WO2024071134A1

WO2024071134A1 - Mutant of cyanobacterium and production method thereof

Info

Publication number: WO2024071134A1
Application number: PCT/JP2023/034980
Authority: WO
Inventors: 隆一廣田; 章夫黒田
Original assignee: 国立大学法人広島大学
Priority date: 2022-09-30
Filing date: 2023-09-26
Publication date: 2024-04-04

Abstract

The present invention addresses the problem of providing a mutant of a cyanobacterium having predation resistance. This problem is solved by a mutant of a cyanobacterium in which the function of the spoIID gene is reduced or defective compared to the spoIID gene in the wild type.

Description

Cyanobacterial mutants and methods for producing same

The present invention relates to a cyanobacterial mutant and a method for producing the same.

In recent years, from the viewpoint of reducing _CO2 emissions and mitigating the burden on the environment, bioproduction using renewable biological resources has been attracting attention. Cyanobacteria, which are single-celled organisms that perform photosynthesis, can use light energy to produce various substances (e.g., fats and oils, starch, proteins, amino acids, carotenoids, etc.) as metabolic products using water and _CO2 as raw materials. In addition, cyanobacteria are easy to genetically manipulate, and therefore research is being conducted extensively on them as chassis strains for bioproduction.

In order to commercialize bioproduction from microalgae such as cyanobacteria, it is necessary to cultivate the microalgae on a practical scale (especially large-scale cultivation outdoors). However, in large-scale cultivation of microalgae, contamination of the culture system with other biological species is a major problem.

Damage caused by contamination can include damage caused by nutritional competition due to the introduction of protozoa such as algae and bacteria, and parasitic damage due to the introduction of viruses, mold, etc. In particular, damage caused by direct predation of microalgae by the introduced protozoa (predation damage) is a major problem, especially when large-scale cultivation is carried out outdoors, as it can cause a significant decrease in productivity of the culture system (pond crash) (Non-Patent Documents 1 and 2).

In large-scale outdoor cultivation of microalgae, contamination by protozoans is virtually unavoidable. To date, methods have been found to control contamination in small-scale laboratory-level cultivation using physical methods such as ultrasonication, chemical methods such as the addition of chemical substances, and biological methods using competing organisms. However, these methods cannot be said to be effective control methods for large-scale cultivation. At present, no effective method for controlling contamination in large-scale cultivation has been developed, and only diagnostic methods have been developed (Non-Patent Document 3).

In the production of substances using microalgae such as cyanobacteria, it is believed that suppressing damage to the cyanobacteria caused by contaminating protozoans, in particular damage caused by predation by protozoans, can greatly contribute to improving the biomass productivity of cyanobacteria and to their practical application.

However, as mentioned above, with current technology it is difficult to completely prevent contamination of culture systems with protozoa. Therefore, endowing the cyanobacteria being cultured with resistance to predation by predatory organisms is thought to be an effective method for preventing damage caused by contamination.

Non-Patent Document 4 discloses a phenomenon in which a species of heterotrophic bacteria undergoes a morphological change in the presence of a predatory protozoan, improving its resistance to predation.

In light of the above-mentioned circumstances, one aspect of the present invention aims to provide a cyanobacterial mutant that is resistant to predation by predatory protozoans.

In order to solve the above problem, the cyanobacterial mutant according to one embodiment of the present invention is a cyanobacterial mutant in which the function of the spoIID gene is reduced compared to that of the wild-type spoIID gene or is absent.

In addition, the method for producing a cyanobacterial mutant according to one embodiment of the present invention includes a step of reducing the function of the spoIID gene below that of the wild-type spoIID gene or deleting the spoIID gene.

According to one aspect of the present invention, it is possible to provide a cyanobacterial mutant that is resistant to predation by predatory protozoans.

FIG. 1 shows the appearance of a wild-type strain, a mutant strain, and a transformant of the marine cyanobacterium strain 7002. FIG. 1 shows the change in appearance of cultures of wild-type, mutant and transformant strains of strain 7002 after 5 days of culture, depending on the presence or absence of predatory protozoa. 1 is a graph showing the changes in cell number (OD ₇₅₀ value) and chlorophyll content during culture of wild-type, mutant and transformant strains of strain 7002 in the presence or absence of predatory protists. 1 is a graph showing the change in cell number of predatory protists in a culture system in which a wild-type strain, a mutant strain, or a transformant of strain 7002 was co-cultured with the predatory protists.

One embodiment of the present invention is described below, but the present invention is not limited to this. The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments and examples obtained by appropriately combining the technical means disclosed in the different embodiments and examples. In addition, all of the documents described in this specification are incorporated herein by reference. In this specification, when a numerical range is described as "A to B," the description intends "greater than or equal to A and less than or equal to B."

1. Basic principle of the present invention
The present inventors have been conducting research on Synechococcs sp. PCC 7002 (hereinafter referred to as "7002 strain"), a type of cyanobacteria. In the course of research, the inventors have found that, in some culture systems, a mutant strain of cyanobacteria has been generated in which the cell length has dramatically increased to tens to hundreds of μm or more, whereas normal cyanobacteria have a roughly spherical shape of about 10 μm. Furthermore, the inventors have found that the shape of the cells of the mutant strain has also changed significantly, such that, whereas the normal 7002 strain has a single-celled form, multiple cells are connected together without being separated and become filamentous. In addition, the inventors have found that the mutant strain predominates in the culture system in which the mutant strain has been generated, even though it was contaminated with ciliate protozoa, which are known as predatory protozoans. The fact that the mutant strain predominates in the culture system even in the presence of ciliate protozoans, which are predatory protozoans, means that the mutant strain has resistance to predation by predatory protozoans.

The inventors obtained multiple strains of the above mutant strain in independent experiments and determined the mutation points of each, leading to the new finding that the mutations were concentrated in the stage II sporulation protein D gene (hereinafter sometimes abbreviated as "spoIID gene"). As a result of further intensive research based on this finding, they discovered that by reducing or deleting the function of the spoIID gene in cyanobacteria, it is possible to induce changes in characteristics similar to those of the above mutant strain, in other words, it is possible to create a cyanobacterial mutant (transformant) that is resistant to predation, which led to the completion of the present invention.

The cyanobacterial mutants (mutant strains and transformants) discovered by the present inventors themselves have excellent resistance to predation by predatory protozoans, and therefore can suppress damage caused by contamination by predatory protozoans, regardless of the scale of the culture system. Such mutants are extremely useful as chassis strains for bioproduction. Furthermore, the transformants have a larger cell size than normal cyanobacteria, making them highly productive in bioproduction, and because the bacterial cells can be easily collected, they are also extremely useful from the perspective of improving the production efficiency of bioproduction.

In this way, one embodiment of the present invention can contribute to the achievement of the Sustainable Development Goals (SDGs), such as Goal 12 "Responsible Consumption and Production," by contributing to improving the production efficiency of bioproduction.

2. Mutants
A cyanobacterial mutant according to one embodiment of the present invention (hereinafter sometimes referred to as "the mutant"; furthermore, in this specification, "mutant" refers to a cyanobacterial mutant) is a cyanobacterial mutant in which the function of the spoIID gene is reduced compared to that of the wild-type spoIID gene or is missing.

Because this mutant has the above-mentioned structure, it grows to a much larger size than wild-type cyanobacteria (cyanobacteria before the function of the spoIID gene is reduced or deleted). Therefore, it has excellent resistance to predation by predatory protozoans. Furthermore, when this mutant is used for substance production (bioproduction), due to its size, (1) an increase in the production amount of the target substance (bioproduction) can be expected, and (2) it can be easily recovered by sedimentation, etc.

In this specification, the term "cyanobacteria mutant" refers to cyanobacteria that have genetic mutations and exhibit different characteristics (phenotypes) compared to wild-type cyanobacteria. Here, the "gene mutation" in "cyanobacteria mutant" refers to physical or structural changes in the base sequence, as well as structural or quantitative changes in the translation product (e.g., protein). The "gene mutation" in this mutant may be caused by artificial gene introduction, or may be caused by mutation. In other words, this mutant may be a cyanobacteria transformant (sometimes simply referred to as a "transformant") created by artificial gene introduction, or may be a mutant strain of cyanobacteria that has arisen by mutation (sometimes simply referred to as a "mutant strain").

<2-1. Blue-green algae＞
The cyanobacteria that can be used as a host (parent strain) for the present mutant is not particularly limited, and any organism of the Cyanobacteria can be used. Examples of organisms of the Cyanobacteria include organisms of the order Synechococcales, and more particularly, In particular, examples of organisms include those in the Synechococcaceae family, such as Anabaena sp., Synechocystis sp., and Synechococcus sp. More specifically, Synechococcus sp. PCC 7002 (referred to as 7002 strain) and Synechococcus elongatus PCC 7942 (referred to as 7942 strain) can be mentioned. In these cyanobacteria, there are no known causes other than the reduction or deletion of the function of the spoIID gene. Any mutant that has been transformed (for example, to impart a desired bioproduction ability) can also be used. In addition, among wild-type cyanobacteria, there are those that have a unicellular morphology and those that have a multicellular morphology. The unicellular morphology of the cyanobacteria is the host of the present mutant. The above-mentioned strains 7002 and 7942 are cyanobacteria having a unicellular morphology. Since the strains 7002 and 7942 have a unicellular morphology, they are easy to use as research subjects, and their characteristics are A huge amount of data has been accumulated about this, so it can be used favorably in, for example, bioproduction.

If the size of the cyanobacteria that serves as the host for this mutant is, for example, a cell length (longest diameter) of 10 μm or less, the size of this mutant may be, for example, a cell length of 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, 500 μm or more, or 750 μm or more. In this way, the cell length of this mutant can be several to several hundred times larger than that of the host cyanobacteria.

2-2. spoIID gene
In the present specification, the spoIID gene refers to a gene (homologous gene) encoding a SpoIID protein (hereinafter sometimes abbreviated as "SpoIID"), which is a protein having peptidoglycan lysis activity, and its homologue. That is, the spoIID gene in the present specification is a concept that includes its homologue gene. The spoIID gene (and its homologue gene) is a gene that is widely present throughout cyanobacteria, and is encoded, for example, in the 7002 strain at the SYNPCC7002_A1891 locus, and in the 7942 strain at the Synpcc7942_2012 locus. That is, in one embodiment of the present invention, (i) when the 7002 strain is used as a host for the present mutant, the spoIID gene is a gene encoded in the SYNPCC7002_A1891 locus, and (ii) when the 7942 strain is used as a host for the present mutant, the spoIID gene is a gene encoded in the Synpcc7942_2012 locus.

In this specification, "SpoIID and its homologs" refers to proteins having biological functions equivalent to SpoIID, i.e., proteins having the above-mentioned peptidoglycan lytic activity. Therefore, the spoIID gene according to one embodiment of the present invention can be said to be a gene encoding a protein having peptidoglycan lytic activity, or a gene encoding a polynucleotide corresponding to a protein having peptidoglycan lytic activity.

The spoIID gene according to one embodiment of the present invention may include a variant thereof. That is, in one embodiment of the present invention, the spoIID gene may be any of the following genes (a) to (d):
(a) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 or 2.

(b) A polynucleotide that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 2 and encodes a protein having peptidoglycan lytic activity.

(c) A polynucleotide having a base sequence that is 90% or more identical to the base sequence shown in SEQ ID NO: 1 or 2 and that encodes a protein having peptidoglycan lytic activity.

(d) A polynucleotide that consists of a base sequence in which one or more bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 1 or 2, and encodes a protein that has peptidoglycan lytic activity.

With regard to (a) above, the base sequence shown in SEQ ID NO: 1 corresponds to the base sequence of the gene encoded in the SYNPCC7002_A1891 locus of strain 7002, and the base sequence shown in SEQ ID NO: 2 corresponds to the base sequence of the gene encoded in the Synpcc7942_2012 locus of strain 7942. (b) to (d) represent variants of the gene shown in (a).

With regard to (b) above, "hybridizing under stringent conditions" refers to conditions in which hybridization is carried out in a hybridization solution with a salt concentration of 6x SSC at 50-60°C for 16 hours, followed by washing in a solution with a salt concentration of 0.1x SSC, followed by hybridization.

With regard to (c) above, the identity of the base sequences is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The identity of the base sequences can be determined, for example, using BLASTX.

With regard to (d) above, the upper limit of the number of bases substituted, deleted, inserted and/or added may be, for example, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1.

It can also be said that the spoIID gene according to one embodiment of the present invention is a gene encoding SpoIID and its variants. Thus, in one embodiment of the present invention, the spoIID gene may be a gene encoding a protein represented by any one of the following (e) to (g):
(e) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 or 4.

(f) A protein having peptidoglycan lytic activity, comprising an amino acid sequence in which one or more amino acid residues have been substituted, deleted, inserted and/or added from the amino acid sequence shown in SEQ ID NO: 3 or 4.

(g) A protein having an amino acid sequence that has 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3 or 4 and has peptidoglycan lytic activity.

With regard to (e) above, the amino acid sequence shown in SEQ ID NO: 3 corresponds to the amino acid sequence of SpoIID in the 7002 strain, and the amino acid sequence shown in SEQ ID NO: 2 corresponds to the amino acid sequence of the SpoIID homologue protein in the 7942 strain. (f) and (g) represent variants of the protein shown in (e).

With regard to (f) above, the upper limit of the number of amino acid residues substituted, deleted, inserted and/or added may be, for example, 37, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1.

With regard to (g) above, the identity of the amino acid sequence is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The identity of the amino acid sequence can be determined, for example, using BLASTX.

The present inventors have found that the amino acid sequences of SpoIID in strains 7002 and 7942 share 40% or more identity. This suggests that the amino acid sequences of SpoIID (and its homologues) are widely conserved between different cyanobacterial species with approximately 40% or more identity. Therefore, the spoIID gene according to one embodiment of the present invention can be expressed as follows: (1) a gene encoding a protein having peptidoglycan lytic activity and having an amino acid sequence that is 40% or more identical to the amino acid sequence of a protein encoded in the SYNPCC7002_A1891 locus in Synechococcus sp. PCC 7002, or (2) a gene encoding a protein having peptidoglycan lytic activity and having an amino acid sequence that is 40% or more identical to the amino acid sequence of a protein encoded in the Synpcc7942_2012 locus in Synechococcus elongatus PCC 7942.

In this specification, the function of the spoIID gene is reduced compared to the wild-type spoIID gene or is absent means that, due to the introduction of a genetic mutation into the spoIID gene, (1) the expression level of SpoIID encoded by the spoIID gene is reduced or lost compared to the wild-type spoIID gene, and (2) the activity of the expressed SpoIID is reduced or lost compared to the wild-type spoIID gene.

In this specification, "expression" of SpoIID means that a translation product is produced from the spoIID gene encoding the SpoIID and that it is localized at its site of action in a functional state (a state having normal activity). Therefore, a reduction or loss of expression of SpoIID means that the amount of SpoIID present in the cyanobacteria is reduced or lost due to the introduction of a genetic mutation into the spoIID gene. In addition, a reduction or loss of activity of SpoIID means that the normal function (activity) of SpoIID is partially or completely lost in the translation product produced from the spoIID gene.

Here, a decrease in the expression level of SpoIID means that the expression level of SpoIID in the mutant is decreased to 50% or less, preferably 25% or less, and more preferably 10% or less, compared to the expression level of SpoIID in wild-type cyanobacteria. Also, a decrease in the activity of SpoIID means that the activity of SpoIID in the mutant is decreased to 50% or less, preferably 25% or less, and more preferably 10% or less, compared to the activity of SpoIID in wild-type cyanobacteria. The expression level and activity of SpoIID can be measured by known immunological techniques, such as Western blotting and immunohistochemical staining.

In this specification, "wild type" refers to the cyanobacteria that serve as the host for this mutant, in other words, the cyanobacteria before the function of the spoIID gene is reduced or deleted. "Wild-type spoIID gene" refers to the spoIID gene in the wild-type cyanobacteria, and refers to the spoIID gene before the function of the gene is reduced or deleted. In one embodiment of the present invention, the "wild-type cyanobacteria" that serves as the host for this mutant may be a wild-type cyanobacteria strain, an artificially bred cyanobacteria strain, a transformant that has been further subjected to any transformation other than the reduction or deletion of the function of the spoIID gene, or a mutant.

As a mutant according to one embodiment of the present invention, a strain that has been internationally deposited under the Budapest Treaty with the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation (address: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under the accession number NITE BP-03734 (deposit date: August 24, 2022), NITE BP-03735 (deposit date: August 24, 2022), NITE BP-03736 (deposit date: August 24, 2022) or NITE BP-03737 (deposit date: August 24, 2022) can also be used. All of the above strains are mutant strains created by the present inventors using the 7002 strain as a host and reducing or deleting the function of the spoIID gene compared to the wild-type spoIID gene.

3. Method for producing mutants
In one embodiment of the present invention, there is provided a method for producing a cyanobacterial mutant (i.e., the present mutant), which comprises a step of reducing the function of the spoIID gene below that of the wild-type spoIID gene or deleting the spoIID gene. Note that in this specification, "reducing the function of the spoIID gene below that of the wild-type spoIID gene" may be abbreviated as "reducing the function of the spoIID gene".

In the method for producing this mutant, methods for reducing or eliminating the function of the spoIID gene include introducing a mutation into one or more bases in the base sequence of the spoIID gene, i.e., deleting part or all of the bases in the base sequence of the spoIID gene, or substituting or inserting another base sequence into the spoIID base sequence.

In the method for producing this mutant, the region in which a mutation is introduced to reduce or eliminate the function of the spoIID gene may be the transcription region of the spoIID gene, as well as a transcription regulatory region such as the promoter, enhancer (transcription activation region) or terminator of the spoIID gene, with the transcription region being preferred.

The transcriptional regulatory region of the spoIID gene can be a region within 1000 bases, preferably within 500 bases, upstream from the 5' end and downstream from the 3' end of the transcriptional region of the spoIID gene on the chromosomal DNA.

In the method for producing this mutant, the mutation introduced into the bases in the transcription region of the spoIID gene is not limited in type and number of bases, so long as it reduces or eliminates the function of the spoIID gene, and may be, for example, a deletion of one or more bases, a substitution of one or more bases, an addition of one or more bases, or any combination of these. More specifically, a mutation that reduces or eliminates the function of the spoIID gene is a mutation in which one or more amino acids are added, substituted, and/or deleted from the amino acid sequence encoded by the spoIID gene by the introduction of the mutation, and is preferably a mutation in which a frameshift mutation or a stop codon mutation is introduced.

Furthermore, the amino acid sequence of SpoIID is relatively highly conserved between species in a region of about 50 amino acids on the C-terminus (e.g., the region from positions 320 to 372 of SEQ ID NO: 3, or the region from positions 350 to 407 of SEQ ID NO: 4). From this, it is believed that the N-terminal region of SpoIID is the region that is largely involved in the part of SpoIID activity that affects the morphology of cyanobacteria. Therefore, from the viewpoint of more efficiently reducing or eliminating SpoIID activity, it is preferable that the mutation that reduces or eliminates the function of the spoIID gene introduced in the method for producing this mutant is a mutation that affects the amino acid sequence on the C-terminus of SpoIID (e.g., the amino acid sequence from positions 320 to 372 of SEQ ID NO: 3, or the amino acid sequence from positions 350 to 407 of SEQ ID NO: 4). More specifically, the mutation is preferably one in which one or more amino acids are added, substituted, and/or deleted from the amino acid sequence within 50 amino acids from the C-terminus of SpoIID, or a frameshift mutation or a stop codon mutation is introduced, resulting in partial or complete loss of a region consisting of the amino acid sequence within 50 amino acids from the C-terminus of SpoIID from the translation product.

Specific examples of mutations that reduce or eliminate the function of the spoIID gene include the following mutations (a1) to (a10) and (b1):
(a1) a mutation in which the glycine residue at position 342 in the amino acid sequence shown in SEQ ID NO: 3 is replaced with an arginine residue;
(a2) a mutation in which a histidine residue and a cysteine residue are inserted between the glutamine residue at position 349 and the glycine residue at position 350 in the amino acid sequence shown in SEQ ID NO: 3, and the glycine residue at position 350 is deleted;
(a3) a mutation in which the isoleucine residue at position 351 in the amino acid sequence shown in SEQ ID NO: 3 is replaced with a leucine residue;
(a4) a mutation that causes a frameshift at the asparagine residue at position 352 in the amino acid sequence shown in SEQ ID NO: 3;
(a5) a mutation that causes a frameshift at the tyrosine residue at position 353 in the amino acid sequence shown in SEQ ID NO: 3;
(a6) a mutation that causes a frameshift at the phenylalanine residue at position 82 in the amino acid sequence shown in SEQ ID NO: 3;
(a7) a mutation that causes a frameshift at the valine residue at position 19 in the amino acid sequence shown in SEQ ID NO: 3;
(a8) a mutation in which the glutamine residue at position 340 in the amino acid sequence shown in SEQ ID NO: 3 is replaced with an arginine residue;
(a9) a mutation that causes a frameshift at the isoleucine residue at position 54 in the amino acid sequence shown in SEQ ID NO: 3;
(a10) a mutation that causes a frameshift at the alanine residue at position 27 in the amino acid sequence shown in SEQ ID NO: 3;
(b1) A mutation that causes a frameshift in the amino acid sequence of SEQ ID NO:4 in which the proline residue at position 342 is replaced with a valine residue.

In other words, the mutant may be a mutant in which one or more of the mutations (a1) to (a10) described above have been introduced into the 7002 strain, or a mutant in which the mutation (b1) described above has been introduced into the 7942 strain.

The cyanobacterial strain internationally deposited under the Budapest Treaty with the accession number NITE BP-03734 is a cyanobacterial strain obtained by introducing the above-mentioned (a1) mutation into the 7002 strain, the cyanobacterial strain internationally deposited under the Budapest Treaty with the accession number NITE BP-03735 is a cyanobacterial strain obtained by introducing the above-mentioned (a6) mutation into the 7002 strain, the cyanobacterial strain internationally deposited under the Budapest Treaty with the accession number NITE BP-03736 is a cyanobacterial strain obtained by introducing the above-mentioned (a8) mutation into the 7002 strain, and the cyanobacterial strain internationally deposited under the Budapest Treaty with the accession number NITE BP-03737 is a cyanobacterial strain obtained by introducing the above-mentioned (a9) mutation into the 7002 strain.

A method for introducing a base mutation into the spoIID gene can be, for example, a method of preparing a mutant spoIID gene with a base mutation and then homologously recombining the mutant spoIID gene with the spoIID gene in a host cell. Another example is a method that utilizes bacteriophage or conjugation. In addition, in the method for producing this mutant, it is also possible to obtain a mutant in which the function of the spoIID gene is reduced or lost by subjecting the host to a mutation treatment such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS), ultraviolet light, or radiation treatment, analyzing the genes of the obtained mutant strain, and screening for mutant strains having a mutation in the spoIID gene.

The inventors have also found that this mutant, i.e., a mutant in which the function of the spoIID gene is reduced or deleted, can also be obtained by co-culturing the host cyanobacteria with a predatory protist (e.g., ochrophytes or ciliates) in an appropriate ratio (cell number ratio). In other words, in the method for producing this mutant, a method of co-culturing the host cyanobacteria with a predatory protist (e.g., ochrophytes or ciliates) in an appropriate ratio (cell number ratio) can also be used as a method for reducing or deleting the function of the spoIID gene.

(others)
An embodiment of the present invention may have the following configuration.

[1] A cyanobacterial mutant in which the function of the spoIID gene is reduced compared to that of the wild-type spoIID gene or the spoIID gene is absent.

[2] The cyanobacterium mutant described in [1], wherein the spoIID gene is a gene encoding a protein having an amino acid sequence that is 40% or more identical to the amino acid sequence of a protein encoded in the SYNPCC7002_A1891 locus in Synechococcus sp. PCC 7002 and has peptidoglycan lytic activity.

[3] The cyanobacterium mutant according to [1] or [2], wherein the spoIID gene is represented by any one of the following (a) to (d):
(a) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 or 2.

[4] The cyanobacterium mutant according to any one of [1] to [3], wherein the spoIID gene is a gene encoding a protein represented by any one of the following (e) to (g):
(e) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 or 4.

[5] A mutant cyanobacterium according to any one of [1] to [4], wherein (i) the cyanobacterium is Synechococcus sp. PCC 7002 and the spoIID gene is a gene encoded at the SYNPCC7002_A1891 locus, or (ii) the cyanobacterium is Synechococcus elongatus PCC 7942 and the spoIID gene is a gene encoded at the Synpcc7942_2012 locus.

[6] A mutant of Synechococcus sp. PCC 7002, the cyanobacterium mutant according to any one of [1] to [5], having the accession number NITE BP-03734, NITE BP-03735, NITE BP-03736 or NITE BP-03737.

[7] A method for producing a cyanobacterial mutant, comprising a step of reducing the function of the spoIID gene below that of the wild-type spoIID gene or deleting the gene.

Below, one embodiment of the present invention will be explained in more detail using examples, but the present invention is not limited to these examples.

1. Identification of gene mutations that lead to cell elongation
When marine cyanobacteria Synechococcus sp. PCC 7002 (7002 strain) and a marine ciliate Uronema marinum, a predatory protist, were co-cultured at a specific cell number ratio, the cell morphology changed significantly, and multiple mutant strains appeared in which the cell size extended to several tens to hundreds of μm in length. These mutant strains dominated the culture system even in the presence of predatory protists, and were considered to be mutant strains with predation resistance. A total of 12 such mutant strains were obtained in independent experimental systems. Next-generation sequence analysis (NGS) was performed on these mutant strains using a next-generation sequencer (Illumina MiSeq) according to a known method, and the genetic mutation points were determined. In addition, a mutant strain of S. elongatus PCC7942 (7942 strain) was obtained by co-cultivation with a predatory protozoan (Poterioochromonas malhamensis), and the mutant strain was also subjected to next-generation sequencing (NGS) to determine the genetic mutation points. The results of the next-generation sequencing analysis are shown in Table 1.

As is clear from Table 1, it was shown that a mutation occurred in the spoIID gene in 11 of the 12 mutant strains of 7002 (mutants Y1 to Y12). It was also shown that a mutation occurred in the spoIID (homolog) gene in the mutant strain of 7942 (mutant Y1'). These results suggest that the spoIID gene is strongly involved in the transformation of cyanobacteria into a filamentous form. Note that multiple gene mutations occurred in the mutant strains described above, and therefore other gene mutations may also be involved, but it is believed that the mutation in the spoIID gene has the greatest functional impact.

2. Preparation of transformants by mutation of spoIID gene
To investigate the involvement of the SpoIID gene in the cell morphological change, the SpoIID gene (SYNPCC7002_A1891, sometimes abbreviated as A1891) of the 7002 strain was disrupted. The A1891 gene sequence (SEQ ID NO: 1) of PCC7002 was obtained from the genome data of the 7942 strain published in the Kazusa Genome Resource (Cyanobase: http://genome.microbedb.jp/cyanobase/). These genes were then disrupted using homologous recombination. The gene disruption was carried out by creating a DNA fragment (Gm ^R ) in which a gentamicin resistance gene (SEQ ID NO: 5) was inserted between about 1 kb of each of the upstream and downstream sequences of A1891 (SEQ ID NO: 1) by overlap extension-PCR, and transforming the 7002 strain with about 0.1 μg of each of the DNA fragments created. The primers used were those shown in Table 2. 1891 Primer1_F and 1891 Primer3_R (sequence numbers 6 and 7) were used to amplify the A1891 upstream fragment, 1891 Primer6_F and 1891 Primer2_R (sequence numbers 8 and 9) were used to amplify the A1891 downstream fragment, and 1891 Primer4_F and 1891 Primer5_R (sequence numbers 10 and 11) were used to amplify the gentamicin resistance gene fragment.

The DNA fragment (SEQ ID NO: 12) for disrupting A1891 obtained by the above procedure was introduced into the 7002 strain by natural transformation. Specifically, the 7002 strain was first cultured in MA2 medium (50 mL), and after the OD750 reached about 0.7 to 1.0, the cells were harvested by centrifugation (6000 rpm, 5 min), and the harvested cells were resuspended in 1.0 mL of MA2 medium. About 0.1 μg of the DNA fragment (SEQ ID NO: 12) obtained by the above procedure was added to 400 μL of this suspension, and the mixture was mixed for 12 hours using a shaker in a 30° C. incubator while shielding from light with aluminum foil.

Then, the aluminum foil was removed and the mixture was mixed for another hour to obtain a mixture of bacteria and plasmid. This mixture was spread on an MA2 plate containing gentamicin (25 μg/mL) and cultured in a plant incubator (illuminance: 2000-3000 Lux, temperature: 30°C). Colonies that appeared after about 10 days were picked with a colony picker and streaked onto an MA2 plate having the same composition as the above plate. Gene disruption was confirmed by PCR, and it was confirmed that all A1891 genes present on the chromosome, of which multiple copies exist in the 7002 strain, had been disrupted. The resulting gene-disrupted strain was cultured in MA2 liquid medium supplemented with gentamicin and used for experiments such as morphological observation.

As a control, a strain was prepared by disrupting the SYNPCC7002_A1638 gene (SEQ ID NO: 13, sometimes abbreviated as A1638) in the 7002 strain by the same method. The DNA fragment used for gene disruption was prepared by inserting a spectinomycin resistance gene (SEQ ID NO: 14) between approximately 1 kb of the upstream and downstream sequences of A1638 (SEQ ID NO: 13). The primers used were those shown in Table 3. 1638 Primer1_F and 1638 Primer3_R (SEQ ID NOs: 15 and 16) were used to amplify the A1638 upstream fragment, 1638 Primer6_F and 1638 Primer2_R (SEQ ID NOs: 17 and 18) were used to amplify the A1638 downstream fragment, and 1638 Primer4_F and 1638 Primer5_R (SEQ ID NOs: 19 and 20) were used to amplify the spectinomycin resistance gene fragment. The obtained DNA fragment (Spc ^R , SEQ ID NO: 21) was used for transformation. The concentration of spectinomycin added to the medium was 40 μg/mL in all cases.

The appearance of the prepared transformants observed under a microscope is shown in FIG. 1. In FIG. 1, (1) WT indicates the appearance of the wild-type 7002 strain, (2) Mutant Y1 indicates the appearance of a mutant of 7002 strain (Mutant Y1) that arose in co-culture with a predatory protist, (3) Transformant 1 indicates the appearance of a transformant (gene-disrupted strain) of 7002 strain into which Gm ^R was introduced by the above-mentioned homologous recombination, and (4) Control indicates the appearance of a transformant of 7002 strain into which Spc ^R was introduced by the above-mentioned homologous recombination. As is clear from FIG. 1, the wild-type (upper left of FIG. 1) and control (lower right of FIG. 1) cells had a unicellular shape and their cell length was less than 20 μm. In contrast, in transformant 1 (lower panel of FIG. 1), like the cells of mutant strain Y1 (upper right panel of FIG. 1), individual cells were not separated but were connected and had a filamentous shape, and further, the cell length was elongated to 100 μm or more. This result showed that the mutation of the spoIID gene can induce the transformation of cyanobacteria into a filamentous form. In addition, the transformant obtained by mutation of the spoIID gene (transformant 1) had a shape similar to that of mutant strain Y1, which can dominate the culture system even in the presence of predatory protozoans, suggesting that it has predation resistance.

<3. Examination of predation resistance of transformants obtained by mutation of spoIID gene>
In order to examine the predation resistance of the cyanobacterial strains (mutant Y1 and transformant 1) exhibiting a filamentous morphology obtained by the above-mentioned mutation of the spoIID gene, the mutant and transformant were co-cultured with the marine ciliate Metanophrys sinensis (hereinafter sometimes abbreviated as "Metanophrys sinensis"), a marine predatory protist. The specific procedure was as follows.

50 mL of Medium A2 (MA2) medium was added to a filter tube (intended to be an adapter made by processing a culture test tube, stuffed with cotton wool, fitted with a silicone stopper, and set in the culture test tube), and a cyanobacterial culture solution with OD ₇₅₀ = 1 was added to the filter tube to make 0.5% volume (0.25 mL), thereby inoculating each cyanobacterial strain (WT, mutant strain Y1, transformant 1, and control) into the MA2 medium. Here, the OD ₇₅₀ value of the cyanobacterial culture solution was a value measured using a spectrophotometer, and the bacterial cell concentration of the cyanobacterial culture solution when the OD ₇₅₀ value = 1 is about 10 ⁸ cells·mL ^-1 . 2 μL of a culture solution of Metanophrys sinensis with 2000 cells was further added to this MA2 medium. The cell count of Metanophrys sinensis was measured directly using a fluorescent microscope (OLYMPUS BX51) and Cellsens (OLYMPUS). The filter tube was placed on a light irradiation shelf, and the temperature of the medium was adjusted to about 30°C under light irradiation of 50 μE/ ^m2 /s (4000 lx). Co-culture (culture of blue-green algae and Metanophrys sinensis in the filter tube) was performed under a state in which 2% _CO2 gas was aerated into the medium using a gas mixing device KOFLOC BR-2C. This culture system was a culture system with predators.

In addition, as a control (culture system without predation), each cyanobacterial strain was cultured (monoculture) under the same conditions as the above culture, except that Metanophrys sinensis was not inoculated.

The Medium A2 (MA2) medium used in the examples was prepared according to the following composition.
_NaNO3 : _1.49 g, _KH2PO4 : 50 mg _, _NaCl : 18 g _, MgSO4.7H2O: ₅ g, CaCl2.2H2O: 0.37 g _, KCl: 0.6 g, _Na2EDTA.2H2O _: ₃₂ mg, FeCl3.6H2O _: 8 mg _, H3BO3 _: 34 mg, _MnCl2.4H2O _: _4.3 mg, ZnCl2 _: 0.32 mg, _Na2MoO4.2H2O : ₅₀ μg, CuSO4.5H2O _: 3.0 μg, CoCl2.6H2O: _{0.2 mg} _. O: 12 μg, cobalamin: 4.0 μg, tris(hydroxymethyl)aminomethane: 8.3 mM, distilled water: 1 L.

During the culture period, a portion of the culture was collected and fixed every two days, and the OD ₇₅₀ value, the amount of chlorophyll, and the number of predators (Metanophrys sinensis) in the culture were measured. The culture was fixed by adding 10 μL of formaldehyde to 500 μL of the collected culture to a final concentration of 2%, and leaving it to stand for 15 minutes. The fixed culture was stored at 4 ° C. until measurement. The OD ₇₅₀ value of the culture was measured using a spectrophotometer, and the amount of chlorophyll was measured by the following procedure. 1 mL of the culture was placed in a 1.5 mL tube, the supernatant was removed, and only the bacterial components were collected. Next, 1 mL of 100% methanol was placed in the tube and stirred with a vortex mixer. After stirring, the mixture was left to stand for about 5 minutes, and then centrifuged at room temperature, 12,000 rpm, and 10 min, and the OD 665 of the resulting supernatant was measured. The OD665 measurement value was multiplied by 13.4 to obtain the amount of chlorophyll (μgChl/mL). The predators were measured by dropping 2 μL of the immobilized culture onto a highly water-repellent microslide glass (MATSUNAMI, TF0410) with a hole, and measuring the total number of cells in the culture using cellsens (OLYMPUS). Measurements were performed four times for each type of culture, and the cell density of the predators (cells/ml) was calculated as the average value of the four measurements.

Figure 2 shows the appearance of each culture system 5 days after the start of culture, Figure 3 shows the changes in OD ₇₅₀ value and chlorophyll content in each culture system during the culture period, and Figure 4 shows the changes in the number of predators in each culture system during the culture period.

2 and 3, in the culture system without predators, the WT and control strains having normal morphology had dark green filter tubes, and the OD ₇₅₀ value and chlorophyll content were also high, so that they could grow without any problems. On the other hand, in the culture system with predators, the WT and control strains lost color from the filter tubes, and the OD ₇₅₀ value and chlorophyll content were also very low, so that it can be seen that the cell number is greatly reduced by predation. On the other hand, the transformant 1 obtained by mutation of the spoIID gene, like the mutant strain Y1, had dark filter tubes regardless of the presence or absence of predators, and the elongation of the cells could be confirmed with the naked eye. In addition, the OD ₇₅₀ value and chlorophyll content also increased over time regardless of the presence or absence of predators. In other words, it can be seen that the transformant 1 (and mutant strain 1) can increase the cell number without being affected by predators.

Furthermore, from the change in predator cell number during the culture period shown in Figure 4, it can be seen that the predator significantly increased the cell number in the culture system co-cultured with the WT or control strain. On the other hand, it can be seen that the cell number was not increased in the culture system co-cultured with the mutant strain Y1 or transformant 1. The reason for the increase in the predator cell number in the culture system co-cultured with the WT or control strain is thought to be that the predator was able to obtain a large amount of resources by preying on these cyanobacterial strains. On the other hand, in the culture system co-cultured with the mutant strain Y1 or transformant 1, it is thought that the cell number was not increased because it was not able to prey on these cyanobacterial strains.

The above results show that transformant 1, a mutant obtained by mutating the spoIID gene, is not preyed upon by predators and can increase cell number even in the presence of predators, i.e., it is a mutant with predation resistance.

The present invention can be suitably used in fields requiring cyanobacterial mutants, particularly in the field of substance production using cyanobacterial mutants.

[Rule 26, amendment 23.10.2023]

Claims

A mutant of cyanobacteria in which the function of the spoIID gene is reduced compared to that of the wild-type spoIID gene or is absent.
The cyanobacterium mutant according to claim 1, wherein the spoIID gene is a gene encoding a protein having an amino acid sequence that is 40% or more identical to the amino acid sequence of a protein encoded at the SYNPCC7002_A1891 locus in Synechococcus sp. PCC 7002 and has peptidoglycan lytic activity.
The cyanobacterium mutant according to claim 1 or 2, wherein the spoIID gene is represented by any one of the following (a) to (d):
(a) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 or 2.
(b) a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 2 and encodes a protein having peptidoglycan lytic activity.
(c) a polynucleotide having a base sequence having 90% or more identity to the base sequence shown in SEQ ID NO: 1 or 2, and encoding a protein having peptidoglycan lytic activity.
(d) A polynucleotide consisting of a base sequence in which one or more bases have been deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 or 2, and encoding a protein having peptidoglycan lytic activity.
The cyanobacterium mutant according to claim 1 or 2, wherein the spoIID gene is a gene encoding a protein represented by any one of the following (e) to (g):
(e) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 or 4.
(f) A protein consisting of an amino acid sequence in which one or more amino acid residues have been substituted, deleted, inserted and/or added from the amino acid sequence shown in SEQ ID NO: 3 or 4, and having peptidoglycan lytic activity.
(g) A protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3 or 4, and having peptidoglycan lytic activity.
(i) the cyanobacterium is Synechococcus sp. PCC 7002, and the spoIID gene is a gene encoded by the SYNPCC7002_A1891 locus; or
(ii) The cyanobacterium mutant according to claim 1 or 2, wherein the cyanobacterium is Synechococcus elongatus PCC 7942 and the spoIID gene is a gene encoded by the Synpcc7942_2012 locus.
The cyanobacterium mutant according to claim 1 or 2, which is a mutant strain of Synechococcus sp. PCC 7002 and has the accession number NITE BP-03734, NITE BP-03735, NITE BP-03736 or NITE BP-03737.
A method for producing a cyanobacterial mutant, comprising a step of reducing the function of the spoIID gene below that of the wild-type spoIID gene or deleting the gene.