JP2021023234A - Method for breeding algae strains highly accumulating oil under conditions of nitrogen source presence, highly oil-accumulating algae strains under conditions of nitrogen source presence, and oil and fat manufacturing method using the same - Google Patents

Abstract

To provide algae strains capable of highly accumulating fats and oils even in the presence of a nitrogen source.SOLUTION: The present invention for solving the above subject is a method for breeding algae strains highly accumulating oil, the method comprising: (A) irradiating algae belonging to the genus Chlamydomonas with an ion beam to introduce a random mutation; (B) culturing the mutant-introduced strain obtained by the step (A) under conditions of nitrogen source presence; and (C) separating a mutant strain having an oil and fat content of 30 wt.% or more from a mutant cell population. The invention also includes highly oil-accumulating algae strains under conditions of nitrogen source presence, in which the oil and fat content is 10 wt.% or more under conditions of nitrogen source presence, where a medium having nitrogen contained as a nutrient source and the concentration of the nitrogen of 1 mg-N/L to 270 mg-N/L is used.SELECTED DRAWING: None

Description

The present invention relates to a method for breeding a highly oil-accumulating alga strain under a nitrogen source presence condition, an oil highly accumulating alga strain under a nitrogen source presence condition, and a method for producing oil and fat using the same.

Attention is being paid to basic technologies for bioenergy production using algae and aquatic microorganisms. In the hydrosphere, there are many algae that have a high ability to accumulate lipids and sugars, are rich in diversity, and have a high growth ability. Algae have excellent carbon dioxide fixation ability, and photosynthetic ability is said to be several tens of times that of land plants. Such algae have been industrially cultivated for more than half a century, and are in high demand as raw materials for fuels, feeds, fine chemicals, health foods, and the like. In particular, due to concerns about the depletion of fossil fuels in the future, the need for early search for alternative fuels has increased, and interest in algae production as a source of biofuels has increased.

As a method for producing fats and oils using algae, a method using a specific marine green alga, Chlamydomonas species, which has high production efficiency, is known (see Patent Document 1). The Chlamydomonas species strain (JSC4) disclosed in Patent Document 1 is an excellent algae strain that achieves both high growth and fat content under photoautotrophic and seawater salt concentration conditions, and is considered to be a promising material for biofuels. Has been done. However, the current situation is that the demand for algae as a source of biofuel is further increasing, and there is a demand for Chlamydomonas species strains with higher production efficiency.

International Publication No. 2015/025553

Under such circumstances, the present inventors have found that in the conventional marine green alga Chlamydomonas species strains such as JSC4, fats and oils are produced and accumulated as a nitrogen source in the medium. We focused on the fact that the depletion of green algae is required and that the growth rate of cells decreases under such nitrogen source depletion conditions. Then, if we could create an algae strain capable of highly accumulating fats and oils even in the presence of a nitrogen source, we thought that the efficiency of fats and oils production could be further increased, and proceeded with the research. Therefore, an object of the present invention is to provide an algae strain capable of highly accumulating fats and oils even in the presence of a nitrogen source.

In order to solve the above problems, the present inventors have performed random mutagenesis by ion beam irradiation on algae strains derived from a conventionally known specific strain of Chlamydomonas species (JSC4 strain). , We carried out selective breeding combined with high-speed screening by flow cytometry, and created a strain with a sufficiently high fat accumulation rate even under the presence of a nitrogen source, and completed the present invention. That is, the gist of the present invention is as follows.

[1] (A) A step of irradiating algae belonging to the genus Chlamydomonas with an ion beam to introduce a random mutation.
(B) A step of culturing the mutant-introduced strain obtained in the above step (A) under the presence of a nitrogen source.
(C) A method for breeding a highly oil-accumulating algae strain, which comprises a step of separating a mutant strain having an oil / fat content of 10% by weight or more when cultured in a nitrogen source presence condition from a mutant cell population.
[2] The condition for the presence of a nitrogen source in the step (B) above is a condition for maintaining the concentration of nitrogen contained in the medium as a nutrient source at 1 mg-N / L to 270 mg-N / L, according to [1]. Breeding method for oil-accumulating algae strains.
[3] Nitrogen having a fat content of 10% by weight or more when cultured under the condition of a nitrogen source in which the concentration of nitrogen contained as a nutrient source in the medium is maintained at 1 mg-N / L to 270 mg-N / L. Oil high accumulation algae strain under source presence conditions.
^{[4] The oil and fat production rate is 100 (g / m 3} /) when cultured under the condition of a nitrogen source in which the concentration of nitrogen contained as a nutrient source in the medium is maintained at 1 mg-N / L to 270 mg-N / L. d) The above-mentioned oil-accumulated algae strain under the nitrogen source presence condition according to [3].
[5] The oil-accumulating algae strain under the nitrogen source presence condition according to [3] or [4], wherein the algae are Chlamydomonas species.
[6] The oil-accumulating alga strain under the nitrogen source presence condition according to [5], wherein the Chlamydomonas species is Chlamydomonas species KAC1801 strain (Chlamydomonas sp. KAC1801 / Receipt number: FERM ABP-22376).
[7] Chlamydomonas Species KAC1801 strain (Chlamydomonas sp. KAC1801 / Receipt number: FERM ABP-22376), which has a high oil and fat storage capacity under the presence of a nitrogen source.
[8] A method for producing and culturing oils and fats, which comprises continuously culturing in the presence of a nitrogen source using the oil-accumulating alga strain under the condition of the presence of a nitrogen source according to any one of [3] to [7].
[9] The oil-accumulating algae strain according to any one of [3] to [8] is cultured in a nitrogen source-containing medium, and the nitrogen concentration contained in the medium as a nutrient source is 1 mg-N. The oil-fat production culture method according to [8], which is characterized by maintaining the content at / L to 270 mg-N / L.

Since the oil-accumulating algae strain of the present invention can accumulate a large amount of fat and oil even in the presence of a nitrogen source, the growth rate is lowered by sufficiently supplementing the nutrient source. Oil and fat production can be continued without any problems. Therefore, according to the oil-accumulating algae strain under the nitrogen source presence condition of the present invention, it is possible to increase the efficiency of oil and fat production. Further, under nitrogen source depletion conditions, the cell condition deteriorates due to nutrient depletion and the risk of opportunistic infection increases. However, the algae strain of the present invention capable of highly accumulating fats and oils even under conditions where a nitrogen source is present. In, it is not necessary to deplete the nutrient source, so that the above risk can be reduced.

FIG. 1 is an electron micrograph of a parent strain (KOR1 strain), which is a conventional algae strain, and an oil-accumulating mutant strain (KAC1710 strain, KAC1801 strain) of the present invention when cultured under the presence of a nitrogen source. FIG. 2 is a diagram showing the concentrations of sodium nitrate contained in the culture broth supernatant of the parent strain (KOR1 strain) and the highly oil-accumulating mutant strains of the present invention (KAC1710 strain, KAC1801 strain). FIG. 3 is a diagram showing the amount of biomass per culture solution of the parent strain (KOR1 strain) and the highly oil-accumulating mutant strains of the present invention (KAC1710 strain, KAC1801 strain). FIG. 4 is a diagram showing the oil and fat content (%) of the parent strain (KOR1 strain) and the highly oil-accumulating mutant strains of the present invention (KAC1710 strain, KAC1801 strain). FIG. 5 is a diagram showing the amount of oil and fat produced (mg / L) per culture solution of the parent strain (KOR1 strain) and the highly oil-accumulating mutant strains of the present invention (KAC1710 strain, KAC1801 strain). FIG. 6 is a diagram showing the fat production rate (mg / L / day) of the parent strain (KOR1 strain) and the highly oil-accumulating mutant strain of the present invention (KAC1710 strain, KAC1801 strain) in each culture period.

Hereinafter, the algae oil-accumulating mutant strain of the present invention will be described in detail. Unless otherwise specified, the molecular biology experiment in the present specification can be carried out by the method described in a general experiment document known to those skilled in the art or a method similar thereto. In addition, the terms used herein are to be interpreted in the meaning commonly used in the art unless otherwise specified.

<Oil high accumulation algae strain under nitrogen source presence condition>
The oil-accumulating algae strain under the nitrogen source presence condition of the present invention is a strain showing a sufficient oil / fat accumulation rate and a high oil / fat production efficiency even when cultured under the nitrogen source presence condition.

Here, in the present invention, the nitrogen source presence condition refers to a condition in which the concentration of nitrogen contained as a nutrient source in the medium when culturing the algae strain is maintained so as not to be 0 mg-N / L or less. The concentration of nitrogen contained therein as a nutrient source is preferably maintained at 1 mg-N / L to 270 mg-N / L, more preferably maintained at 1 mg-N / L to 130 mg-N / L, and 1 mg. It is more preferably maintained at −N / L to 60 mg—N / L. The method for maintaining the concentration of nitrogen contained as a nutrient source in the medium within the above numerical range is not particularly limited, but a method by supplementing a new medium at an appropriate timing in culturing the algae strain can be considered. Examples of the nitrogen source in the medium include inorganic nitrogen sources such as ammonium salts and nitrates, and organic nitrogen sources such as urea, amino acids and peptone. Specifically, sodium nitrate, ammonium chloride and the like are mentioned as preferable nitrogen sources. For example, when a medium containing sodium nitrate is used as the nitrogen source, the nitrogen source presence condition means a condition in which the sodium nitrate concentration is maintained so as not to be 0 mg / L or less, and is 1 mg / L to 1600 mg / L. It is preferably 1 mg / L to 800 mg / L, and even more preferably 1 mg / L to 350 mg / L.

[Algae]
Algae is a general term for plants with anabolic pigments that live in water, and is a green algae plant, a yellow plant (including diatomaceae), a yellowish brown plant, an indigo plant, a brown algae plant, a green algae plant (including axle algae), and red Includes algae plants. From the viewpoint of producing fat and oil components with high efficiency, in the present invention, algae of the genus Chlamydomonas belonging to the green algae plant are preferable.

Chlamydomonas is a genus consisting of unicellular flagellates belonging to the order Chlamydomonas (or Chlamydomonas). Most of Chlamydomonas are freshwater, but some grow in seawater. The preferred algae of the genus Chlamydomonas in the present invention are marine, marine, and can grow in media containing seawater and salt.

[Oil high accumulation algae strain under conditions of nitrogen source presence]
The oil-accumulating algae strain under the presence of a nitrogen source in the present invention was obtained by performing selective breeding of conventional algae by combining random mutagenesis by ion beam irradiation and high-speed screening by flow cytometry. It is a stock. As the conventional algae that can be used as such a parent strain, as described above, the algae belonging to the genus Chlamydomonas belonging to the green algae plant are preferable, and among the chlamydomonas, the Chlamydomonas Species JSC4 strain that produces fat and oil components with high efficiency is particularly preferable. (Chlamydomonas sp. JSC4: also simply referred to as "JSC4 strain" in the present specification) and strains derived from JSC4 strain are preferable.

(Chlamydomonas Species JSC4 strain)
Here, the Chlamydomonas species JSC4 strain is a strain obtained by the following procedure. That is, only one cell was isolated and sterilized by a conventional method from a brackish water sample collected on the coast of the Midwestern part of Taiwan. This is cultured on HSM agar medium at 20 ° C., 8 to 15 μmol feet / m ² / s, 12 hours light period and 12 hours dark period under light conditions, and the algae strain is subcultured once every two weeks. It was established and identified as a green alga of the genus Chlamydomonas by morphological observation and others, and was named JSC4 strain. This JSC4 strain was issued to the Patent Organism Depositary Center (Kazusakamatari 2-5-8, Kisarazu City, Chiba Prefecture) on March 5, 2014, under the provisions of the Putabest Convention, with the accession number FERM BP- It has been deposited internationally as 22266. The phycological properties of the Chlamydomonas species JSC4 strain are as follows.

Morphological properties (1) The vegetative cells are elliptical and have a size of about 10 μm. In vegetative cells, it has two flagella that are about the same size as the cell length. The vegetative cells have motility.
(2) The vegetative cells are surrounded by a cell wall, and one nucleus and one chloroplast are present inside, and mitochondria, Golgi apparatus, vacuoles, starch granules, oil droplets, etc. are observed. It has a pyrenoid at the base of the chloroplast.
Reproductive mode (1) Two to eight endospores are formed in vegetative cells and are evenly distributed in the cells. Endogenous spores have one nucleus and one chloroplast in their cells.
(2) Proliferation by dichotomy is also performed.
Physiology / biochemical properties (1) Culture solution: Can grow in culture solutions containing marine products, steam products, and sea salt.
(2) Photosynthetic ability: Photoautotrophic growth by photosynthesis is possible.
(3) Containing pigments: chlorophyll a, chlorophyll b, and other carotenoids (4) Anabolic storage substances: starch, fats and oils (5) Growth temperature range: 15 ° C to 35 ° C (optimal temperature 25 ° C)
(6) Growth pH range: pH 6.0 to 10.0 (optimal pH is 7.0)
The highly oil-accumulating mutant strain of the present invention has a high oil-and-fat accumulation rate even under day and night conditions such as outdoors, and an oil-and-fat production rate of 150 g / m ³ / day or more under day and night conditions.

(Acquisition of oil-accumulating algae strain under the presence of nitrogen source of the present invention)
(1) Introduction of mutation The breeding of the oil-accumulating algae strain (KAC) under the condition of the presence of a nitrogen source can be carried out using the conventional algae strain as the parent strain, and the parent strain is not particularly limited, but the oil is more efficient. From the viewpoint of obtaining a highly accumulating algae mutant strain, it is preferable to use the above-mentioned Chlamydomonas species JSC4 strain (Chlamydomonas sp. JSC4) or a strain further selected from the JSC4 strain as the parent strain. Mutation can be introduced by irradiating a cell population with an ion beam. The ion beam randomly cleaves the DNA double strand as it passes through the cell nucleus. It is said that cells reconnect DNA strands by their own repair function, and at that time, various mutations such as DNA deletion occur. The ion beam to be irradiated is not particularly limited as long as it can introduce a mutation, and examples thereof include carbon (C), helium (He), neon (Ne), argon (Ar), and the like, and to algae. From the viewpoint of efficiency of mutagenesis, ¹² C ⁵⁺ is preferable. When algae belonging to the genus Chlamydomonas ^{are irradiated with a 12} C5 ⁺ ion beam, the dose range is preferably 10 to 250 Gy, more preferably 20 to 75 Gy. After irradiation with an ion beam, after recovery culture for several days, the obtained cell population can be used as a mutant library for screening described later. The recovery culture is carried out by allowing the cells to stand for 3 days or more under conditions such as a neutral white fluorescent lamp having an appropriate light intensity.

(2) Screening The primary screening of oil-accumulating algae strains under the presence of a nitrogen source can be performed using a fluorescently activated cell sorter (FACS). Intracellular oil droplets of cells cultured under the presence of a nitrogen source are stained with fluorescent dyes BODIPY, Nile Red, etc., and cells with strong fluorescence intensity are separated by FACS. The intensity of fluorescence derived from staining of intracellular oil droplets such as BODIPY fluorescence and Nile Red fluorescence of individual cells and autofluorescence of chlorophyll (used as an index of cell size) was analyzed by FACS, and BODIPY fluorescence per chlorophyll autofluorescence was analyzed. , Nile red fluorescence and other cells with high fluorescence derived from staining of intracellular oil droplets (top 1.5 to 0.5%) can be sorted. It is also possible to concentrate the target cells by repeating the culture under the above-mentioned nitrogen source presence condition and the fractionation by FACS a plurality of times.

The light conditions during culturing can be adjusted with a fluorescent lamp, and the photon flux density during lighting is usually 50 μmol photons / m ² · sec to 2,000 ^{μmol photos / m 2} ·, as conditions close to sunlight. in the range of seconds, in the range of 60μmol photons / ^{m 2} · sec ~ 1,000μmol photons / ^{m 2} · sec is preferably in the range of 80μmol photons / ^{m 2} · sec ~ 500μmol photons / ^{m 2} · sec Is more preferable.

After sorting, the cells are seeded on an agar medium and statically cultured under a neutral white fluorescent lamp having ^{a light intensity of about 50 μmol photons / m for 2 seconds until colonies are formed.} Candidate strains obtained by primary screening are cultured on a microwell plate, and fats and oils are extracted from cells and analyzed by gas chromatography-mass spectrometry, GC-MS. Perform secondary screening for highly oil-accumulating algae strains under conditions. In the secondary screening, the amount of biomass and fats and oils are measured according to the method described later, the fats and oils content is calculated, and finally, a mutant strain that highly accumulates oil by culturing in the presence of a nitrogen source is selected. Can be done.

(Characteristics of oil-accumulating algae strain under the presence of nitrogen source of the present invention)
(1) Culture Each oil-accumulating alga strain obtained by culturing is under 1% to 5% CO ₂ conditions, preferably 1.5% to 2.5% CO ₂ conditions, and more preferably 2% CO ₂ conditions. , 15 ° C to 40 ° C, preferably 20 ° C to 35 ° C, more preferably 25 ° C to 30 ° C, suspended in a medium described later in an incubator such as a flask, and under light conditions capable of photosynthesis. Pre-culture is performed for 2 to 7 days, preferably 3 to 5 days, more preferably about 4 days, and when the cells have grown satisfactorily and the number of cells is sufficient, expanded culture is performed, preferably 10 to 20 days. The main culture is carried out under the above-mentioned nitrogen source presence conditions for 12 to 16 days, more preferably about 14 days. The nitrogen source presence condition is as described above, and refers to a condition in which the concentration of nitrogen contained as a nutrient source in the medium for culturing the algae strain is maintained so as not to be 0 mg-N / L or less. The nitrogen concentration in the medium is preferably maintained at 1 mg-N / L to 270 mg-N / L, more preferably maintained at 1 mg-N / L to 130 mg-N / L, and 1 mg-N / L to 1 mg-N / L. It is more preferably maintained at 60 mg-N / L. The method for maintaining the concentration of nitrogen contained as a nutrient source in the medium within the above numerical range is not particularly limited, but a method by supplementing a new medium at an appropriate timing in culturing the algae strain can be considered. Examples of the nitrogen source in the medium include inorganic nitrogen sources such as ammonium salts and nitrates, and organic nitrogen sources such as urea, amino acids and peptone. Specifically, sodium nitrate, ammonium chloride and the like are mentioned as preferable nitrogen sources. For example, when a medium containing sodium nitrate is used as the nitrogen source, the nitrogen source presence condition means a condition in which the sodium nitrate concentration is maintained so as not to be 0 mg / L or less, and is 1 mg / L to 1600 mg / L. It is preferably 1 mg / L to 800 mg / L, and even more preferably 1 mg / L to 350 mg / L.

As for the light conditions, it is preferable that the amount of fats and oils that can be finally recovered is large, and continuous culture may be performed under conditions that allow photosynthesis, or 12 hours of light with a neutral white fluorescent lamp of about ^{100 to 250 μmol photos / m 2 seconds.} It may be cultured under day and night cycle conditions such as a period and a dark period of 12 hours. The oil-accumulating algae strain of the present invention is preferably a strain that can obtain a sufficient amount of oil-and-fat accumulation even under day and night periodic conditions assuming outdoor culture.

As the culturing method used in the present invention, a static culturing method can be used, but considering the algae body productivity and the productivity of fat and oil components of algae, culturing by shaking culturing method or deep aeration stirring culturing method is performed. preferable. The shaking culture may be reciprocating shaking or rotary shaking.

The medium used for the above culture is not particularly limited as long as it is a medium in which algae belonging to the genus Chlamydomonas can grow, but a medium containing seawater salt containing seawater, concentrated seawater, or artificial seawater produces fats and oils. It is preferable because it improves the ability. For example, examples of the basal medium include Modified Bold 12N (MB12N) medium, TAP medium, HSM medium, BG-11 medium, BBM medium, and the like, and MB12N medium is more preferable because it can produce fat and oil components with high efficiency. Further, those obtained by adding 0.5 to 5% by weight, preferably 2 to 5% by weight, more preferably 2 to 3% by weight of sea salt (sea salt) to these basal media can be used. When a large-scale culture of algae is assumed, convenient seawater can be used as the base of the medium.

Examples of sea salt (sea salt) that can be used in the present invention include known and commonly used sea salt. The seawater salt used in the present invention may be obtained by evaporating and drying seawater, or seawater or a concentrated solution of seawater may be used, but in order to adjust the concentration contained in the medium, , It is more preferable to use seawater salt which is a solid content of seawater.

In the present invention, the concentration of the nitrogen source contained in the medium is _{a method of measuring the concentration of nitrate contained in the medium using the optical density (OD 220)} at a wavelength of 220 nm as an index, a method of measuring absorbance with an ion sensor, a coloring reagent, or the like. Can be measured by.

(2) Amount of biomass In the present invention, the amount of biomass can be calculated using the dry weight of cells as an index. The amount of biomass can be measured by a method known to those skilled in the art, and the method is not limited, but can be measured, for example, as follows. That is, the cells of the oil-accumulating algae strain obtained by the above culture are collected in a required amount in a weighed microtube, washed with distilled water, and then freeze-dried overnight. After drying, the weight of the microtube is measured again, and the dry weight (mg) of the recovered dried algae is obtained by subtracting the weight of the empty microtube. Further, the amount of biomass (g / L) contained in the culture solution can be calculated by dividing this by the amount of the culture solution used for the measurement. After weighing, the dried algae are used for measuring fats and oils and pigments described later. The amount of biomass of the oil-accumulating algae strain of the present invention is sufficient even when cultured in the presence of a nitrogen source.

(3) Oils and fats The oil-accumulating algae strain of the present invention is characterized by a high oil-and-fat content, and the oil-and-fat content is usually 10% by weight or more and 15% by weight or more in the presence of a nitrogen source. It is more preferably 20% by weight or more, and even more preferably 25% by weight or more. The oil-accumulating algae strain of the present invention is also characterized by a high oil-fat production rate, and the oil-fat production rate in the presence of a nitrogen source is 100 g / m ³ / day or more, and 150 g / m ³ / day or more. It is preferably 200 g / m ³ / day or more, more preferably 250 g / m ³ / day or more, and ^{particularly preferably 300 g / m 3} / day or more.

In the present invention, the fat and oil can be measured by a method known to those skilled in the art, and the method is not limited, but can be performed as follows, for example. For the measurement of fats and oils, dried algae prepared in the above biomass measurement experiment are used. The dried algae are weighed in a microtube dedicated to crushing and used for measurement. 0.5 mm diameter glass beads are added to the microtube and the cells are disrupted by a multi-bead shocker device. Fatty acid in cells is methylated using a fatty acid methylation kit (manufactured by Nakarai Co., Ltd., etc.), and the fatty acid methyl ester produced thereby is quantified by gas chromatograph mass spectrometry (gas chromatography-mass spectrum, GC-MS) and dried. The fat content (% by weight) per algae and the fat (oil) production per culture solution (mg / L) can be calculated. The amount of fat (oil) produced per culture solution (mg / L) is calculated by multiplying the fat content by the amount of biomass. In addition, oil-accumulating algae strains are collected over time, and changes in fat content (% by weight) per dry alga and fat (oil) production per culture solution (mg / L) over time are confirmed. be able to. Further, by measuring the amount of fats and oils obtained from the unit culture solution (L) per day, the fats and oils (oil) production rate (mg / L / day (days)) can be calculated. , Can be calculated by dividing the amount of oil and fat production (lipid production, mg / L) by the number of days of culture.

In the present invention, the "fat production rate in the presence of a nitrogen source" means that the sodium nitrate concentration of the main culture is in the range of 1 mg / L to 1600 mg / L when a medium containing sodium nitrate is used as the nitrogen source. The oil and fat production rate achieved during the period. Further, the "fat and oil content under the nitrogen source presence condition" is achieved on the day when the sodium nitrate concentration of the main culture is in the range of 1 mg / L to 1600 mg / L when a medium containing sodium nitrate is used as the nitrogen source. It refers to the oil and fat content per dried algae.

Examples of fats and oils accumulated by the oil-accumulating algae strain of the present invention include triglycerides composed of fatty acids such as palmitic acid, linoleic acid, stearic acid, linolenic acid, and oleic acid, which have high combustion efficiency, biodiesel fuel, and the like. As useful as.

In the oil-accumulating algae strain of the present invention, the fat-and-fat production rate in the presence of a nitrogen source is 200 g / m ³ / day or more after the third day after the start of cultivation, and the fat-and-fat content is 10% by weight or more under the same conditions. Variant strains of Cramidmonas Species KOR1 strain are preferable, specifically, KAC1710 strain and KAC1801 strain are more preferable. The KAC1801 strain received as receipt number FERM ABP-22376 after making a deposit application at is more preferred.

<Manufacturing method of oils and fats using highly oil-accumulating algae strains>
The present invention also includes the above-mentioned method for producing fats and oils using the highly oil-accumulating alga strain under the nitrogen source presence condition of the present invention. The method for producing fats and oils of the present invention is characterized by including a step of continuously culturing a highly oil-accumulating alga strain under the conditions of presence of a nitrogen source of the present invention and a step of recovering the produced fats and oils. To do.

(Step of continuously culturing the oil-accumulating alga strain under the nitrogen source presence condition of the present invention)
For this step, also refer to the description of culturing in the section of characteristics of the oil-accumulating algae strain of the present invention described above. Specifically, for example, KAC1710 strain, KAC1801 strain and the like are taken from an agar medium in appropriate amounts, and pre-culture is carried out for 3 days under the above-mentioned nitrogen source presence conditions. Subculture _{is performed so that the OD 750} becomes about 0.04, and pre-culture is carried out for 3 days under the above-mentioned nitrogen source presence conditions. Subculture _{so that the OD 750} is about 0.04, and carry out the main culture for another 14 days under the above-mentioned nitrogen source presence conditions. At the time of the main culture, for example, the algae can be partially harvested on the 5th, 7th, 9th days and the like from the start of the culture, and a new medium containing nitrogen can be replenished. By replenishing a new medium containing nitrogen in this way and subculturing at an appropriate timing, the oil-rich algae strain of the present invention under the nitrogen source presence condition can be continuously subjected to a good cell condition. It can be cultivated, and finally it is possible to efficiently produce more fats and oils as compared with the conventional strain.

(Culture conditions)
Uses a two-stage flask Medium: MB12N + 2% sea salt
CO ₂ : 2% CO ₂
Light: 250 μmol photons / m ² / s Neutral white fluorescent lamp Temperature: 30 ° C
Stirring: 100 rpm

(Process of collecting produced fats and oils)
As a method for extracting the fat and oil component from the oil-accumulating algae strain obtained in the above culture step, a normal fat and oil extraction method can be used, and in particular, chloroform / methanol represented by the solvent method and the Bligh-Dyer method. It is possible, but not limited to, a general extraction method using an organic solvent such as a system.

<Breeding method for highly oil-accumulating algae strains under conditions of nitrogen source presence>
The present invention also includes a method for breeding a highly oil-accumulating algae strain. That is, the method for breeding a highly oil-accumulating algae strain of the present invention is obtained by (A) a step of irradiating algae belonging to the genus Chlamydomonas with an ion beam to introduce a random mutation, and (B) the above-mentioned (A). It is characterized by including a step of culturing the mutant-introduced strain in the presence of a nitrogen source, and (C) a step of separating the mutant strain having a fat content of 30% by weight or more from the mutant cell population. In the present invention, the mutant strain obtained above may be further separated from the mutant strain by repeating the above steps (A), (B) and (C). According to the method of the present invention, a useful algae strain belonging to the genus Chlamydomonas, which has a high fat content and high fat production efficiency even when cultured in the presence of a nitrogen source, can be obtained.

((A) A step of irradiating algae belonging to the genus Chlamydomonas with an ion beam to introduce a random mutation)
In this step, the cell population of algae belonging to the genus Chlamydomonas is irradiated with an ion beam. The ion beam to be irradiated is not particularly limited as long as it can introduce a mutation, and examples thereof include carbon (C), helium (He), neon (Ne), argon (Ar), and the like, and to algae. From the viewpoint of efficiency of mutagenesis, ¹² C ⁵⁺ is preferable. When algae belonging to the genus Chlamydomonas ^{are irradiated with a 12} C5 ⁺ ion beam, the dose range is preferably 10 to 250 Gy, more preferably 20 to 75 Gy. After irradiation with an ion beam, after recovery culture for several days, the obtained cell population can be used as a mutant library for screening described later. The recovery culture is carried out, for example, by allowing it to stand for 3 days or more under conditions such as a neutral white fluorescent lamp having an appropriate light intensity.

((B) A step of culturing the mutant-introduced strain obtained in the above step (A) under the presence of a nitrogen source)
In this step, the mutant-introduced strain obtained in the above step (A) is cultured under the above-mentioned nitrogen source presence conditions. For a specific culturing method, refer to the above-mentioned description of culturing in the section on characteristics of oil-accumulating algae strains of the present invention.

((C) Step of separating a mutant strain having a high fat content from a mutant cell population)
In this step, a mutant strain that can maintain a high fat content even by culturing in step (B) is isolated from the mutant cell population obtained in step (A) above. At this time, the mutant strain having an oil / fat content of preferably 20% by weight or more, more preferably 30% by weight or more is separated. As a specific separation method, a highly oil-accumulating algae strain can be separated using a fluorescently activated cell sorter (FACS). Intracellular oil droplets of a mutant cell population cultured under target conditions, for example, conditions in which a nitrogen source is present, are stained with fluorescent dyes BODIPY, Nile Red, etc., and cells having strong fluorescence intensity are separated by FACS. The intensity of fluorescence derived from staining of intracellular oil droplets such as BODIPY fluorescence and Nile Red fluorescence of individual cells and autofluorescence of chlorophyll (used as an index of cell size) was analyzed by FACS, and BODIPY fluorescence per chlorophyll autofluorescence was analyzed. , Nile red fluorescence and other cells with high fluorescence derived from staining of intracellular oil droplets (top 1 to 0.5%) can be sorted. It is also possible to concentrate the target cells by repeating the culture under the above conditions and the fractionation by FACS a plurality of times.

Here, among the culture conditions set in the present invention, the nitrogen source presence condition is as described above. Specifically, the sodium nitrate concentration in the medium when culturing the algae strain is 0 mg /. It refers to a condition that does not become L or less, and the sodium nitrate concentration in the medium is preferably 1 mg / L to 1600 mg / L, more preferably 1 mg / L to 800 mg / L, and 1 mg / L to 350 mg / L. It is more preferable to have.

The light conditions during culturing can be adjusted with a fluorescent lamp, and the photon flux density during lighting is usually 50 μmol photons / m ² · sec to 2,000 ^{μmol photos / m 2} ·, as conditions close to sunlight. in the range of seconds, in the range of 60μmol photons / ^{m 2} · sec ~ 1,000μmol photons / ^{m 2} · sec is preferably in the range of 80μmol photons / ^{m 2} · sec ~ 500μmol photons / ^{m 2} · sec Is more preferable.

The separated cells are seeded on an agar medium and statically cultured under a neutral white fluorescent lamp having a light intensity of about 50 μmol photons / m for ^{2 seconds until colonies are formed.} Candidate strains obtained by primary screening are cultured on a microwell plate, and fats and oils are extracted from cells and analyzed by gas chromatography-mass spectrometry, GC-MS. Perform secondary screening for highly oil-accumulating algae strains under conditions. In this step, mutant strains having a fat content of 30% by weight or more can be separated. In the secondary screening, the amount of biomass and fats and oils are measured according to the method described later, the fats and oils content is calculated, and finally, a mutant strain that highly accumulates oil by culturing in the presence of a nitrogen source is selected. Can be done.

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

1. 1. Mutation breeding of oil-rich algae strains (1) Introduction of mutations For breeding of oil-rich algae strains (KAC) under the presence of a nitrogen source, KOR1 strain (Chlamydomonas sp. KOR1) derived from Chlamydomonas species JSC4 strain was used. It was carried out by two-step mutation breeding as a parent strain. The mutation was introduced by irradiating the parent strain with an ion beam at the ion irradiation research facility (TIARA: Takasaki Ion accelerators for Advanced Radiation Application) of the Takasaki Quantum Applied Research Institute, National Research and Development Corporation.

Specifically, an appropriate amount of algae was taken from the agar medium and pre-cultured for 3 days under the following conditions. Subculture was performed so that the optical density (OD ₇₅₀ ) at a wavelength of 750 nm was 0.04, and the main culture was carried out for another 2 days under the following conditions. After culturing, the _{mixture was diluted with TAP medium so that OD 750} was 0.5, and 100 μL of the diluted solution was applied to TAP agar medium. Algae cells on an agar medium were irradiated with an ion beam ( ¹² C ^{5+, 220 MeV) accelerated by AVF cytocron at a dose of 50 Gy.} After irradiation with the heavy ion beam, recovery culture was carried out by allowing the cells to stand for 3 days or more under a neutral white fluorescent lamp having a light intensity of 50 μmol photons / m ^{2 / s.} This was used in the following experiments as a mutant library of KOR1.

(Culture conditions)
Uses a two-stage flask Medium: 70 mL TAP
CO ₂ : 2% CO ₂
Light: 100 μmol photons / m ² / s Fluorescent lamp Temperature: 30 ° C
Stirring: 100 rpm

(2) Primary screening The primary screening of oil-accumulating algae strains in the presence of a nitrogen source was carried out using a fluorescently activated cell sorter (FACS). Intracellular oil droplets of cells cultured in the presence of a nitrogen source were stained with the fluorescent dye BODIPY, and cells with strong fluorescence intensity were separated by FACS.

An appropriate amount of algae was taken from the agar medium of the mutant library prepared above, and pre-culture was carried out for 3 days under the following conditions. Subculture _{was performed so that the OD 750} was 0.04, and the main culture was carried out for another 7 days under the following conditions. After culturing, the cells were collected and suspended in PBS at ^{5 × 10 6 cells / mL.} The fluorescent dye BODIPY was added to this so as to have a concentration of 50 μM, and the cells were placed in a dark place for 5 minutes to fluorescently stain intracellular oil droplets. The intensity of BODIPY and chlorophyll autofluorescence (used for standardization as an index of cell size) of individual cells was analyzed by FACS (SH800 manufactured by SONY), and cells with high BODIPY fluorescence per chlorophyll autofluorescence (top 1.5 to 0.5%) was separated. The filter set used to detect fluorescence is shown below. The cells after sorting were recovered and cultured under the following culture conditions by changing the medium to TAP medium and until the cells proliferated sufficiently. Preculture was performed by subculturing an appropriate amount of proliferated cells, and the pre-culture operation according to the above procedure was repeated a total of 5 times (seed from agar medium → pre-culture → main culture → pre-culture by FACS → recovery culture → pre-culture. → ...). After the fifth preparative operation, the cells were seeded on TAP agar medium and statically cultured under a neutral white fluorescent lamp having ^{a light intensity of 50 μmol photons / m 2 / s until colonies were formed.}

(Culture conditions)
Uses a two-stage flask Medium: 70 mL MB12N + 2% sea salt
CO ₂ : 2% CO ₂
Light: 100 μmol photons / m ² / s Fluorescent lamp Temperature: 30 ° C
Stirring: 100 rpm

(FACS analysis conditions)
BODIPY fluorescence: excitation: 488 nm laser
Fluorescence: PE filter (570nm-630nm)
Chlorophyll fluorescence: excitation: 488 nm laser
Fluorescence: PerCP-Cy5.5 filter (690nm-750nm)

(3) Secondary screening By culturing the candidate strain on a microwell plate, extracting fats and oils from the cells, and analyzing them by gas chromatography-mass spectrometry (GC-MS), the conditions under the presence of a nitrogen source are obtained. Secondary screening of oil-accumulating algae strains was performed.

An appropriate amount of the algae of the candidate mutant strain obtained in the primary screening was taken from the agar medium, and pre-culture was carried out for 3 days under the following conditions. Subculture _{was performed so that the OD 750} was 0.1, and the main culture was carried out for another 3 days under the following conditions. After culturing, 2 mL of the culture solution was collected from each well, and the fat content was measured according to the procedures of "biomass measurement" and "fat measurement" described later. Finally, KAC1710 was obtained as a mutant strain that highly accumulates oil by culturing in the presence of a nitrogen source.

(Culture conditions)
Use 12-well plate Medium: 2.5 mL MB12N + 2% sea salt
CO ₂ : 2% CO ₂
Light: 100 μmol photons / m ² / s white LED
Temperature: 30 ° C
Stirring: 100 rpm

The above steps (1) to (3) were carried out using the KAC1710 strain as the parent strain, and finally KAC1801 was obtained as a mutant strain that highly accumulates oil by culturing in the presence of a nitrogen source.

2. 2. Evaluation of oil-accumulated algae strains (1) Culture for evaluation of oil-accumulated algae strains Each oil-accumulated algae strain KAC obtained by mutated breeding is cultured under the common culture conditions shown in this section, and is described below. Evaluated by method.

An appropriate amount of algae was taken from the agar medium and cultured for 3 days before and after under the following conditions. Subculture _{was performed so that the OD 750} was 0.04, and preculture was further carried out for 3 days under the following conditions. Subculture _{was performed so that the OD 750} was 0.04, and the main culture was carried out for another 14 days under the following conditions.

(Culture conditions)
Uses a two-stage flask Medium: MB12N + 2% sea salt
CO ₂ : 2% CO ₂
Light: 250 μmol photons / m ² / s Neutral white fluorescent lamp Temperature: 30 ° C
Stirring: 100 rpm

(2) Electron microscopic analysis For the observation of oil droplets in cells, electron microscopic analysis combining a chemical fixation method and a resin-embedded ultrathin section method was used. For the analysis, KOR1, KAC1710, and KAC1801 cells prepared in the above procedure (1) on the 4th day of the main culture were used. The cultured cells were suspended in a fixative (2% paraformaldehyde, 2% glutaraldehyde, 50 mM cacodylic acid buffer pH = 7.4) and fixed at 4 ° C. overnight. An electron micrograph of each cell is shown in FIG. Nitrogen remains in the medium on the 4th day of the main culture, and the nitrogen nutritional environment is satisfied.

As shown in FIG. 1, on the 4th day of the main culture, under the condition that a sufficient nitrogen source remained in the medium, almost no oil droplets were observed in the parent strain KOR1, whereas oil droplets appeared in KAC1710. Was confirmed. In addition, more oil droplets were accumulated in KAC1801 than in KAC1710.

_{(3) Measurement of nitrogen source Sodium nitrate (NaNO 3} ) is the only nitrogen source contained in the MB12N + 2% sea salt medium used in the culture experiment for the evaluation of the oil-accumulated algae strain, and the MB6N + 2% sea salt medium. Contains twice as much sodium nitrate. Since sodium nitrate absorbs light having a wavelength of 220 nm well, the optical density (OD ₂₂₀ ) at a wavelength of 220 nm was used as an index in measuring the amount of sodium nitrate contained in the medium. The culture supernatant was prepared by removing the cells by centrifugation. The culture broth supernatant was appropriately diluted with distilled water, and OD ₂₂₀ was measured. A calibration curve was prepared using MB12N medium containing sodium nitrate having a known concentration, and the concentration of sodium nitrate contained in the culture medium supernatant was calculated based on the calibration curve.

As shown in FIG. 2, the sodium nitrate contained in the culture broth supernatant was completely consumed in KOR1 by the 6th day of culturing. In addition, KAC1710 was completely consumed by the 8th day of culture. On the other hand, in KAC1801, although the sodium nitrate concentration in the culture broth supernatant decreased until about the 8th day of the culture, a constant concentration level of sodium nitrate was maintained until at least the 13th day thereafter.

(4) Measurement of biomass The amount of biomass was indexed by the dry weight of cells. The required amount of cells cultured in the culture experiment for evaluation of the above oil-accumulated algae strain was collected in a weighed microtube, washed once with distilled water, and then freeze-dried overnight. After drying, the weight of the microtube was measured again, and the dry weight (mg) of the recovered cells was determined by subtracting the weight of the empty microtube. Further, the amount of biomass (g / L) contained in the culture solution was obtained by dividing this by the amount of the culture solution used for the measurement. The results are shown in FIG. After weighing, the dried algae were used for the measurement of fats and oils described later.

As shown in FIG. 3, the amount of biomass changed in a state where the parent strain KOR1 was slightly larger than the other two strains. Until the 6th day of culturing when sodium nitrate was depleted, it remained at the same level as the parent strain KOR1. No significant difference was found between KAC1710 and KAC1801.

(5) Measurement of fats and oils The dried algae prepared in the above biomass measurement experiment were used for the measurement of fats and oils. Approximately 3 mg of dried algae was weighed in a microtube dedicated to crushing and used for measurement. 0.5 mm diameter glass beads were added to the microtube and the cells were disrupted by a multi-bead shocker device. Fatty acid in cells was methylated using a fatty acid methylation kit (manufactured by Nakarai Co., Ltd.), and the fatty acid methyl ester produced thereby was quantified by gas chromatograph mass spectrometry (GC-MS). The results of the oil and fat content per dried algae of each strain from Day 3 to Day 6 are shown in FIG. In addition, the amount of fat (oil) produced per culture solution (mg / L) and the rate of fat (oil) production (mg / L / day (day)) in each culture period are shown in FIGS. 5 and 6, respectively.

As shown in FIG. 4, the oil and fat content in the dried algae is a very low value of less than 10% up to day4 in the parent strain KOR1, and the nitrogen source is completely depleted on day5 when the nitrogen source starts to be depleted. When it became day6, it increased to around 10 to 15%. On the other hand, in KAC1710 and KAC1801, the value in day3 already exceeded 10%, and in day4, the value in day6, which was the highest content rate in KOR1, was already exceeded. After that, the fat and oil contents increased remarkably with day5 and day6. In particular, in KAC1801, the increase in the fat content rate on day5 and day6 was remarkable. Further, as shown in FIG. 5, it was shown that the amount of fats and oils produced per culture solution of KAC1710 and KAC1801 was remarkably higher than that of the parent strain KOR1. Further, as shown in FIG. 6, the fat production rate during the culture period of day 3 to day 5 was significantly higher in KAC1801 than in the other two strains. That is, when producing fats and oils using KAC1801, it is interpreted that it is preferable to maintain the culture conditions of day 3 to day 5 in this experiment.

3. 3. Oil production and culture method by semi-continuous culture A method for efficiently producing oil and fat using the oil-accumulating algae strain under nitrogen-sufficient conditions of the present invention will be investigated. That is, an appropriate amount of the KOR1 strain and the KAC1801 strain are taken from the agar medium, and the pre-culture is carried out for 3 days under the following conditions. Subculture _{is performed so that the OD 750} becomes 0.04, and preculture is carried out for 3 days under the following conditions. Subculture _{so that the OD 750} becomes 0.04, and carry out the main culture for another 14 days under the following conditions. During the 14-day main culture, the algae are partially harvested on the 5th, 7th, and 9th days, and a new medium containing nitrogen is replenished. By the above-mentioned method, the change with time of the biomass and sodium nitrate concentration can be confirmed.

(Culture conditions)
A two-stage flask was used.
Medium: MB12N + 2% sea salt
CO ₂ : 2% CO ₂
Light: 250 μmol photons / m ² / s Neutral white fluorescent lamp Temperature: 30 ° C
Stirring: 100 rpm

When the oil-accumulating algae strain under the nitrogen source presence condition of the present invention is used, more fats and oils can be efficiently produced while maintaining good cell condition by continuously culturing while supplementing the nitrogen source-containing medium. Can be expected to be produced.

Since the highly oil-accumulating algae strain of the present invention can accumulate a large amount of oil and fat even in the presence of a nitrogen source, the oil and fat production is carried out without lowering the growth rate by supplementing the nutrient source. be able to. Therefore, according to the oil-accumulating algae strain under the nitrogen source presence condition of the present invention, it is possible to increase the efficiency of oil and fat production. Further, under nitrogen source depletion conditions, the cell condition deteriorates due to nutrient depletion and the risk of opportunistic infection increases. However, the algae strain of the present invention capable of highly accumulating fats and oils even under conditions where a nitrogen source is present. In, it is not necessary to deplete the nutrient source, so that the above risk can be reduced.

Claims (9)

(A) A step of irradiating algae belonging to the genus Chlamydomonas with an ion beam to introduce a random mutation.
(B) A step of culturing the mutant-introduced strain obtained in the above step (A) under the presence of a nitrogen source.
(C) A method for breeding a highly oil-accumulating algae strain, which comprises a step of separating a mutant strain having an oil / fat content of 10% by weight or more when cultured in a nitrogen source presence condition from a mutant cell population. The high oil accumulation according to claim 1, wherein the nitrogen source presence condition in the step (B) is a condition for maintaining the nitrogen concentration contained in the medium as a nutrient source at 1 mg-N / L to 270 mg-N / L. Breeding method for algae strains. Nitrogen source existence condition that the fat content is 10% by weight or more when culturing under the nitrogen source existence condition in which the nitrogen concentration contained as a nutrient source in the medium is maintained at 1 mg-N / L to 270 mg-N / L. Lower oil high accumulation algae strain. The fat production rate when culturing in the presence of a nitrogen source in which the concentration of nitrogen contained as a nutrient source in the medium is maintained at 1 mg-N / L to 270 mg-N / L is 100 (g / m ³ / d) or more. The oil-accumulated alga strain under the nitrogen source presence condition according to claim 3. The oil-accumulating algae strain under the nitrogen source presence condition according to claim 3 or 4, wherein the algae are Chlamydomonas species. The oil-accumulating algae strain according to claim 5, wherein the Chlamydomonas species is Chlamydomonas species KAC1801 strain (Chlamydomonas sp. KAC1801 / Receipt number: FERM ABP-22376). Chlamydomonas Species KAC1801 strain (Chlamydomonas sp. KAC1801 / Receipt number: FERM ABP-22376), which has a high oil and fat storage capacity under the presence of a nitrogen source. A method for producing and culturing oils and fats, which comprises continuously culturing in the presence of a nitrogen source using the oil-accumulating alga strain under the condition of the presence of a nitrogen source according to any one of claims 3 to 7. The oil-accumulating algae strain according to any one of claims 3 to 7 is cultured in a nitrogen source-containing medium, and the concentration of nitrogen contained in the medium as a nutrient source is 1 mg-N / L to 1. The oil-fat production culture method according to claim 8, wherein the amount is maintained at 270 mg-N / L.