WO2022045110A1

WO2022045110A1 - Method of producing monoploid unicellular red alga, and culture medium for monoploid unicellular red alga

Info

Publication number: WO2022045110A1
Application number: PCT/JP2021/030937
Authority: WO
Inventors: 夢國分; 岳江原; 進也宮城島; 俊亮廣岡; 崇之藤原
Original assignee: Dic株式会社; 大学共同利用機関法人情報・システム研究機構
Priority date: 2020-08-24
Filing date: 2021-08-24
Publication date: 2022-03-03
Also published as: JPWO2022045110A1

Abstract

A method for producing a monoploid unicellular red alga, said method comprising culturing unicellular red alga cells in a culture medium containing 80 mM or more of an osmotic pressure regulator. A culture medium for a monoploid unicellular red alga, said culture medium containing 80 mM or more of an osmotic pressure regulator.

Description

Method for producing monoploid unicellular red algae and medium for monoploid unicellular red algae

The present invention relates to a method for producing a monoploid unicellular red alga and a medium for a monoploid unicellular red alga.
This application claims priority based on Japanese Patent Application No. 2020-141201 filed in Japan on August 24, 2020, the contents of which are incorporated herein by reference.

Since microalgae have a high carbon dioxide fixation capacity compared to land plants, and because their habitat does not compete with agricultural products, some species are mass-cultured to feed, functional foods, and cosmetic materials. It is used industrially as such.
When microalgae are used industrially, it is desirable that they are microalgaes that can be mass-cultured outdoors from the viewpoint of cost. However, in order to be a microalgae that can be mass-cultured outdoors, it must be resistant to environmental changes (light, temperature, etc.), can be cultivated under conditions where other organisms cannot survive, and can grow to high densities. Conditions such as that are required.

Some unicellular red algae preferentially grow in sulfuric acid acidic hot springs. Such unicellular red algae may be characterized in that they can be cultivated in an environment in which other organisms such as high salt concentration, high temperature, and low pH are difficult to grow. Therefore, such unicellular red algae are considered to be suitable for industrial use. Further, if a desired trait can be imparted to unicellular red algae by gene modification technology or the like, it becomes possible to produce a cell line more suitable for industrial use.

Generally, haploid cells are considered to be suitable for gene modification. In nature, most unicellular red algae exist as polyploid (eg, diploid) cells. Therefore, if monoploid cells can be produced from polyploid cells, it is considered that gene modification will be facilitated. For example, Patent Document 1 describes that diploid cells could be produced from diploid cells in the algae of Cyanidiophyceae, which are unicellular red algae.

International Publication No. 2019/107385

With the method described in Patent Document 1, it is difficult to stably maintain haploid cells produced from diploid cells for a long period of time. Therefore, a technique capable of stably maintaining haploid cells for a long period of time is required.

Therefore, it is an object of the present invention to provide a method for producing a single-layered unicellular red algae capable of stably maintaining single-layered cells, and a medium for single-sized single-celled red algae.

The present invention includes the following aspects.
[1] A method for producing a unicellular single-celled red alga, which comprises culturing single-celled red algae in a medium containing an osmotic pressure regulator of 80 mM or more.
[2] A method for producing unicellular single-celled red algae, which comprises culturing single-celled red algae cells in a medium having an osmotic pressure of 150 mOsm / kg or more.
[3] The method for producing a unicellular single-celled red alga according to [1] or [2], wherein the osmotic pressure adjusting agent is at least one selected from the group consisting of sugar, sugar alcohol, and amino acid.
[4] The method for producing a unicellular single-celled red alga according to any one of [1] to [3], wherein the single-celled red alga is a polyploid cell.
[5] The method for producing a unicellular unicellular red alga according to [4], wherein the unicellular red alga is a diploid cell.
[6] The method for producing a unicellular single-celled red alga according to any one of [1] to [3], wherein the single-celled red alga is a monoploid cell.
[7] The method for producing a unicellular unicellular red alga according to [6], which is a method for maintaining unicellular red algae cells in a haploid state.
[8] The method for producing a unicellular unicellular red alga according to [6], which is a method for growing unicellular unicellular red algae cells.
[9] A medium for monoploid unicellular red algae containing 80 mM or more of an osmotic pressure regulator.
[10] Medium for monoploid unicellular red algae having an osmotic pressure of 150 mOsm / kg or more.
[11] The medium for single-cell single-cell red algae according to [9] or [10], wherein the osmotic pressure regulator is at least one selected from the group consisting of sugar, sugar alcohol, and amino acid.
[12] The monoploid single-cell red alga according to any one of [9] to [11], which is used to produce a monoploid single-cell red alga from a polyploid single-cell red alga. Algae medium.
[13] The medium for monoploid single-celled red algae according to [12], wherein the polyploid single-celled red algae cells are diploid single-celled red algae cells.
[14] The medium for haploid unicellular red algae according to any one of [9] to [11], which is used to maintain haploid unicellular red algae cells as haploid. ..
[15] The medium for monoploid unicellular red algae according to any one of [9] to [11], which is used for proliferating monoploid unicellular red algae cells.

According to the present invention, there is provided a method for producing a monoploid unicellular red alga that can stably maintain monoploid cells, and a medium for monoploid unicellular red algae.

An example of a haploid colony resulting from a diploid unicellular red algae cell is shown. A photograph of an agar plate in which monoploid cells of the CCCryo127-00 strain were subcultured and cultured on an 18% sorbitol + Gross 1.5% agar medium is shown. The photograph of the plate which cultured the haploid cell of CCCryo127-00 strain for one month in 18% sorbitol + Gross 1.5% agar medium is shown. The photograph of the plate which cultured the haploid cell of CCCryo127-00 strain for 2 weeks in 1% sorbitol + Gross 1.5% agar medium is shown. Return to diploid cells was confirmed. An example in which haploid cells of the CCCryo127-00 strain proliferated in 18% sorbitol + Gross 1.5% agar medium is shown. The photo on the left is the plate at the start of culture, and the photo on the right is the plate after 3 weeks of culture. An example in which haploid cells were produced from a diploid CCCryo127-00 strain on 18% sorbitol + Gloss 1.5% agar medium is shown. Ploidy cells were also maintained on the inoculated agar medium. An example in which haploid cells of CCCryo127-00 strain were grown in 18% sorbitol + Gross liquid medium is shown.

The cells described herein can be isolated. "Isolated" means a state isolated from the natural state.

<Manufacturing method of monoploid unicellular red algae>
A first aspect of the present invention is a method for producing a monocellular single-celled red alga, which comprises culturing single-celled red algae cells in a medium containing an osmotic pressure regulator of 80 mM or more.
An object of the present invention is to provide a method for producing a haploid unicellular red alga that can stably maintain haploid cells. In the present invention, when the haploid can be maintained for more than 2 weeks, it can be determined that "the haploid cells can be stably maintained".

(Unicellular red algae)
"Unicellular red algae" refers to algae belonging to the phylum Red algae (Rhodophyta), which are unicellular. Examples of single-celled red algae include Cyanidiophyceae, Stylonematophyceae, Porphyridiophyceae, and Rhodellophyceae. Among these, Cyanidiophyceae is preferable because it is easy to stably maintain the haploid. The genus Cyanidioschyzon, the genus Cyanidio, and the genus Galdieria are known as Cyanidiophyceae. The genus Cyanidioschyzon melolae exists as a diploid in nature, whereas the genus Cyanidioschyzon and the genus Garderia exist as diploids in nature. Therefore, among the Cyanidiophyceae, the genus Cyanidium and the genus Garderia are preferable, and the genus Garderia is more preferable.
The genus Garderia includes, for example, G.I. sulphuraria, G.M. Partita, G.M. daedala, G.M. Examples include, but are not limited to, maxima and the like. As for the genus Garderia, G. Sulfuraria is particularly preferred.
Examples of the genus Cianidium include C.I. Caldarium, C.I. sp. Examples include, but are not limited to, Monte Rotaro.
Examples of the algae strain of Cyanidiophyceae include those shown in FIG. 10 of International Publication No. 2019/107385.

In the method of this embodiment, the unicellular red algae cells used at the start of culture may be polyploid (for example, diploid) or monoploid.
When polyploid cells are cultured as monocellular red algae cells by the method of this embodiment, the polyploid cells undergo meiosis during the culture to give rise to monoploid cells. By continuing the culture by the method of this embodiment, the haploid cells can be maintained as haploid without returning to the polyploid. Therefore, the method of this embodiment includes a method for producing a unicellular unicellular red alga from a polyploid unicellular red alga.
When a haploid cell is cultured as a unicellular red alga by the method of this embodiment, the haploid cell is maintained as a haploid without returning to the polyploid cell during the culture. Therefore, the method of this embodiment includes a method of maintaining monoploid unicellular red algae cells.
By the method of this embodiment, monoploid unicellular red algae cells proliferate as haploid during culture. Therefore, the method of this embodiment includes a method of growing a monoploid unicellular red alga.

(Culture medium)
The medium used in the method of this embodiment is a medium containing 80 mM or more of an osmotic pressure adjusting agent.
"Osmotic pressure regulator" refers to a chemical substance that can adjust the osmotic pressure. The osmotic pressure adjusting agent is not particularly limited as long as it is a chemical substance whose osmotic pressure can be adjusted by adding it to the medium. Examples of the osmotic pressure adjusting agent include sugars, sugar alcohols, amino acids, metal salts, ureas, proteins, betaines, inositol, polysaccharides and the like. Among these, sugars, sugar alcohols, amino acids, and metal salts are preferable.

Examples of sugars include dihydroxyacetone, glyceraldehyde, elittlerose, erythrose, treose, ribulose, xylrose, ribose, arabinose, xylose, liquisource, deoxyribose, psicose, fructose, sucrose, tagatose, allose, altorose, glucose and mannose. , Growth, idose, galactose, tarose, fucose, fructose, ramnorse, sedhepturose and other monosaccharides (either D-form or L-form, or a mixture of D-form and L-form); sucrose, lactose. , Lactose, Martose, Trehalose, Cellobiose, Kojibiose, Nigerose, Isomartose, β, β-trehalose, α, β-trehalose, Sophorose, Laminaribiose, Genthiobiose, Turanose, Marzulose, Palatinose, Genthiobiulose, Mannobious , Meribiulose, neolactos, galactosucrose, silaviose, neohesperidos, lucinose, lucinulose, bicianose, xylobiose, primeberos, trehalosamine, martitol, cellobionic acid, lactosamine, lactosediamine, lactobionic acid, lactitol, hyalobiuronic acid, sucrose. Sugars; trisaccharides such as nigerotriose, maltotriose, meregitos, maltotriulose, raffinose, kestose; tetrasaccharides such as nistose, nigerotetraose, stakiose; and lactose-fructose oligosaccharides, lactosucrose, maltooligosaccharides, isomaltooligo Examples thereof include, but are not limited to, sugars, genthio-oligosaccharides, nigerooligosaccharides, fructose-oligosaccharides, galactooligosaccharides, mannan oligosaccharides, xylooligosaccharides, soybean oligosaccharides and the like. As the sugar, monosaccharides or disaccharides are preferable.
Monosaccharides include dihydroxyacetone, glyceraldehyde, erythrose, erythrose, treose, ribulose, xylrose, ribose, arabinose, xylose, lyxose, deoxyribose, psicose, fructose, sorbose, tagatose, allose, altrose, glucose, mannose, Growth, idose, galactose, tarose, fucose, fuclos, ramnorse, sedoheptulose are preferred, dihydroxyacetone, glyceraldehyde, elittlerose, erythrose, ribulose, ribose, arabinose, xylose, deoxyribose, fructose, glucose, mannose, galactose, or sedoheptulose. Is more preferred, and glucose is even more preferred.
Disaccharides include sucrose, lacturose, lactose, maltose, trehalose, cellobiose, cozybiose, nigerose, isomaltose, β, β-trehalose, α, β-trehalose, sophorose, laminaribiose, gentiobiose, turanoth, malturose, palatinose, Genthioviulose, Mannoviose, Meribiose, Meribiulose, Neolactos, Galac sucrose, Syrabios, Neohesperidos, Lucinose, Lucinulose, Visianose, Xylobiose, Primeberose, Trehalosemin, Martinol, Cerobionic acid, Lactosamine, Lactosediamine, Lactobionic acid. , Hyalobiuronic acid, or sucrose is preferred, sucrose, lacturose, lactose, sucrose, trehalose, or cellobiose is more preferred, and sucrose is even more preferred.

Examples of the sugar alcohol include trivalent sugar alcohols such as glycerol; tetravalent sugar alcohols such as erythritol, D-trateol, and L-treitol; D-arabinitol, L-arabinitol, xylitol, rivitol, adonitol and the like. Pentavalent sugar alcohols; hexavalent sugar alcohols such as D-iditol, galactitol, darsitol, D-glucitol, sorbitol, mannitol; Examples include, but are not limited to, octavalent sugar alcohols such as octitol; 9-valent sugar alcohols such as isomalt, lactitol, and martitol; and mixtures of sugar alcohols such as HSH and reduced water candy sugar. As the sugar alcohol, a trivalent sugar alcohol, a tetravalent sugar alcohol, a pentavalent sugar alcohol, a hexavalent sugar alcohol, a nine-valent sugar alcohol, or a mixture of sugar alcohols is preferable, and a trivalent sugar alcohol or a mixture of sugar alcohols is preferable. Valuable sugar alcohols are more preferred, and hexavalent sugar alcohols are even more preferred.
As the sugar alcohol, glycerol, erythritol, xylitol, sorbitol, mannitol, isomalt, lactitol, maltitol, HSH, or reduced water candy are preferable, and mannitol or sorbitol is more preferable.

The amino acid may be either D-form or L-form, or may be a mixture of D-form and L-form. The amino acid may be any of α-amino acid, β-amino acid, γ-amino acid, and δ-amino acid. Amino acids include, for example, alanine, aspartic acid, aspartic acid, cysteine, glutamic acid, glutamine, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, arginine, serine, treonine, serenocysteine, valine, tryptophan, tyrosine, 2-aminoadipic acid, 3-aminoadipic acid, 2-aminobutanoic acid, 2,4-diaminobutanoic acid, 2-aminohexanoic acid, 6-aminohexanoic acid, β-alanine, 2-aminopentanoic acid, 2,3 -Diaminopropanoic acid, 2-aminopimeric acid, 2,6-diaminopimeric acid, citrulin, cysteine acid, 4-carboxyglutamic acid, 5-oxoproline, pyroglutamic acid, homocysteine, homoserine, homoserine lactone, 5-hydroxylysine, allohydroxy Examples thereof include lysine, 3-hydroxyproline, 4-hydroxyproline, alloisolysin, norleucine, nolvalin, ornithine, sarcosin, allotoreonin, tyrosin and the like. Amino acids are preferably alanine, aspartic acid, aspartic acid, cysteine, glutamic acid, glutamine, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, arginine, serine, treonine, selenocysteine, valine, tryptophan, or tyrosine. , Glycine, proline, or arginine are more preferred.

Examples of metal salts include alkali metals (sodium, potassium, etc.) or alkaline earth metals (magnesium, calcium, etc.) and inorganic acids (hydrogen, sulfuric acid, carbonic acid, sulfite, nitrate, etc.) or organic acids (lactic acid, succinic acid, etc.). , Acetic acid, etc.) and salts. As the metal salt, a salt of an alkali metal or an alkaline earth metal and an inorganic acid is preferable, potassium chloride, sodium sulfate or the like is more preferable, and potassium chloride is further preferable.

Among these, the osmotic pressure adjusting agent is preferably at least one selected from the group consisting of sugars, sugar alcohols, and amino acids because it is easy to add to the medium to adjust the osmotic pressure. Suitable sugars include glucose and sucrose. Suitable sugar alcohols include hexavalent sugar alcohols (eg, mannitol, sorbitol). Suitable amino acids include glycine, proline and arginine.
The osmotic pressure adjusting agent may be used alone or in combination of two or more.

The medium is not particularly limited as long as it contains 80 mM or more of the osmotic pressure adjusting agent. The medium can be prepared, for example, by adding an osmotic pressure adjusting agent to a medium known as a medium for unicellular algae so as to be 80 mM or more. The medium for single-celled algae is not particularly limited, and examples thereof include an inorganic salt medium containing a nitrogen source, a phosphorus source, and trace elements (zinc, boron, cobalt, copper, manganese, molybdenum, iron, etc.). For example, examples of the nitrogen source include ammonium salts, nitrates, nitrites and the like, and examples of the phosphorus source include phosphates and the like. Examples of such a medium include Gross medium, 2 × Allen medium (Allen MB. Arch. Microbiol. 1959 32: 270-277.), M-Alllen medium (Minoda A et al. Plant Cell Physiol. 2004 45: 667-71.), MA2 medium (Ohnuma M et al. Plant Cell Physiol. 2008 Jan; 49 (1): 117-20.), Modified M-Alllen medium, etc., but is not limited thereto.

The single-celled red algae may be autotrophically cultured under light irradiation, or may be heterotrophically cultured in the dark. In the case of heterotrophic culture, a carbon source (glucose or the like) may be added to the above-mentioned inorganic salt medium.

The concentration of the osmotic pressure regulator in the medium is not particularly limited as long as it is 80 mM or more. By setting the concentration of the osmotic pressure adjusting agent to 80 mM or more, haploid cells can be stably maintained regardless of the type of the osmotic pressure adjusting agent. The concentration of the osmotic pressure regulator is 100 mM or more, 110 mM or more, 120 mM or more, 130 mM or more, 140 mM or more, 150 mM or more, 160 mM or more, 170 mM or more, 180 mM or more, 190 mM or more, 200 mM or more, 210 mM or more, 220 mM or more, 230 mM or more, It may be 240 mM or more, 250 mM or more, 260 mM or more, 270 mM or more, 280 mM or more, 290 mM or more, 300 mM or more, 350 mM or more, 360 mM or more, 370 mM or more, 380 mM or more, 390 mM or more, or 400 mM or more. The upper limit concentration of the osmotic pressure adjusting agent is not particularly limited and may be a limit value that can be dissolved in the medium. From the viewpoint of cell growth rate, the upper limit concentration of the osmotic pressure regulator is, for example, 2M or less, 1.5M or less, 1.4M or less, 1.3M or less, 1.2M or less, 1.1M or less, or 1M. It can be as follows. Examples of the concentration range of the osmotic pressure adjusting agent in the medium include 80 mM to 2 M. The lower limit value and the upper limit value can be arbitrarily combined. As the concentration range of the osmotic pressure adjusting agent, for example, 100 mM to 1.5 M is preferable, 200 mM to 1.4 M is more preferable, 300 mM to 1.3 M is further preferable, and 400 mM to 1.3 M is particularly preferable.
Unless otherwise specified, the concentration of the osmotic pressure regulator in the medium is the concentration before the start of culture. When two or more kinds of osmotic pressure adjusting agents are used in combination, the total content of the two or more kinds of osmotic pressure adjusting agents may be 80 mM or more. The same applies to the range exemplified as the concentration of the osmotic pressure adjusting agent.

For example, when the osmotic pressure regulator is glucose, the glucose concentration in the medium includes, for example, 200 mM to 2 M, preferably 250 mM to 1.7 M, and more preferably 270 mM to 1.5 M. Alternatively, the glucose concentration in the medium is preferably 4 to 40% by mass, more preferably 5 to 30% by mass, based on the total mass (100% by mass) of the medium.
For example, when the osmotic pressure adjusting agent is sucrose, the sucrose concentration in the medium includes, for example, 80 mM to 1.1 M, preferably 80 mM to 800 mM, and more preferably 80 mM to 600 M. Alternatively, the concentration of sucrose in the medium is preferably 2 to 40% by mass, more preferably 3 to 30% by mass, still more preferably 3 to 20% by mass, based on the total mass (100% by mass) of the medium.
For example, when the osmotic pressure adjusting agent is glycerol, the glycerol concentration in the medium may be, for example, 200 mM to 800 mM, preferably 300 mM to 600 mM. Alternatively, the glycerol concentration in the medium is preferably 3 to 6% by mass with respect to the total mass (100% by mass) of the medium.
For example, when the osmotic pressure adjusting agent is mannitol, the mannitol concentration in the medium includes, for example, 180 mM to 1.5 M, preferably 200 mM to 1.2 M, and more preferably 250 mM to 1 M. Alternatively, the concentration of mannitol in the medium is preferably 4 to 20% by mass, more preferably 5 to 18% by mass, based on the total mass (100% by mass) of the medium.
For example, when the osmotic pressure adjusting agent is sorbitol, the sorbitol concentration in the medium includes, for example, 200 mM to 2 M, preferably 400 mM to 1.5 M, and more preferably 430 mM to 1.5 M. Alternatively, the sorbitol concentration in the medium is preferably 5 to 40% by mass, more preferably 8 to 27% by mass, based on the total mass (100% by mass) of the medium.
For example, when the osmotic pressure adjusting agent is glycine, the glycine concentration in the medium includes, for example, 100 mM to 2 M, preferably 120 mM to 1.5 M, and more preferably 130 mM to 1 M. Alternatively, the glycine concentration in the medium is preferably 0.5 to 10% by mass, more preferably 1 to 8% by mass, based on the total mass (100% by mass) of the medium.
For example, when the osmotic pressure adjusting agent is proline, the proline concentration in the medium includes, for example, 80 mM to 2 M, preferably 500 mM to 1.5 M, and more preferably 600 mM to 1.5 M. Alternatively, the propane concentration in the medium is preferably 1 to 20% by mass, more preferably 7 to 10% by mass, based on the total mass (100% by mass) of the medium.
For example, when the osmotic pressure adjusting agent is arginine, the arginine concentration in the medium includes, for example, 20 mM to 2 M, preferably 30 mM to 1.5 M, and more preferably 50 mM to 1 M. Alternatively, the arginine concentration in the medium is preferably 0.5 to 30% by mass, more preferably 1 to 20% by mass, based on the total mass (100% by mass) of the medium.
For example, when the osmotic pressure adjusting agent is potassium chloride, the potassium chloride concentration in the medium includes, for example, 50 mM to 1.5 M, preferably 100 mM to 1 M, and more preferably 130 mM to 500 mM. Alternatively, the potassium chloride concentration in the medium is preferably 0.5 to 10% by mass, more preferably 1 to 5% by mass, based on the total mass (100% by mass) of the medium.

The medium preferably has an osmotic pressure of 150 mOsm / kg or more. By setting the osmotic pressure of the medium to 150 mOsm / kg or more, haploid cells can be stably maintained regardless of the type of osmotic pressure adjusting agent. The osmotic pressure is 200 mOsm / kg or more, 210 mOsm / kg or more, 220 mOsm / kg or more, 230 mOsm / kg or more, 240 mOsm / kg or more, 250 mOsm / kg or more, 260 mOsm / kg or more, 270 mOsm / kg or more, 280 mOsm / kg or more, 290 mOsm. / Kg or more, 300 mOsm / kg or more, 310 mOsm / kg or more, 320 mOsm / kg or more, 330 mOsm / kg or more, 340 mOsm / kg or more, 350 mOsm / kg or more, 360 mOsm / kg or more, 370 mOsm / kg or more, 380 mOsm / kg or more, 390 mOsm It may be / kg or more, or 400 mOsm / kg or more. The upper limit of the osmotic pressure is not particularly limited, and may be a limit value at which the osmotic pressure adjusting agent can be dissolved in the medium. From the viewpoint of cell proliferation rate, the upper limit of osmotic pressure can be, for example, 2000 mOsm / kg or less, 1500 mOsm / kg or less, or 1400 mOsm / kg or less. The lower limit value and the upper limit value can be arbitrarily combined. The range of osmotic pressure of the medium includes, for example, 150 to 2000 mOsm / kg. As the osmotic pressure range, for example, 200 to 1500 mOsm / kg is preferable, 250 to 1400 mOsm / kg is more preferable, 300 to 1400 mOsm / kg is further preferable, and 400 to 1400 mOsm / kg is particularly preferable.
The osmotic pressure of the medium is a value before the start of culture unless otherwise specified. The osmotic pressure of the medium can be measured using an osmometer.

The medium may be a liquid medium or a solid medium. As the solid medium, for example, an agar medium can be used. In the case of a solid medium, the concentration and osmotic pressure of the above-mentioned osmotic pressure adjusting agent may be those in the liquid medium before the addition of the solidifying agent (for example, agar).

The above-exemplified medium can be used for producing haploid cells from polyploid cells, maintaining haploid cells, and proliferating haploid cells.
When producing haploid cells from polyploid cells, for example, the concentration of the osmotic pressure adjusting agent is preferably 50 mM to 2 M, more preferably 100 mM to 1.5 M. The osmotic pressure of the medium is preferably 150 to 2610 mOsm / kg, more preferably 300 to 1700 mOsm / kg. The medium may be a liquid medium or a solid medium, but it is preferable to use a solid medium because it is easy to determine that haploids have been formed.
When maintaining haploid cells, for example, the concentration of the osmotic pressure regulator is preferably 50 mM to 2 M, more preferably 100 mM to 1.5 M. The osmotic pressure of the medium is preferably 150 to 2610 mOsm / kg, more preferably 300 to 1700 mOsm / kg. The medium may be a liquid medium or a solid medium, but it is preferable to use a solid medium because it is easy to maintain a stable medium for a long period of time.
When growing haploid cells, for example, the concentration of the osmotic pressure regulator is preferably 50 mM to 2 M, more preferably 100 mM to 1.5 M. The osmotic pressure of the medium is preferably 150 to 2610 mOsm / kg, more preferably 300 to 1700 mOsm / kg. The medium may be a liquid medium or a solid medium, but it is preferable to use a liquid medium because cells can easily grow.

(Culture conditions)
The method of this embodiment comprises culturing unicellular red algae cells in a medium containing 80 mM or more of an osmotic pressure regulator. The culture conditions in the above culture are not particularly limited, and conditions usually used as culture conditions for unicellular red algae can be used. Examples of the culture conditions include pH 1 to 8, temperature 10 to 50 ° C., CO ₂ concentration 0.3 to 3%, and the like. Light conditions may be dark when heterotrophic culturing. In the case of autotrophic culture, the light conditions include, for example, 5 to 2000 μmol / m ² s.
The culture conditions are not limited to those exemplified above, and can be appropriately selected depending on the type of unicellular red algae. For example, when the unicellular red alga is Cyanidiophyceae, the pH conditions include pH 1.0 to 6.0, preferably pH 1.0 to 5.0, and more preferably pH 1.0 to 3.0. Examples of the temperature condition include 15 to 50 ° C, preferably 30 to 50 ° C, and more preferably 35 to 50 ° C. Examples of the light intensity include 5 to 2000 μmol / m ² s, and 5 to 1500 μmol / m ² s is preferable. It may be cultured with continuous light, or a light-dark cycle (10L: 14D, etc.) may be provided. In addition, in the case of heterotrophic culture, it can also be cultured in a dark place.

The culture period is not particularly limited. When the unicellular red algae cells used at the start of culture are polyploid (for example, diploid), the cells are cultured until at least monoploid cells are generated. In the method of this embodiment, haploid cells can be generated in a short period of time by using a medium containing 80 mM or more of an osmotic pressure regulator. When producing a haploid from a polyploid, for example, the culture period is preferably 5 days or longer, more preferably 10 days or longer, still more preferably 14 days or 15 days or longer. In the method of this embodiment, the haploid cells generated during the culture are stably maintained. Therefore, the upper limit of the culture period is not particularly limited.
When the unicellular red algae cells used at the start of culture are haploid, the culture period is not particularly limited. In the method of this embodiment, since the haploid cells are stably maintained, the culture may be continued for a period in which the haploids need to be maintained.

During the culture period, unicellular red algae cells may be subcultured as appropriate. In the method of this embodiment, the haploid can be stably maintained for 2 weeks or more in the same medium. Therefore, the interval between passages can be two weeks or more. For example, by subculturing haploid unicellular red algae cells at intervals of once every 1 to 3 months, haploid cells can be maintained more stably. The passage interval is preferably 1 to 1.5 months. When growing haploid cells, passage may be performed at shorter intervals in order to increase the growth efficiency. For example, the passage interval for proliferation is preferably 14 to 60 days, more preferably 14 to 42 days.

(How to check haploid)
In the method of this embodiment, haploid cells can be produced from polyploid unicellular red algae cells, and haploid cells can be stably maintained for 2 weeks or more. The method for confirming that the unicellular red algae cells are haploid is not particularly limited, and a known method can be used.

For example, the determination of haploid can be made by confirming the number of copies of the same locus. That is, if the number of copies of the same locus is 1, it is determined to be haploid.
A next-generation sequencer can also be used to determine that it is haploid. For example, sequence reads of the entire genome are acquired by a next-generation sequencer, the sequence reads are assembled, and then the sequence reads are mapped to the sequence obtained by assembling. In diploid, differences in bases for each allele can be found in various regions on the genome, but in diploid, only one allele exists, so such a region cannot be found.
When the cell is homodiploid, it can be determined whether the cell is diploid or diploid by measuring the DNA content of the cell. The DNA content of haploid cells is ½ of the DNA content of diploid cells.

In addition, when the diploid cell does not have a strong cell wall and the diploid cell is a single-celled red alga cell (for example, Ideyukogome class) having a strong cell wall, by observing the cell morphology. You can distinguish haploid cells. For example, in haploid cells, the cell wall is usually not observed when observed with an optical microscope (for example, at a magnification of 600 times). Therefore, if the cell wall is not observed by an optical microscope, it can be determined that the cell is a haploid cell.
Further, since the haploid cells of the unicellular red algae cells as described above do not have a strong cell wall, they are treated relatively mildly (neutralization treatment, hypotonic treatment, freeze-thaw treatment, surfactant treatment). Etc.) can destroy cells. For example, if cells are suspended in a medium containing 2% by mass of a surfactant and the cells disintegrate immediately to 5 minutes after the addition of the surfactant, it can be determined that the cells are haploid. can. Examples of the surfactant include sodium dodecyl sulfate. More specifically, sodium dodecyl sulfate is added to the culture medium of algae of unicellular red algae so as to be 2% by mass, and if the cells are disrupted within 5 minutes after the addition, the cells are diploid. It can be determined that there is. Whether or not the cells have collapsed can be confirmed by observing the cells with an optical microscope.
In addition, when culturing in a solid medium, it is possible to determine whether the cells are haploid cells based on the shape of the colonies. Since haploid cells do not have a strong cell wall, they are flatter and have a shape that spreads on the surface of a solid medium as compared with a colony of diploid cells. When a colony having such a shape appears on a solid medium, it can be determined to be a haploid colony.

According to the method of this embodiment, a ploidy single-celled red alga can be produced from a polyploid single-celled red alga, and a monoploid single-celled red alga can be stably maintained. In addition, monoploid unicellular red algae can be grown as they are.
When the monoploid monoploid of a single-celled red alga is cultured in a normal medium, cells that return to the polyploid (for example, diploid) appear, and the polyploid cells proliferate. Therefore, it is necessary to select haploid cells at intervals of about 5 days and repeat the passage. On the other hand, according to the method of this embodiment, the appearance of cells regressing to polyploid is suppressed, and monoploid cells are allowed to grow for more than 2 weeks (preferably 1 month or more) without subculture. Can be maintained.

Since the haploid unicellular red alga produced, maintained or propagated by the method of this embodiment has only one set of genomes, gene modification can be easily performed. Therefore, the method of this embodiment can be suitably used for producing unicellular red algae for gene modification.

<Medium for monoploid unicellular red algae>
A second aspect of the present invention is a medium for monoploid unicellular red algae containing 80 mM or more of an osmotic pressure regulator.

The medium of this embodiment is the same as that described in the above "<Method for producing monoploid unicellular red algae>". The medium of this embodiment can be used to produce haploid single-celled red algae cells from polyploid (eg, diploid) single-celled red algae cells. It can also be used to maintain haploid unicellular red algae cells as haploid. In addition, monoploid unicellular red algae cells can be used for proliferation.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.

<Unicellular red algae>
As the unicellular red alga, Galdia sulphuraria CCCryo127-00 strain (hereinafter, also referred to as "CCCryo127-00 strain") was used.

<Medium>
Gross medium was used as the basal medium. The composition of the Gloss medium is shown in Table 1. The compositions of Fe-EDTA Solution and Trace Elements used in the Gross medium are shown in Tables 2 and 3, respectively.

<How to check haploid>
The haploid cells of unicellular red algae cells do not have a strong cell wall (International Publication No. 2019/107385). Therefore, the cells were suspended in a Gloss medium containing 2% by mass of a surfactant (sodium dodecyl sulfate (SDS)), and the disintegrating cells were judged to be haploid. Cell disintegration was confirmed by observation using an optical microscope. Observation with a light microscope was performed immediately after the addition of SDS. In addition, colonies dominated by diploid cells are flatter and have a shape that spreads on the surface of the agar medium as compared with colonies formed from diploid cells (see FIG. 1: arrow is 1 times larger). Colony of the body, with some diploids remaining in its central part). Therefore, the morphology of the colonies on the agar medium was also used to determine whether the colonies were haploid.

(1) Cultivation of primal monocellular red algae cells Permeation of medium using osmotic pressure as an osmotic pressure regulator to examine the possibility that osmotic pressure may affect the stable maintenance of primal cells. The pressure was adjusted. Ploid cells were harvested from haploid colonies of the CCCryo127-00 strain and subcultured on 18% sorbitol + Gloss 1.5% agar medium. Then, the cells were cultured at 40 ° C. in a dark place and in an atmospheric atmosphere for 1 to 2 months. During the culture period, haploid colonies were maintained (Fig. 2). From this result, it was confirmed that the haploid cells could be maintained stably for more than 2 weeks by increasing the osmotic pressure in the medium.

(2) Examination of medium capable of maintaining haploid cells The osmotic pressure adjusting agent shown in Table 4 was added to Gross medium or 1% glucose + Gross medium so as to be 1 to 50% by mass. Further, 1.5% by mass of agar was added to each of the above-mentioned media to prepare 1.5% agar media. The monopoly of CCCryo127-00 strain was inoculated on these agar media and cultured at 40 ° C. in a dark place and in an atmospheric atmosphere for 1 month or more. During the culture period, the morphology of the colonies on the agar medium was observed to confirm whether the monoploid colonies were maintained. Also, the proliferation of haploid cells was evaluated based on the size of the colonies. The results are shown in Table 4 based on the following evaluation criteria.

<Evaluation criteria>
A: The maintenance period of 1-ploid is 1 month or more B: The maintenance period of 1-ploid is more than 2 weeks and less than 1 month C: The maintenance period of 1-ploid is within 2 weeks N: Does not grow D: Cannot be cultured (dead)
-: Not implemented

An example in which a haploid colony is maintained is shown in FIG. FIG. 3 is a plate cultured for 1 month in 18% sorbitol + Gloss 1.5% agar medium.
An example of returning to a diploid colony is shown in FIG. FIG. 4 is a plate cultured in 1% sorbitol + Gloss 1.5% agar medium for 2 weeks.
An example in which a haploid colony proliferates is shown in FIG. FIG. 5 is a plate cultured on 18% sorbitol + Gloss 1.5% agar medium. The photo on the left is the plate at the start of culture, and the photo on the right is the plate after 3 weeks of culture.

In Table 4, "Gross" indicates a Gloss medium, and "1% Glc + Gloss" indicates a 1% glucose Gloss medium. The values in parentheses indicate the molar concentration of the osmotic pressure regulator.

Table 5 shows the results of measuring the osmotic pressure of the medium before the addition of agar for each medium shown in Table 4. The osmotic pressure of the medium was measured with an osmotic meter (product name: automatic osmotic pressure analyzer Ozmo Station OM-6060, manufacturer: Arcley Co., Ltd.). In Table 5, the values in [] indicate the osmotic pressure (mOsm / kg).

From the results in Table 4, it was confirmed that haploid cells could be maintained in more than 2 weeks in a medium supplemented with an osmotic pressure regulator of about 80 mM or more. When the concentration of the osmotic pressure regulator was high, the growth tended to be slowed down, but even when the osmotic pressure regulator was added up to the upper limit of the solubility, haploid cells could be maintained for generally more than 2 weeks. Considering the growth rate, it was considered appropriate that the upper limit of the concentration of the osmotic pressure regulator was about 1.5 M.

From the results in Table 5, it was confirmed that haploid cells can be maintained for more than 2 weeks in a medium having an osmotic pressure of about 150 mOsm / kg or more. When the osmotic pressure was high, the proliferation tended to be slow, but even when the osmotic pressure was high, haploid cells could be maintained for about one month or more. Considering the growth rate, it was considered appropriate that the upper limit of the osmotic pressure of the medium was about 1500 Osm / kg.

(3) Production of monoploid single-celled red algae cells by the medium examined in (2) The diploid cells of the CCCryo127-00 strain were seeded on 18% sorbitol + Gross 1.5% agar medium. Then, the cells were cultured at 40 ° C. in a dark place and in an atmospheric atmosphere. It was confirmed whether monoploid colonies appeared during the culture period.
As a result, haploid colonies appeared in about 2 weeks (Fig. 6, arrows are haploid colonies). Cells were harvested from haploid colonies and subcultured on 18% sorbitol + Gross 1.5% agar medium. It was confirmed that haploid colonies could be maintained for more than 1 month even in the subcultured medium (Fig. 6).

From this result, it was shown that haploid cells can be efficiently produced from diploid cells by using the medium examined in (2).

(4) Liquid culture with the medium examined in (2) The haploid cells of the CCCryo127-00 strain were seeded in 18% sorbitol + Gross liquid medium. Then, the cells were cultured at 40 ° C. in a dark place and in an atmospheric atmosphere. As a result, it was confirmed that the cells proliferate well while maintaining the haploid cells (Fig. 7).

Although the preferred embodiments of the present invention have been described and illustrated above, it should be understood that these are illustrative of the present invention and should not be regarded as limiting. Additions, omissions, replacements, and other modifications may be made without departing from the spirit or scope of the invention. Therefore, the present invention is not considered to be limited by the above description, but only by the scope of the appended claims.

Claims

A method for producing unicellular single-celled red algae, which comprises culturing single-celled red algae cells in a medium containing an osmotic pressure regulator of 80 mM or more.
A method for producing unicellular single-celled red algae, which comprises culturing single-celled red algae cells in a medium having an osmotic pressure of 150 mOsm / kg or more.
The method for producing a monoploid single-celled red alga according to claim 1 or 2, wherein the osmotic pressure adjusting agent is at least one selected from the group consisting of sugar, sugar alcohol, and amino acid.
The method for producing a unicellular single-celled red alga according to any one of claims 1 to 3, wherein the single-celled red alga is a polyploid cell.
The method for producing a unicellular single-celled red alga according to claim 4, wherein the unicellular red alga is a diploid cell.
The method for producing a unicellular single-celled red alga according to any one of claims 1 to 3, wherein the single-celled red alga is a monoploid cell.
The method for producing unicellular unicellular red algae according to claim 6, which is a method for maintaining unicellular red algae cells as haploid.
The method for producing a unicellular unicellular red alga according to claim 6, which is a method for growing unicellular unicellular red algae cells.
Medium for monoploid unicellular red algae containing 80 mM or more of an osmotic pressure regulator.
Medium for monoploid unicellular red algae having an osmotic pressure of 150 mOsm / kg or more.
The medium for single-cell single-celled red algae according to claim 9 or 10, wherein the osmotic pressure adjusting agent is at least one selected from the group consisting of sugar, sugar alcohol, and amino acid.
The medium for monoploid single-celled red algae according to any one of claims 9 to 11, which is used for producing monoploid single-celled red algae cells from polyploid single-celled red algae cells.
The medium for monoploid single-celled red algae according to claim 12, wherein the polyploid single-celled red algae cell is a diploid single-celled red algae cell.
The medium for haploid unicellular red algae according to any one of claims 9 to 11, which is used to maintain haploid unicellular red algae cells as haploid.
The medium for monoploid unicellular red algae according to any one of claims 9 to 11, which is used for proliferating monoploid unicellular red algae cells.