount of protein contained in the filtered culture supernatant is quantified by the bicinchoninic acid (BCA) method. This series of operations is performed for each of the three independently cultured cultures, and the average value and standard deviation of the protein amount extracellularly secreted from the slr1841-suppressing strain of Sample 1 are determined. Also for the strains of Samples 2 and 3, the proteins in the three culture solutions are quantified under the same conditions, and the average value and standard deviation of the protein amounts in the three culture solutions are obtained.

FIG. 8 shows the results of the protein secretion productivity test. FIG. 8 is a graph showing protein amounts in culture media of modified cyanobacteria of samples 1, 2, and 3; In this graph, the number of data n is 3, and error bars indicate standard deviation (SD).

As shown in FIG. 8, in both the slr1841-suppressed strain of sample 1 and the slr0688-suppressed strain of sample 2, the protein amount (mg/L) secreted into the culture supernatant was lower than that of the control strain of sample 3. , can be improved by a factor of about 25.

Although description of data is omitted, in both the slr1841-suppressed strain of sample 1 and the slr0688-suppressed strain of sample 2, the amount of secreted protein per 1 g of cell dry weight (mg protein/g cell dry weight) was about 36-fold improvement compared to the Control strain. The amount of secreted protein per 1 g of dry cell weight can be determined by measuring the absorbance of the culture solution at a wavelength of 730 nm.

As shown in FIG. 8, the amount of protein secreted into the culture supernatant of the slr0688-suppressed strain of sample 2 was higher than that of the slr1841-suppressed strain of sample 1. more than quantity. This is thought to be related to the fact that the number of covalent sugar chains on the cell wall surface is greater than the number of SLH domain-retaining outer membrane protein Slr1841 in the outer membrane. That is, in the slr0688-suppressed strain of sample 2, the binding amount and binding strength between the outer membrane and the cell wall are lower than the slr1841-suppressed strain of sample 1, and the amount of secreted protein is lower than that of the slr1841-suppressed strain of sample 1. It is thought that it is likely to be more than .

As described above, by suppressing the functions of proteins involved in the binding between the outer membrane and the cell wall, the binding between the outer membrane and the cell wall of cyanobacteria is partially weakened, and the outer membrane partially detaches from the cell wall. can be confirmed. In addition, it can also be confirmed that the weakened bond between the outer membrane and the cell wall makes it easier for the proteins produced in the cyanobacterial cells to leak out of the cells. Therefore, according to the modified cyanobacterium and the production method thereof according to the present embodiment, the protein secretion productivity can be greatly improved.

(5) Identification of secreted protein The secreted protein contained in the culture supernatant obtained in (4-2) above can be identified by liquid chromatography-mass spectrometry (LC-MS/MS).

(5-1) Sample preparation Cold acetone was added in an amount 8 times the volume of the culture supernatant of each sample, allowed to stand at 20°C for 2 hours, and centrifuged at a centrifugal force of 20,000 g for 15 minutes. , to obtain a protein precipitate. 100 mM Tris with a pH of 8.5 and 0.5% by weight sodium dodecanoate (SDoD) are added to this precipitate and the proteins are dissolved by an internal sonicator. After adjusting the protein concentration to 1 μg/mL, dithiothreitol (DTT) with a final concentration of 10 mM is added and allowed to stand at 50° C. for 30 minutes. Subsequently, iodoacetamide (IAA) with a final concentration of 30 mM is added, and the mixture is allowed to stand at room temperature for 30 minutes while shielded from light. In order to stop the IAA reaction, cysteine is added to a final concentration of 60 mM, and the mixture is allowed to stand at room temperature for 10 minutes. Add 400 ng of trypsin and leave overnight at 37°C. This allows the protein to be peptide fragmented. After adding 5% by mass of Trifluoroacetic Acid (TFA), the mixture is centrifuged at room temperature with a centrifugal force of 15,000 g for 10 minutes to obtain a supernatant. This work removes SDoD. After desalting using a C18 spin column, the sample is dried using a centrifugal evaporator. After that, 3% by mass of acetonitrile and 0.1% by mass of formic acid are added, and the sample is dissolved using a closed ultrasonic crusher. Prepare a peptide concentration of 200 ng/μL.

(5-2) LC-MS/MS analysis
Using an LC-MS/MS device UltiMate 3000 RSLCnano LC System, the sample obtained in (5-1) above is analyzed under the following conditions.

Sample injection amount: 200 ng
Column: CAPCELL CORE MP 75 μm × 250 mm
A solvent: 0.1 wt% formic acid aqueous solution B solvent: a solution of 0.1 wt% formic acid and 80 wt% acetonitrile Gradient program: 4 min after sample injection, 8% B solvent, 27 min after sample injection, 44% B solvent, 28 B solvent 80% after 34 minutes, end of measurement after 34 minutes

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

Software: Scaffold DIA
Database: UniProtKB/Swiss Prot database ( Synechocystis sp. PCC 6803)
Fragmentation: HCD
Precursor Tolerance: 8ppm
Fragment Tolerance: 10ppm
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

Table 4 shows 10 proteins among the identified proteins in descending order of relative quantification value.

All 10 types of proteins are contained in the culture supernatants of the sample 1 slr1841-suppressed strain and the sample 2 slr0688-suppressed strain. All of these proteins retain translocation signals for the periplasm, the space between the outer and inner membranes. From these results, it was found that in the modified strains of samples 1 and 2, organic substances such as proteins in the periplasm tend to leak out of the outer membrane, that is, out of the cells, due to partial detachment of the outer membrane from the cell wall. It is confirmed. Therefore, the modified cyanobacterium according to the present embodiment has significantly improved protein secretion productivity.

(6) Comparison with conventional examples described in Non-Patent Documents 5 and 6 Below, Samples 4 and 5, which are conventional examples described in Non-Patent Documents 5 and 6, and Samples 1 and 1, which are the present embodiment 2 will be described.
(Sample 4)
As sample 4, a modified cyanobacterium lacking slr1908 (so-called slr1908-deficient strain) was obtained based on the description in Non-Patent Document 5.
(Sample 5)
As sample 5, a modified cyanobacterium lacking slr0042 (so-called slr0042-deficient strain) was obtained based on the description in Non-Patent Document 6.

(6-1) Total amount of proteins involved in binding between outer membrane and cell wall are cultured in the same manner as in (3-1) above. After that, the culture solution was centrifuged at a centrifugal force of 5,000×g for 10 minutes to obtain a cell pellet. Cells are disrupted with an ultrasonicator, and centrifuged at a centrifugal force of 5,000×g for 10 minutes to precipitate and remove uncrushed cells. Thereafter, the centrifugation supernatant is further centrifuged at 20,000×g for 30 minutes to obtain a cell-derived membrane fraction pellet. Components other than the outer membrane are solubilized by incubating the membrane fraction pellet in 2% SDS at 37° C. for 15 minutes, followed by centrifugation at 20,000×g for 30 minutes. This yields an outer membrane fraction pellet. After quantifying the amount of protein contained in the outer membrane fraction pellet by the BCA method described in (4-2) above, 5 μg protein equivalent was subjected to electrophoresis SDS-PAGE, and the protein contained in the outer membrane pellet fraction Analyze the ingredients.

FIG. 9 shows the results of electrophoresis showing the amounts of proteins (slr1841, slr1908, and slr0042) involved in the binding of the outer membrane to the cell wall in the modified cyanobacteria of sample 1 and the modified cyanobacteria of samples 3-5. FIG. 9(a) is an electrophoretic image showing the amount of proteins involved in binding between the outer membrane and the cell wall in each sample. (b) of FIG. 9 is an enlarged view of the dashed line area Z. FIG. The intensity (darkness and thickness) of the bands in the electropherogram of FIG. 9 represents the amount of each protein. In FIG. 9, (A) is the molecular weight marker, (B) is the sample 3, (C) is the sample 5, (D) is the sample 1, and (E) is the electrophoretic image of the sample 4. Band intensities are quantified using ImageJ software. Comparison of band intensities revealed that in the modified cyanobacteria of sample 1, the total amount of proteins (slr1841, slr1908, and slr0042) involved in the binding of the outer membrane to the cell wall was reduced by suppressing the expression of the slr1841 protein. Compared with the modified cyanobacteria of a certain sample 3, it is reduced to about 30%.

On the other hand, in the modified cyanobacteria of sample 5, as shown in the enlarged photograph on the right side of FIG. The total amount of slr1841, slr1908, and slr0042) decreased by only a few percent compared to the parent strain. In the modified cyanobacteria of sample 4, the amount of slr1841 protein is increased instead of lacking slr1908 protein. increased by about 10% compared to the parent strain. A phenomenon in which deficiency of a specific outer membrane protein leads to an increase in another similar protein is a phenomenon commonly seen in other bacteria.

(6-2) Electron Micrograph The state of the outer membrane of the modified cyanobacteria of Samples 4 and 5 was observed using a transmission electron microscope under the same conditions as in (3) above. 10A and 10B show TEM images of the modified cyanobacteria of sample 4. FIG. FIG. 10B shows a magnified image of the area enclosed by the dashed line in FIG. 10A. 11A and 11B show TEM images of modified cyanobacteria of sample 5. FIG. FIG. 11B shows a magnified image of the area enclosed by the dashed line in FIG. 11A. As shown in FIGS. 10A, 10B, 11A, and 11B, the outer membrane structure of the modified cyanobacteria of samples 4 and 5 is almost the same as that of the parent strain, Comparative Example 1.

(6-3) Amount of Protein Secreted and Produced When the modified cyanobacteria samples 1 to 5 are cultured, the amount of protein secreted and produced in the culture supernatant is measured in the same manner as in (4-2) above. The results are shown in FIG. As can be seen from FIG. 12, only the modified cyanobacteria of samples 1 and 2 secrete and produce a large amount of protein into the culture medium.

(6-4) Amount of pyruvic acid Pellets of modified cyanobacterial cell-derived membrane fractions of samples 2 and 3 are obtained in the same manner as in (3-1) above. After boiling in 2% SDS for 1 hour, the cell wall fraction is sedimented by centrifugation at a centrifugal force of 40,000×g for 60 minutes. Cell wall fractions are suspended in 0.5 M HC and hydrolyzed at 100° C. for 30 minutes. After adjusting the pH to 7.0 by adding NaOH, the amount of pyruvic acid contained in this hydrolyzate is quantified using a commercially available pyruvic acid quantification kit. FIG. 13 shows the results of quantification of the amount of pyruvic acid. In FIG. 13, (A) shows the results of sample 3, and (B) shows the results of sample 2. FIG. It can be seen that in the modified cyanobacteria of sample 2, the amount of pyruvic acid is reduced to about 50% of that of sample 3, which is the parent strain. From this, it is considered that the amount of the cell wall-pyruvate modifying enzyme, which is a protein involved in binding between the outer membrane and the cell wall, is also suppressed to about 50% of the protein in the parent strain.

(7) Consideration According to samples 1 and 2, it can be confirmed that the modified cyanobacteria of the present embodiment secrete proteins present inside the cells (inside the periplasm here) to the outside of the cells. The modified cyanobacteria of the present embodiment can be genetically modified to produce other proteins instead of the proteins identified above, that is, the proteins originally produced in the cells. Therefore, it becomes possible to efficiently produce an organic substance such as a desired protein. In addition, since cyanobacteria have a high photosynthetic ability, they can easily obtain the necessary proteins when they are needed by culturing them with light, water, air, and a trace amount of inorganic substances. There is no need to use complicated equipment for In addition, proteins tend to lose their functions during processing into supplements, for example. Therefore, according to the modified cyanobacteria of the present disclosure, it is possible to provide proteins while maintaining their functions. Due to the above advantages, the modified cyanobacteria of the present disclosure are expected to be applied in various fields.

In addition, according to "(3-3) Observation by electron microscope", in the modified strain that suppresses the expression of the gene encoding the protein related to the binding between the outer membrane and the cell wall, the binding between the outer membrane and the cell wall is weakened. It can be confirmed that the adventitia was partially peeled off and that the adventitia was partially swollen. This is because the bond between the outer membrane and the cell wall weakens, and the osmotic pressure makes it easier for the culture medium to flow from the outside of the cell into the place where the outer membrane has partially detached from the cell wall. is thought to have partially expanded. Since the difference between the protein concentration in the culture medium and the protein concentration in the periplasm is large, the culture medium flowing from the outer membrane into the periplasm does not stop, and the outer membrane, which cannot withstand the internal pressure, ruptures and peels off. It is thought that it fell off, that is, peeled off.

In addition, Table 3 lists only 10 proteins among all proteins identified by LC-MS/MS analysis in descending order of relative quantification value. Then, in "(5-3) Data analysis", it was stated that all the 10 types of proteins listed in Table 3 were contained in the culture supernatants of the slr1841-suppressed strain of sample 1 and the slr0688 strain of sample 2. was similarly found in the culture supernatants of samples 1 and 2 for all other proteins identified by LC-MS/MS analysis. It is also confirmed that the secreted proteins include proteins originally present in the periplasm and proteins transported from the cytoplasm to the periplasm and functioning in the periplasm.

[5. Biofilm production method]
A method for producing a biofilm according to this embodiment will be described. A biofilm is obtained, for example, by planarly aggregating modified cyanobacteria contained in a culture medium. Here, in the modified cyanobacteria, the total amount of proteins involved in binding between the outer membrane and the cell wall in cyanobacteria is suppressed to 30% to 70% of the total amount of the proteins in the parent strain. In other words, the biofilm according to this embodiment is obtained by planarly aggregating the modified cyanobacteria according to this embodiment.

FIG. 14 is a diagram showing an example of a biofilm production method. As shown in FIG. 14, a base material 30 is used in this manufacturing method. First, culture solution 15 containing modified cyanobacteria 10 is allowed to exist around substrate 30 . For example, the substrate 30 is arranged inside the container 40 and the culture solution 15 is accommodated inside the container 40 so as to be in contact with the substrate 30 . The modified cyanobacteria 10 are then deposited onto the substrate 30 . As a result, the modified cyanobacteria 10 agglomerate in a plane to obtain a biofilm 20 . In the modified cyanobacterium 10, the total amount of proteins involved in binding between the outer membrane and the cell wall in cyanobacteria is suppressed from 30% to 70% of the total amount of the proteins in the parent strain.

The method of attaching the modified cyanobacteria 10 onto the substrate 30 is not limited to a particular method. For example, the modified cyanobacteria 10 can adhere to the substrate 30 by allowing the modified cyanobacteria 10 to settle in the culture medium 15 . The modified cyanobacteria 10 may adhere to the substrate 30 by arranging the substrate 30 so as to create a flow of the culture solution 15 inside the container 40 and resist the flow.

As shown in FIG. 14 , the culture solution 15 may be irradiated with light when the modified cyanobacteria 10 are adhered onto the substrate 30 . For example, the culture solution 15 is irradiated with light from the light source 50 . As a result, the culture of the modified cyanobacteria 10 is promoted in the culture solution 15 , and the modified cyanobacteria 10 easily adhere to the substrate 30 to form the biofilm 20 . Light irradiation conditions are not limited to specific conditions. The irradiated light may be continuous light or monochromatic light. The light source 50 may be an artificial light source or sunlight. The photon flux density of the irradiated light is not limited to a specific value.

When the modified cyanobacteria 10 are attached onto the substrate 30, the space containing the culture solution 15 may be ventilated. This makes it easier for many modified cyanobacteria 10 to adhere to the base material 30 in the culture solution 15, and the productivity of the biofilm 20 tends to be improved. As shown in FIG. 14, container 40 has inlet 41 and outlet 42 . For example, air is supplied to the interior 45 of the container 40 through the inlet 41 and the air in the interior 45 is discharged to the outside of the container 40 through the outlet 42 . Thereby, the space in which the culture solution 15 is stored is ventilated. When the modified cyanobacteria 10 are deposited on the substrate 30, aeration may be performed continuously over the entire period, continuously during a part of the period, or intermittently. may be broken. When the modified cyanobacteria 10 are adhered onto the substrate 30, the space containing the culture medium 15 may not be ventilated. In this specification, "space" means a place where a substance exists and a phenomenon occurs. Aeration includes actively sending air into the space in which the culture solution 15 is accommodated.

Substrate 30 is not limited to a particular substrate, as long as modified cyanobacteria 10 adhere to substrate 30 . Substrate 30 includes, for example, silicone rubber or polyvinyl chloride. This makes it easier for the modified cyanobacteria 10 to adhere onto the substrate 30 and to produce the biofilm 20 easily. Moreover, when the base material 30 contains silicone rubber, the biofilm 20 of good quality is likely to be obtained.

As long as the modified cyanobacteria 10 adhere to the substrate 30, the material of the container 40 is not limited to a specific material. For example, when the culture solution 15 is irradiated with light, the container 40 transmits the light. The container 40 may be made of, for example, inorganic glass, organic glass, resin, or plastic.

As long as the modified cyanobacteria 10 can adhere to the substrate 30, the components contained in the culture solution 15 are not limited to specific components. The culture solution 15 may contain known components necessary for culturing cyanobacteria.

The temperature of the culture solution 15 when the modified cyanobacteria 10 are adhered onto the substrate 30 is not limited to a specific value as long as the biofilm 20 can be produced. The temperature is, for example, a temperature at which the modified cyanobacteria 10 can be cultured.

FIG. 15 is a diagram showing another example of a biofilm production method. In this manufacturing method, biofilm 20 can be manufactured without using substrate 30 . In this manufacturing method, first, a culture solution 15 containing modified cyanobacteria 10 is stored such that an air-liquid interface 17 exists between the culture solution 15 and air. For example, the culture solution 15 is stored in the interior 47 of the container 46 and air exists above the air-liquid interface 17 . After that, the modified cyanobacteria 10 are aggregated in a plane along the gas-liquid interface 17 . Thereby, the biofilm 20 can be manufactured.

As shown in FIG. 15, the culture medium 15 may be irradiated with light when the modified cyanobacteria 10 are planarly aggregated along the air-liquid interface 17 . For example, the culture solution 15 is irradiated with light from the light source 50 . As a result, the culture of the modified cyanobacteria 10 is promoted in the culture solution 15 , and the modified cyanobacteria 10 easily aggregate in a plane along the air-liquid interface 17 to form a biofilm 20 . Light irradiation conditions are not limited to specific conditions. The irradiated light may be continuous light or monochromatic light. The light source 50 may be an artificial light source or sunlight. The photon flux density of the irradiated light is not limited to a specific value.

The modified cyanobacteria 10 are statically cultured in the culture solution 15 when the modified cyanobacteria 10 are agglutinated in a plane along the air-liquid interface 17 . This makes it easier for the modified cyanobacteria 10 to aggregate in a plane along the gas-liquid interface 17 . In this static culture, interior 47 of container 46 is typically not aerated.

The pH of the culture solution 15 is not limited to a specific value as long as the modified cyanobacteria 10 can be planarly aggregated along the air-liquid interface 17 . The pH of the culture solution 15 is, for example, 6.5 or more and 8.4 or less. In this case, the modified cyanobacteria 10 tend to aggregate in a plane along the gas-liquid interface 17 . The pH of the culture medium 15 means, for example, the value immediately before the culture of the modified cyanobacteria 10 .

[6. biofilm]
A biofilm 20 can be provided as shown in FIGS. 14 and 15 by the method for producing a biofilm according to the present embodiment. Biofilm 20 contains modified cyanobacteria 10 . In the modified cyanobacterium 10, the total amount of proteins involved in binding between the outer membrane and the cell wall in cyanobacteria is suppressed from 30% to 70% of the total amount of the proteins in the parent strain.

The biofilm 20 may be provided on a substrate 30 as shown in FIG. 14, or may be provided without a substrate. For example, the biofilm 20 can be provided along the air-liquid interface 17 between the culture solution 15 and the air, as shown in FIG. In this case, the biofilm 20 is provided together with a container containing a liquid such as culture medium. The biofilm 20 may be provided in a state arranged on a substrate, or may be provided as a single biofilm 20 .

[7. Method for producing organic matter using biofilm]
A method for producing an organic substance using a biofilm according to this embodiment will be described. In this production method, the modified cyanobacteria 10 of the biofilm 20 produce organic substances and secrete the organic substances to the outside of the modified cyanobacteria 10 . In this case, biofilm 20 may or may not be placed on a substrate. In addition, the biofilm 20 may be arranged along the air-liquid interface 17 between the culture medium 15 and the air, or may be arranged in another state. Organics are, for example, proteins, peptides, polypeptides, enzymes, amino acids, nucleic acids, lipids, carbohydrates, or other biomolecules.

When biofilms 20 are used to produce organic matter, modified cyanobacteria 10 are typically cultured. Cultivation of cyanobacteria can generally be carried out based on liquid culture using the BG-11 medium shown in Table 2 or a modification thereof. Therefore, culturing of modified cyanobacteria 10 may be carried out in the same manner. Cultivation of the modified cyanobacteria 10 is performed, for example, by static culture. Thereby, the shape of the biofilm 20 is easily maintained. In culturing the modified cyanobacteria 10 when producing an organic matter using the biofilm 20, the space around the biofilm 20 may or may not be ventilated.

Part or all of the medium may be replaced when the biofilm 20 is used to produce organic matter. For example, the medium can be replaced by collecting a predetermined amount of the supernatant of the culture medium and supplying a new medium. By collecting the supernatant of the culture medium, the produced organic matter can be collected. Also, by supplying new culture medium, organic matter can be continuously produced using the biofilm 20 . By using biofilm 20, organic matter can be produced over a long period of time, for example, 35 days or longer.

When producing an organic substance using the biofilm 20, an inducer such as aTc is supplied to the medium for culturing the modified cyanobacteria 10 at a predetermined timing. As a result, organic matter can be continuously produced using the biofilm 20 .

When an organic substance is produced using the biofilm 20, stirring and aeration of the culture solution can be made unnecessary, and there are few restrictions for producing the organic substance. In addition, the cell density in the culture medium tends to be stable, and constant monitoring of the cell density in the culture medium is not required. Moreover, since the organic substances produced by collecting the supernatant of the culture solution can be recovered, there is no need to dispose a container for collecting the organic substances in contact with the container containing the culture solution. Therefore, cleaning of the container is easy. Therefore, the method of producing organic matter using the biofilm 20 is suitable for scale-up.

Hereinafter, the biofilm production method, biofilm, and organic matter production method of the present disclosure will be specifically described in Examples, but the present disclosure is not limited only to the following Examples.

(Example 1 and Comparative Example 1)
Synechocystis dCas9 slr0688_sgRNA strain was obtained in the same manner as sample 2 above. This strain was a strain in which the outer membrane was detached from the cell wall by genetic manipulation. Synechocystis dCas9 strain was obtained in the same manner as Sample 3 above. The Synechocystis dCas9 strain corresponds to the parent strain of the Synechocystis dCas9 slr0688_sgRNA strain.

The Synechocystis dCas9 slr0688_sgRNA strain was diluted with BG-11 liquid medium containing 1 μg/mL of aTc, an inducer, so that the cell concentration OD ₇₃₀ was 0.05, and the culture solution according to Example 1 was prepared. did. The pH of the BG-11 liquid medium was 7.6. A culture solution according to Comparative Example 1 was prepared in the same manner as in Example 1, except that the Synechocystis dCas9 strain was used instead of the Synechocystis dCas9 slr0688_sgRNA strain. 1 mL of each of the culture solution according to Example 1 and the culture solution according to Comparative Example 1 was injected into each well of a 4-well plate Nunclon Delta Surface manufactured by Thermo Fisher Scientific, and 100 μmol m ⁻ was added to the culture solution. Static culture was carried out at 30° C. for 7 days while irradiating continuous light having a photon flux density of ² ·s ⁻¹ . No aeration was performed in this static culture.

16A and 16B are photographs showing the state of the culture solution on day 7 from the start of static culture. 16A corresponds to Example 1, and FIG. 20B corresponds to Comparative Example 1. FIG. As a result of confirming the state of the culture solution on the 7th day from the start of the stationary culture, in Example 1, planar cell masses were floating along the air-liquid interface of the culture solution. biofilm was confirmed. In Comparative Example 1, no clumps of cells were present near the air-liquid interface of the culture solution, and the cells were deposited on the bottom of the well.

(Examples 2 and 3 and Comparative Examples 2 to 5)
The Synechocystis dCas9 slr0688_sgRNA strain was diluted with 12 mL of BG-11 liquid medium containing 1 μg/mL of aTc as an inducer to prepare a culture stock solution so that the cell concentration OD ₇₃₀ was 0.1. The pH of this BG-11 liquid medium was 7.6. This undiluted culture solution was placed in an Erlenmeyer flask and cultured with shaking at 30° C. for 3 days while irradiating the undiluted culture solution with continuous light having a photon flux density of 100 μmol m ⁻² ·s ⁻¹ . This culture stock solution was added to BG-11 liquid medium of pH 6.9, pH 7.9, and pH 8.8 containing 1 μg/mL of aTc as an inducer so that the cell concentration OD ₇₃₀ was 0.1. After dilution, culture solutions according to Example 2, Example 3, and Comparative Example 2 were prepared. Further, instead of the Synechocystis dCas9 slr0688_sgRNA strain, except for using the Synechocystis dCas9 strain, in the same manner as in Example 2, Example 3, and Comparative Example 2, the culture solutions according to Comparative Examples 3, 4, and 5, respectively was prepared. 1 mL of each of the culture solutions according to Examples 2 and 3 and the culture solutions according to Comparative Examples 2, 3, 4, and 5 was injected into each well of a 4-well plate Nunclon Delta Surface manufactured by Thermo Fisher Scientific, and cultured. The liquid was statically cultured at 30° C. for 6 days while being irradiated with continuous light having a photon flux density of 100 μmol m ⁻² ·s ⁻¹ . No aeration was performed in this static culture.

The state of each culture solution after 6 days of stationary culture was confirmed. As a result, in the culture solutions according to Examples 2 and 3, planar cell masses were floating along the air-liquid interface of the culture solutions, and biofilms according to Examples 2 and 3 were confirmed. On the other hand, in the culture solution according to Comparative Example 2, cell clusters were deposited on the bottom of the well. In the culture solutions according to Comparative Examples 3, 4, and 5, no cell clumps were observed, and cells were sedimented on the bottom of the wells.

(Comparative Example 1 and Comparative Reference Example 1)
A culture vessel having a bottom surface of 18.1 cm ² in which a substrate can be sandwiched was prepared. This culture vessel had an inlet and an outlet. The inlet of the culture vessel was located at a height of 2.5 cm from the substrate when the substrate was sandwiched between the bottom of the culture vessel. The outlet, on the other hand, was located at a height of 9.2 cm from the substrate. A silicon rubber Shin-Etsu Silico Sheet BA grade (economy type) manufactured by Shin-Etsu Chemical Co., Ltd. was arranged as a base material on the bottom surface of the culture vessel. The thickness of this substrate was 0.3 mm.

The Synechocystis dCas9 slr0688_sgRNA strain was diluted with 150 mL of BG-11 liquid medium containing 1 μg/mL anhydrotetracycline (aTc), which is an inducer, so that the cell concentration OD ₇₃₀ was 0.1. Such a culture medium was prepared. OD730 means optical density at a wavelength of ₇₃₀ nm. The pH of the BG-11 liquid medium was 7.6. In the description below, the pH of the BG-11 liquid medium is 7.6, unless otherwise specified. A culture solution according to Comparative Reference Example 1 was prepared in the same manner as the culture solution according to Reference Example 1, except that the Synechocystis dCas9 strain was used instead of the Synechocystis dCas9 slr0688_sgRNA strain. The culture solution according to Reference Example 1 and the culture solution according to Comparative Reference Example 1 were each injected into the culture vessel, and continuous light having a photon flux density of 100 μmol m ⁻² ·s ⁻¹ was applied to the culture solution. The aeration culture was carried out at 30° C. for 2 days while irradiating. In this aeration culture, air was supplied from the inlet into the culture vessel at a flow rate of 3.6 L/min.

Observation of the culture solution according to Reference Example 1 after 2 days of aeration culture confirmed that bacterial cells adhered to the base material and aggregated in a plane, and biofilms according to Reference Example 1 were confirmed. After 2 days of aeration culture, the bacterial cell concentration OD ₇₃₀ in each culture solution was measured as the floating cell concentration, and the value of the floating cell concentration was multiplied by the volume of the culture solution, 150 mL, to give an indicator of the number of floating cells A [OD ₇₃₀ • mL] was determined. Further, in each culture solution, the entire amount of the culture solution was removed, and the cells adhering to the substrate were detached and suspended in a new BG-11 liquid medium to obtain a suspension. The bacterial cell concentration OD ₇₃₀ of the resulting suspension was measured as the biofilm cell concentration, and the value of the biofilm cell concentration was multiplied by the volume of the suspension to give an index of the number of biofilm cells B [OD ₇₃₀ • mL] was determined. Using these values, calculation of {value of index B/(value of index A + value of index B)}×100 was performed, and the calculation result was determined as biofilm cell ratio [%]. Table 5 shows the results. Table 5 shows the average ± standard deviation of the results of five measurements for Reference Example 1, and the average ± standard deviation of the results of three measurements for Comparative Reference Example 1.

As shown in Table 5, the value of index A for the number of suspended cells in Comparative Reference Example 1 was greater than the value of index A for the number of suspended cells in Reference Example 1. The p-value for this statistical hypothesis test was 0.15. On the other hand, the value of index B for the number of biofilm-forming cells in Reference Example 1 was greater than the value of index B for the number of biofilm-forming cells in Comparative Reference Example 1. The p-value in this statistical hypothesis test was 0.8×10 ⁻⁵ . In addition, the value of the biofilm-forming cell ratio in Reference Example 1 was approximately 35 times the value of the biofilm-forming cell ratio in Comparative Reference Example 1.

(Reference Example 2 and Comparative Reference Example 2)
Comparative Reference Example 2 and Comparative Reference Example 2 were prepared in the same manner as in Reference Example 1 and Comparative Reference Example 1, respectively, except that a polyvinyl chloride sheet vinyl film sheet provided by AS ONE was used as the base material instead of the silicone rubber. A culture solution according to was prepared. In addition, instead of the culture solutions according to Reference Example 1 and Comparative Reference Example 1, the culture solutions according to Reference Example 2 and Comparative Reference Example 2 were used, respectively, in the same manner as in Reference Example 1 and Comparative Reference Example 1. Aeration culture of each culture solution was performed. Observation of the culture solution according to Reference Example 2 after 2 days of aeration culture revealed that bacterial cells adhered to the base material and aggregated in a plane, and a biofilm according to Reference Example 2 was confirmed. In addition, in the same manner as in Reference Example 1 and Comparative Reference Example 1, using each culture medium of aerobic culture for 2 days and the bacterial cells attached to the substrate, index A for the number of floating cells, index B for the number of biofilm cells, and biofilm cell percentage values were determined. Table 6 shows the results. Table 6 shows the average value±standard deviation of the results of three measurements for Reference Example 2, and the average value±standard deviation of the results of eight measurements for Comparative Reference Example 2.

As shown in Table 6, the value of index A for the number of suspended cells in Comparative Reference Example 2 was greater than the value of index A for the number of suspended cells in Reference Example 2. The p-value for this statistical hypothesis test was 0.06. On the other hand, the value of index B for the number of biofilm-forming cells in Reference Example 2 was greater than the value of index B for the number of biofilm-forming cells in Comparative Reference Example 2. The p-value for this statistical hypothesis test was 0.06. In addition, the value of the biofilm-forming cell ratio in Reference Example 2 was approximately five times the value of the biofilm-forming cell ratio in Comparative Reference Example 2.

(Reference example 3)
The Synechocystis dCas9 slr0688_sgRNA strain was diluted with 12 mL of BG-11 liquid medium containing 1 μg/mL aTc as an inducer so that the cell concentration OD ₇₃₀ was 0.1, and the culture solution according to Reference Example 3 was added. prepared. This culture solution was placed in an Erlenmeyer flask and cultured with shaking at 30° C. for 2 days while being irradiated with continuous light having a photon flux density of 100 μmol m ⁻² ·s ⁻¹ . The culture solution after shaking culture for 2 days was aerated as in Reference Example 1 so that the cell count index calculated by multiplying the cell concentration OD ₇₃₀ by the volume of the culture solution was 32 [OD ₇₃₀ mL]. It was injected into the culture vessel used for culture. After that, static culture was carried out at 30° C. for 2 days while irradiating the culture medium with continuous light having a photon flux density of 100 μmol m ⁻² ·s ⁻¹ . In this stationary culture, the inside of the culture vessel was not aerated. In addition, in this stationary culture, silicone rubber was used as a base material in the same manner as in the aerobic culture of Reference Example 1. Observation of the culture solution after static culture for 2 days revealed that bacterial cells adhered to the base material and agglomerated in a plane, and a biofilm according to Reference Example 3 was confirmed.

Using the culture solution after static culture for 2 days and the bacterial cells attached to the substrate, in the same manner as in Reference Example 1, the indicator A for the number of floating cells, the indicator B for the number of biofilm-forming cells, and the biofilm-forming cells Percentage values were determined. Table 7 shows the results. Table 7 shows the mean±standard deviation of three measurement results for Reference Example 3. For comparison, the results of Reference Example 1 are shown again in Table 7.

As shown in Table 7, the biofilm-forming cell ratio in Reference Example 3 was reduced to about 52% of the biofilm-forming cell ratio in Reference Example 1.

(Reference Examples 4 and 5)
The Synechocystis dCas9 slr0688_sgRNA strain was diluted with 150 mL of BG-11 liquid medium containing 1 μg/mL of aTc as an inducer to prepare a culture solution so that the cell concentration OD ₇₃₀ was 0.1. This culture solution was injected into the culture vessel used in Reference Example 1, in which a silicone rubber or polyvinyl chloride sheet was arranged as a base material, and a photon flux density of 100 μmol m ⁻² s ⁻¹ was applied to the culture solution. Aeration culture was carried out at 30° C. for 2 days while irradiating with continuous light. In this aeration culture, air was supplied from the inlet into the culture vessel at a flow rate of 3.6 L/min. An example using a silicone rubber as a base material corresponds to Reference Example 4, and an example using a polyvinyl chloride sheet as a base material corresponds to Reference Example 5.

After 2 days of aerobic culture, the cells adhered to the substrate and agglomerated in a plane to form a biofilm. The culture supernatant was removed from the interior of the culture vessel, leaving a 35 mL portion, and 150 mL of fresh BG-11 medium was added to the interior of the culture vessel. The BG-11 medium contained 1 μg/mL of aTc, an inducer. After that, the inlet and outlet of the culture vessel were sealed with rubber plugs to seal the inside of the culture vessel. In this state, static culture was started without aeration.

After static culture for 14 days, using the culture solution and the cells attached to the substrate, in the same manner as in Reference Example 1, the indicator A for the number of floating cells, the indicator B for the number of biofilm-forming cells, and biofilm formation Cell percentage values were determined. Table 8 shows the results. In addition, the secreted protein concentration was measured in the supernatant of the culture solution immediately before static culture and in the supernatant of the culture solution after static culture for 14 days. Measurement of secretory protein concentration was performed as follows. A solution A and a solution B of BCA protein assay reagents manufactured by Thermo Fisher Scientific were mixed at a volume ratio of 50:1 to prepare 500 μL of working reagent. A sample liquid to be measured and a bovine serum albumin solution of known concentration were each added to the working reagent in an amount of 50 μL. A bovine serum albumin solution is used as a general protein standard solution. A sample in which the sample solution to be measured was added to the working reagent and a sample in which the bovine serum albumin solution was added to the working reagent were incubated at 37°C for 30 minutes and cooled. was measured. A calibration curve was prepared based on the obtained absorbance data of bovine serum albumin solutions with known concentrations, and the protein concentration contained in the sample solution to be measured was calculated based on this calibration curve. The amount of change in secreted protein concentration was determined by subtracting the concentration of secreted protein in the supernatant of the culture immediately before static culture from the concentration of secreted protein in the supernatant of the culture after static culture for 14 days. Table 8 shows the results. In Table 8, each value is shown as the mean±standard deviation of three measurements.

As shown in Table 8, in Reference Examples 4 and 5, no significant difference was observed in the index B of the number of biofilm-forming cells and the amount of change in secretory protein concentration. The p-values in the statistical hypothesis test for the biofilm number index B and the amount of change in secreted protein concentration were 0.14 and 0.98, respectively. On the other hand, the value of index A for the number of suspended cells in Reference Example 5 was lower than the value of index A for the number of suspended cells in Reference Example 4. The p-value for this statistical hypothesis test was 0.02. From these results, it is understood that the protein-producing ability of the bacterial cells in the biofilm is maintained regardless of whether the substrate is a silicone rubber or a polyvinyl chloride sheet. On the other hand, it was suggested that cell proliferation is less likely to be inhibited by using silicone rubber as the base material than when using a polyvinyl chloride sheet as the base material. This is presumably because the base material of silicone rubber is superior in air permeability to the base material of polyvinyl chloride sheet.

After static culture for 14 days, the absorption spectrum of the suspension obtained by peeling and suspending the cells adhering to the substrate was measured. The results are shown in FIG. In FIG. 17 , the solid-line graph indicates the results for Reference Example 4, and the broken-line graph indicates the results for Reference Example 5. From the data shown in FIG. 17, the ratio A ₆₃₀ /A ₇₅₀ of the absorbance A ₆₃₀ at 630 nm derived from phycobiliprotein, which is a major photosynthetic light-harvesting protein, relative to the absorbance A ₇₅₀ at a wavelength of 750 nm was determined. As a result, in Example 4, A ₆₃₀ /A ₇₅₀ was 1.24±0.03, and in Reference Example 5, A ₆₃₀ /A ₇₅₀ was 1.18±0.04. The A _{630 /A 750 value in Reference Example 5 tended to be lower than the A 630 / A 750} value in Reference Example 4. The p-value for this statistical hypothesis test was 0.08.

FIG. 18 shows a photograph of a suspension obtained by peeling off and suspending the cells adhering to the substrate after static culture for 14 days. In FIG. 18, the suspension contained in the two sample bottles on the left side corresponds to Reference Example 4, and the suspension contained in the two sample bottles on the right side corresponds to Reference Example 5. From this photograph, it was confirmed that the color of the suspension according to Reference Example 5 had turned yellow, indicating that chlorosis had occurred. On the other hand, the suspension according to Reference Example 4 exhibited a darker green color than the suspension according to Reference Example 5. These results suggest that the use of silicone rubber as a base material tends to improve cell growth conditions as compared to the use of a polyvinyl chloride sheet as a base material. This is presumably because the base material of silicone rubber is superior in air permeability to the base material of polyvinyl chloride sheet.

(Reference example 6)
FIG. 19 is a cross-sectional view showing a culture vessel 40s that was produced as a trial to examine the possibility of scale-up. The culture container 18 includes a base material 30, a bottom member 40a, a middle frame 40b, a lid member 40c, a sealing member 43, and a sealing member 44. Each of the bottom member 40a, the middle frame 40b, and the lid member 40c is made of acrylic resin. The middle frame 40b is arranged between the bottom member 40a and the lid member 40c. The bottom member 40a, the middle frame 40b, and the lid member 40c are fixed with a snap lock in a state in which the bottom member 40a and the lid member 40c are pressed against the middle frame 40b. The base material 30 is arranged between the bottom member 40a and the middle frame 40b, and is fixed while being pressed by the bottom member 40a and the middle frame 40b. The substrate 30 is a sheet of silicone rubber of the same type as the silicone rubber used in Example 1. An annular groove is formed in the surface of the bottom member 40a in contact with the base material 30, and the seal member 43 is arranged in this groove. Thereby, the gap between the bottom member 40a and the base member 30 is sealed. An annular groove is formed in the surface of the middle frame 40b that contacts the lid member 40c, and a seal member 44 is arranged in this groove. Thereby, the gap between the middle frame 40b and the lid member 40c is sealed.

Inside the culture container 40s, the base material 30 forms a bottom surface of 676 cm ² . An inlet 41 and an outlet 42 are formed at a height of 3 cm from the base material 30 in the middle frame 40b.

The Synechocystis dCas9 slr0688_sgRNA strain was diluted with 1 L of BG-11 liquid medium containing 1 μg/mL of aTc as an inducer so that the cell concentration OD ₇₃₀ was 0.1, and the culture solution according to Reference Example 6 was made. This culture solution is injected into the above culture vessel 40s, and aeration culture is performed at 30° C. for 7 days while irradiating the culture solution with continuous light having a photon flux density of 100 μmol m ⁻² s ⁻¹ . did. In this aeration culture, air was supplied from the inlet 41 into the culture vessel 40s at a flow rate of 30 L/min.

After seven days of aeration culture, the inlet 41 and the outlet 42 of the culture container 40s were filled with cotton plugs, and static culture without aeration was started. On the 0th day, 7th day, 14th day, 23rd day and 34th day from the start of static culture, the suspension cell concentration OD ₇₃₀ and protein concentration of the culture supernatant were measured. The protein concentration was measured by the same method as shown in Reference Examples 4 and 5. In addition, the amount of secreted protein was calculated from the protein concentration of the supernatant of the culture medium and the volume of the supernatant of the culture medium. The results are shown in FIG. On the 0th day, 7th day, 14th day, and 23rd day from the start of static culture, 1 μg/mL of aTc as an inducer was added. On the 14th day after the start of static culture, half of the supernatant of the culture was replaced with fresh BG-11 medium, and on the 23rd day after the start of static culture, 130 mL of fresh BG-11 medium was added. Suspended cell concentration, protein concentration, and volume of the culture supernatant were measured prior to these operations.

As shown in FIG. 20, the suspension cell concentration OD ₇₃₀ was always below 0.5 during the static culture period, indicating that the cells were stably growing as a biofilm. On the other hand, the amount of secreted protein showed that the protein was continuously secreted during the static culture period.

On the 36th day from the start of static culture, the cells adhering to the substrate were peeled off and suspended to prepare a suspension. Using this suspension, in the same manner as in Reference Example 1, the values of the index A for the number of floating cells and the index B for the number of biofilm-forming cells were determined. As a result, the index A for the number of floating cells was 322 [OD ₇₃₀ -mL], and the index B for the number of biofilm-forming cells was 779 [OD ₇₃₀ -mL]. The biofilm cell percentage calculated from these values was 71%. Even when a scaled-up prototype such as the culture vessel 40s was used, it was confirmed that the same proportion of cells as in the small-scale Reference Example 1 contributed to biofilm formation.

The biofilm production method, biofilm, and organic matter production method according to the present disclosure have been described above based on the embodiments, but the present disclosure is not limited to these embodiments. As long as it does not deviate from the gist of the present disclosure, various modifications that a person skilled in the art can think of are applied to the embodiments, 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, an example was described in which a protein produced within a bacterium is leaked out of the bacterium by inhibiting the function of a protein involved in binding between the outer membrane and the cell wall in cyanobacteria. is not limited to For example, by applying an external force to the cyanobacteria, the bond between the outer membrane and the cell wall may be weakened, and the outer membrane may be weakened. Enzymes or agents may also be added to the cyanobacterial culture to weaken the outer membrane.

According to the biofilm production method, biofilm, and organic matter production method of the present disclosure, water, light, air, and a trace amount of inorganic matter are given to modified cyanobacteria for cultivation, thereby efficiently obtaining organic matter. can be done. For example, it is possible to obtain raw materials for food ingredients or enzymes for producing compounds, diagnostic enzymes or therapeutic enzymes in the medical field, or feed enzymes in the field of agriculture, fisheries and livestock.

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 organism channel protein 9 cell wall-pyruvate modifying enzyme 10 modified cyanobacteria 15 culture medium 20 biofilm 30 substrate

Claims (9)

A culture medium containing a modified cyanobacterium in which the total amount of proteins involved in binding between the outer membrane and the cell wall in cyanobacteria is suppressed to 30% to 70% of the total amount of the proteins in the parent strain is placed between the culture medium and air. storing such that there is a gas-liquid interface of
Aggregating the modified cyanobacteria into a planar shape along the gas-liquid interface;
Methods of producing biofilms. The method for producing a biofilm according to claim 1, wherein the culture solution is irradiated with light when the modified cyanobacteria are planarly aggregated along the air-liquid interface. 3. The method for producing a biofilm according to claim 1, wherein the modified cyanobacteria are statically cultured in the culture medium when the modified cyanobacteria are planarly aggregated along the air-liquid interface. The method for producing a biofilm according to any one of claims 1 to 3, wherein the culture solution has a pH of, for example, 6.5 or more and 8.4 or less. The method for producing a biofilm according to any one of claims 1 to 4, wherein the protein is at least one of a surface layer homology domain-retaining outer membrane protein and a cell wall-pyruvate modifying enzyme. A biofilm comprising modified cyanobacteria wherein the total amount of proteins involved in the binding of the outer membrane to the cell wall in cyanobacteria is suppressed to 30% to 70% of the total amount of said proteins in the parental strain. 7. The biofilm of claim 6, disposed along an air-liquid interface between the medium and air. A biofilm containing a modified cyanobacterium in which the total amount of proteins involved in the binding of the outer membrane and the cell wall in the cyanobacteria is suppressed to 30% to 70% of the total amount of the proteins in the parent strain, producing organic matter in the modified cyanobacteria. and secreting the organic substance to the outside of the modified cyanobacterium,
Organic production method. 9. The method for producing organic matter according to claim 8, wherein the biofilm is arranged along an air-liquid interface between the culture solution and the air.

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