Oil and fat production method

Abstract

To produce oil and fat using microalgae in the absence of salt stress.SOLUTION: A method for producing oil and fat includes the step of culturing microalgae, which have an enhanced carbohydrate decomposition rate, in the absence of salt stress.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to methods for producing fats and oils, and more particularly, to methods for producing fats and oils using microalgae with enhanced carbohydrate degradation rates, compositions for producing fats and oils, and systems for producing fats and oils. [Background technology]

[0002] Fundamental technologies for bioenergy production using algae and aquatic microorganisms are attracting attention. In bioenergy production using microalgae, it is known that the lipid content of cells is increased by salt stress in many microalgae, such as Botryococcus, Chlamydomonas, Chlorella, and Nannochloropsis. On the other hand, salt stress has an adverse effect on cell growth, leading to a decrease in lipid productivity. In addition, under salt stress, the risk of opportunistic infection due to bacterial contamination increases, and culture instability becomes a problem. On the other hand, it is also known that improving salt stress tolerance through mutation breeding reduces lipid content, and so there is a need to achieve both high lipid accumulation and high salt stress tolerance. Summary of the Invention [Means for solving the problem]

[0003] The present inventors have found that by using microalgae with an enhanced carbohydrate decomposition rate, a high fat / oil content can be achieved even under culture conditions without salt stress. Based on this finding, the present disclosure provides a method for producing fat / oil using microalgae with an enhanced carbohydrate decomposition rate.

[0004] Thus, the present disclosure provides: (Item 1) A method for producing fats and oils, comprising culturing a microalga that has an enhanced carbohydrate decomposition rate in the absence of salt stress. (Item 2) The method according to any of the preceding claims, wherein the enhancement of the carbohydrate decomposition rate is achieved by genetic modification of the microalgae. (Item 3) The method according to any one of the preceding claims, wherein the genetic modification includes improving the degradability of carbohydrates produced by the microalgae and / or improving the carbohydrate decomposition ability of the microalgae. (Item 4) Item 11. The method of any one of the preceding items, wherein the carbohydrates include starch and glycogen. (Item 5) The method according to any one of the above items, wherein the genetic modification comprises suppression or deficiency of the enzyme activity of starch debranching enzyme (DBE), and / or enhancement of the enzyme activity of starch branching enzyme (BE), amylase, and / or starch phosphorylase. (Item 6) The method according to any one of the preceding items, wherein the enhancement of the enzyme activity is achieved by increasing the gene copy number, using a high-expression promoter, and / or introducing a highly active enzyme gene derived from another organism. (Item 7) The method according to any one of the above items, wherein the production of the oil or fat is carried out under freshwater conditions. (Item 8) The method according to any one of the preceding items, wherein the microalgae include a mutant strain KOR1 derived from Chlamydomonas sp. JSC4 strain, and a strain derived from KOR1. (Item 9) 2. The method according to any one of the preceding claims, further comprising depriving a nitrogen nutrient source. (Item 10) The method according to any one of the preceding claims, wherein the production of the oil or fat is initiated by the lack of the nitrogen nutrient source. (Item 11) The method according to any one of the preceding items, further comprising culturing the microalgae under brackish or seawater conditions, wherein the microalgae further comprises a modification that improves salt stress tolerance. (Item A1) A composition for producing fats and oils, the composition comprising a microalga that has an enhanced rate of carbohydrate degradation in the absence of salt stress. (Item A2) The composition of any one of the preceding claims, wherein the enhanced carbohydrate degradation rate is achieved by genetic modification of the microalgae. (Item A3) The composition of any one of the preceding claims, wherein the genetic modification includes improving the degradability of carbohydrates produced by the microalgae and / or improving the carbohydrate decomposition ability of the microalgae. (Item A4) The composition of any one of the preceding claims, wherein the carbohydrates include starch and glycogen. (Item A5) A composition described in any one of the above items, wherein the genetic modification includes suppression or deficiency of the enzymatic activity of starch debranching enzyme (DBE), and / or enhancement of the enzymatic activity of starch branching enzyme (BE), amylase, and / or starch phosphorylase. (Item A6) The composition according to any one of the above items, wherein the enhancement of the enzyme activity is achieved by increasing the gene copy number, using a high-expression promoter, and / or introducing a highly active enzyme gene from another biological species. (Item A7) The composition according to any one of the preceding claims, wherein the production of the oil or fat is carried out under freshwater conditions. (Item A8) The composition of any one of the preceding claims, wherein the microalgae include a mutant strain KOR1 derived from Chlamydomonas sp. JSC4 strain, and a strain derived from KOR1. (Item A9) The composition according to any one of the preceding claims, wherein the microalgae are cultured under conditions deficient in a nitrogen nutrient source. (Item A10) The composition according to any one of the preceding claims, wherein the production of the oil or fat is initiated by the lack of the nitrogen nutrient source. (Item A11) The composition of any one of the preceding claims, wherein the microalgae further comprises a modification that improves salt stress tolerance and is cultured in brackish water or seawater conditions. (Item B1) A system for producing fats and oils, comprising: 1) Microalgae that exhibit enhanced carbohydrate decomposition rates in the absence of salt stress, 2) a culture medium; 3) Cultivation facilities and A system comprising: (Item B2) The system according to any one of the preceding claims, wherein the enhanced carbohydrate decomposition rate is achieved by genetic modification of the microalgae. (Item B3) The system of any one of the preceding claims, wherein the genetic modification includes improving the degradability of carbohydrates produced by the microalgae and / or improving the carbohydrate decomposition ability of the microalgae. (Item B4) The system of any one of the preceding claims, wherein the carbohydrates include starch and glycogen. (Item B5) The system described in any one of the above items, wherein the genetic modification includes suppression or deficiency of the enzyme activity of starch debranching enzyme (DBE), and / or enhancement of the enzyme activity of starch branching enzyme (BE), amylase, and / or starch phosphorylase. (Item B6) The system according to any one of the above items, wherein the enhancement of the enzyme activity is achieved by increasing the gene copy number, using a high-expression promoter, and / or introducing a highly active enzyme gene from another biological species. (Item B7) The system according to any one of the above items, wherein the production of the oil or fat is carried out under freshwater conditions. (Item B8) The system according to any one of the preceding claims, wherein the microalgae include a mutant strain KOR1 derived from Chlamydomonas sp. JSC4 strain, and a strain derived from KOR1. (Item B9) The system according to any one of the preceding claims, wherein the microalgae are cultured under conditions deficient in a nitrogen nutrient source. (Item B10) The system according to any one of the above items, wherein the production of the oil or fat is initiated by the deficiency of the nitrogen nutrient source. (Item B11) The system according to any one of the preceding claims, wherein the microalgae further comprises a modification that improves salt stress tolerance and is cultured under brackish or seawater conditions.

[0005] It is contemplated that one or more of the above features may be provided in combinations other than those specifically stated.Further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0006] Furthermore, features and notable actions and effects of the present disclosure other than those described above will become apparent to those skilled in the art by referring to the following description of the preferred embodiments of the present invention and the drawings. Effect of the Invention

[0007] According to the present disclosure, by using microalgae with an enhanced carbohydrate decomposition rate, a high lipid content is exhibited even under culture conditions that do not impose salt stress, eliminating the need for salt concentration control for lipid accumulation, as was done with the conventional strain (Chlamydomonas sp. JSC4 strain), thereby simplifying the culture process and reducing costs.

[0008] Furthermore, when cultured in freshwater conditions without salt stress, a higher oil content can be achieved than with the conventional strain (Chlamydomonas sp. JSC4 strain).

[0009] Furthermore, when cultivated under brackish or seawater conditions that cause salt stress, salt tolerance can be imparted while maintaining lipid accumulation, thereby achieving higher productivity than the conventional strain (Chlamydomonas sp. JSC4 strain) and reducing the risk of opportunistic infection due to bacterial contamination. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a graph showing the results of measuring the amount of biomass obtained by culturing microalgae having an enhanced carbohydrate decomposition rate in one embodiment of the present disclosure. [Diagram 2] FIG. 2 is a graph showing the measurement results of the amount of nitrate obtained by culturing microalgae having an enhanced carbohydrate decomposition rate in one embodiment of the present disclosure. [Diagram 3] FIG. 3 is a graph showing the results of measuring the amount of fats and oils obtained by culturing microalgae with an enhanced carbohydrate decomposition rate in one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure will be described below while showing the best mode. Throughout this specification, the expression of the singular form should be understood to include the concept of the plural form, unless otherwise specified. Therefore, the singular article (for example, in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will take precedence.

[0012] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0013] As used herein, "about" means ±10% of the preceding numerical value.

[0014] As used herein, "microalgae" refers to microorganisms that have chlorophyll and perform oxygenic photosynthesis. Microalgae can fix atmospheric CO2 through photosynthesis to synthesize organic matter (e.g., starch), while generating oxygen (O2) from water (H2O). Microalgae may have a unicellular morphology or a colonial morphology (e.g., filament, sheet, or ball). Microalgae may grow in marine, brackish, freshwater, or on land.

[0015] In the present specification, "microalgae" may refer to either prokaryotic cyanobacteria (blue-green algae) or eukaryotic organisms (e.g., green algae, diatoms, dinoflagellates, red algae, prasinophytes, euglena, and true eyespot algae). Examples of cyanobacteria (blue-green algae) include the genera Synechocystis, Arthrospira, Spirulina, Anabaena, Synechococcus, Thermosynechococcus, Nostoc, Prochlorococcus, Microcystis, and Gloeobacter. Examples of eukaryotic organisms include green algae such as those of the genera Chlamydomonas, Chlorella, Dunaliella, Hematococcus, Volvox, and Botryococcus; those of the genera Rhizosolenia, Chaetoceros, Cyclotella, Cylindrotheca, Navicula, and Phaeodactylum. , Thalassiosira, Fistulifera, and other diatoms; Amphidinium, Symbiodinium, and other dinoflagellates; Cyanidioschyzon, Porphyridium, and other red algae; Ostreococcus, and other prasinophytes; Euglena, and other euglenas; Nannochloropsis, and other true eyespots. For example, microalgae microbial species include Synechocystis sp. PCC6803, Synechococcus sp. PCC7002, and other microbial species.PCC7002, Synechococcus elongatus PCC7942, Arthrospira platensis (also called "Spirulina"), Spirulina maxima, Spirulina subsalsa, Anabaena sp. PCC7120, Chlamydomonas reinhardtii, Chlamydomonas sp., Chlorella vulgaris, Chlorella pyrenoidosa, Dunaliella salina, Dunaliella sp., Hematococcus pluvialis, Volvox carteri, Botryococcus braunii, Cyclotella cryptica, Cylindrotheca fusiformis, Navicula saprophila, Phaeodactylum tricornutum, Thalassiosira pseudonana, Fistulifera sp., Amphidinium sp., Symbiodinium microadriaticum microadriaticum, Cyanidioschyzon merolae, Porphyridium sp., Ostreococcus tauri, Euglena gracilis, Nannochloropsis oculata, etc.

[0016] In this specification, "oils and fats" refers to esters of glycerol and fatty acids (e.g., triglycerides, diglycerides, monoglycerides, etc.). In general, substances that are fluid at room temperature are called "oils" and substances that are not fluid are called "fats," but the concept encompasses both of these.

[0017] Specific examples of fats and oils include triglycerides composed of fatty acids such as palmitic acid, linoleic acid, stearic acid, linolenic acid, and oleic acid, and methyl ester compounds of these fatty acids have high combustion efficiency and are useful as biodiesel fuels, etc. These fats and oils may be used alone or in combination of two or more kinds.

[0018] In this specification, "salt stress" refers to a state of tension in cells caused by being placed in an environment of high salt concentration, and refers to stress experienced when exposed to an environment in which growth is inhibited due to an increase in osmotic pressure caused by the accumulation of salts in the soil, medium, or hydroponic solution in which the microalgae grow, and / or due to the intrusion of salts into the cells causing ion toxicity. The concentration of salts is not particularly limited, but may be the salt concentration in soil or medium classified as saline soil or saline medium. The salts that cause salt stress are not particularly limited, but may be salts observed in saline soil, such as phosphates, nitrates, and hydrochlorides. Specific examples of salts include alkali metal salts of phosphoric acid, nitric acid, and hydrochloric acid, such as sodium chloride and magnesium chloride, alkaline earth metal salts, and ammonium salts. The concentration of salt that causes salt stress varies depending on the type of microalgae and salt, but for general microalgae, the amount of sodium chloride can be about 10 mM to about 1 M.

[0019] As used herein, "in the absence of salt stress" refers to any environment and / or situation in which the above-mentioned salt stress does not exist, and stress other than salt stress may be present. "In the absence of salt stress" also includes situations in which the cell state is recognized as being in the absence of salt stress even in an environment of high salt concentrations, such as the acquisition of salt tolerance.

[0020] In this specification, the term "nitrogen nutrient source" refers to a nitrogen compound that can be utilized by microalgae, and a mixture containing such a nitrogen compound, and the concentration and content of the nitrogen compound in the medium are not particularly limited. For example, examples of nitrogen nutrient sources include urea compounds such as urea, thiourea, guanylurea salts, and melamine, nitrogen-containing organic compounds such as metal salts of ethylenediaminetetraacetic acid (EDTA), amine compounds, amide compounds, and imide compounds, livestock manure compost such as chicken manure, cow manure, and pig manure, organic fertilizers such as oil cake, defatted rice bran, and soybean meal, proteins such as casein, collagen, albumin, yeast extract, meat extract, and peptone, amino acid salts, inorganic nitrogen salts such as ammonia and nitric acid, and lime nitrogen, and the like, which can be used in combination of two or more kinds as desired. Preferred nitrogen sources include sodium nitrate, ammonium chloride, and the like. In the stage of growing microalgae, the nitrogen concentration contained as a nutrient source in the medium is preferably maintained so as not to become 0 mg-N / L, and preferably, the nitrogen concentration contained as a nutrient source in the medium is maintained at about 1 mg-N / L to 20 mg-N / L, and more preferably, at about 20 mg-N / L or more. There are no particular limitations on the method for maintaining the nitrogen concentration contained as a nutrient source in the medium within the above numerical range, but a method of supplementing with new medium at an appropriate timing during the cultivation of microalgae is considered.

[0021] As used herein, the term "carbohydrate" refers to a substance whose main component is basically monosaccharide. Examples of carbohydrates that are produced by microalgae through photosynthesis and accumulate within the cells include starch and glycogen.

[0022] As used herein, the "carbohydrate decomposition rate" refers to the rate at which the above-mentioned carbohydrates are decomposed within the cells of microalgae, and can be an index of the ease of decomposition of carbohydrates. In addition, as used herein, the "enhancement of the carbohydrate decomposition rate" can be achieved by enhancing the activity of enzymes involved in the decomposition of carbohydrates and / or by improving the ease of decomposition of carbohydrates through structural changes, or may have a combination of these properties.

[0023] As used herein, "freshwater conditions" refers to a low-salinity aqueous environment having a salt concentration lower than that of seawater or brackish water, typically with a total salt concentration of less than about 0.05% by weight.

[0024] In this specification, "brackish water conditions" refers to an aqueous environment in which the salt concentration is intermediate between brackish water and seawater, and generally has a total salt concentration of about 0.05% by weight to about 3% by weight.

[0025] As used herein, "seawater conditions" refers to an aqueous environment in which the salt concentration is higher than that of freshwater or brackish water, and generally the total salt concentration is about 3% by weight or more.

[0026] In this specification, "deficiency" is interpreted in a broad sense and refers to a process in which a substance, molecule, etc. is reduced and finally ceases to exist. "Deficiency of nitrogen nutrient source" refers to a nitrogen nutrient source being consumed by microalgae cells and disappearing from the medium, and includes a case in which nitrogen is not detected in the medium or is present at a concentration that is almost undetectable by a nitrogen concentration measurement method (such as a turbidity method or a color development method) that is widely used in this field, or a case in which nitrogen is present at a concentration that causes a nitrogen deficiency response in microalgae cells (a concentration that causes a nitrogen deficiency state for microalgae cells).

[0027] (Preferred embodiment) Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. In addition, the following embodiments of the present disclosure can be used alone or in combination.

[0028] In one aspect of the present disclosure, a method for producing fats and oils is provided, comprising culturing a microalga having an enhanced carbohydrate decomposition rate in the absence of salt stress. In one embodiment, the carbohydrates decomposed by the microalga in the method of the present disclosure can include starch and glycogen.

[0029] The microalgae of the present disclosure can exhibit a high fat and oil content even under culture conditions without salt stress. It is known that in Chlamydomonas sp. JSC4, salt stress increases the expression level of genes involved in starch decomposition, and the metabolic flux derived from starch decomposition is distributed to fat and oil synthesis, thereby improving the fat and oil content. In the mutant KOR1 derived from JSC4, the carbohydrates accumulated in the cells are converted from starch to easily decomposable glycogen by disruption of the starch debranching enzyme gene ISA1, promoting carbohydrate decomposition. Therefore, the method of the present disclosure provides a method for producing fat and oil by culturing microalgae with an enhanced carbohydrate decomposition rate.

[0030] In one embodiment, the microalgae is not particularly limited as long as it is a microorganism that has chlorophyll and performs oxygen-producing photosynthesis. Microalgae can fix CO2 in the atmosphere by photosynthesis to synthesize organic matter (e.g., starch), and can also generate oxygen (O2) from water (H2O). Microalgae may have a unicellular form or a colonial form (e.g., filament, sheet, or ball). Microalgae may grow in seawater, brackish water, freshwater, or on land. From the viewpoint of producing oil and fat components with high efficiency, microalgae of the genus Chlamydomonas belonging to the Chlorophyta division can be preferably used in the method of the present disclosure.

[0031] Chlamydomonas is a genus of unicellular flagellates in the Chlorophyceae order Chlamydomonadales (or Vulgaris). Most Chlamydomonas species are found in freshwater, but some grow in seawater. Preferred Chlamydomonas microalgae in the present disclosure are capable of growing in freshwater conditions.

[0032] In one embodiment, microalgae that can be used in the method of the present disclosure can be produced by, for example, genetically modifying the microalgae to enhance the carbohydrate decomposition rate. Such genetic modifications can include improving the decomposition of carbohydrates produced by the microalgae and / or improving the carbohydrate decomposition ability of the microalgae. Specifically, in one embodiment, microalgae that can be used in the method of the present disclosure can be produced by, for example, suppressing or deleting the enzyme activity of starch debranching enzyme (DBE) or enhancing the enzyme activity of starch branching enzyme (BE) to alter the carbohydrates produced by the microalgae and improve their decomposition. In another embodiment, microalgae that can be used in the method of the present disclosure can also be produced by enhancing the enzyme activity of amylase and / or starch phosphorylase to promote the decomposition of carbohydrates accumulated by the microalgae. Such enhancement of enzyme activity can be achieved, for example, by increasing the gene copy number, using a high-expression promoter, and / or by introducing a highly active enzyme gene from another organism.

[0033] (1) Introduction of mutations Such variants, particularly variants in which the enzyme activity of starch debranching enzyme (DBE) is suppressed or deleted, can be obtained, for example, by selective breeding of conventional algae by combining random mutagenesis by ion beam irradiation with high-speed screening by flow cytometry. Although the parent strain is not particularly limited, from the viewpoint of producing oil and fat components with high efficiency, it is preferable to use Chlamydomonas sp. JSC4 or its variant KOR1 strain (JP Patent Publication 2020-195344) as the parent strain. Mutations can be introduced by irradiating a cell population with an ion beam. When an ion beam passes through a cell nucleus, it generates various DNA damages, including DNA double-strand breaks. Cells repair DNA using their own DNA repair mechanisms, but various mutations such as deletions may occur during the process. The ion beam to be irradiated is not particularly limited as long as it can introduce mutations. Examples of the ion beam include carbon (C), helium (He), neon (Ne), and argon (Ar). From the viewpoint of efficiency of introducing mutations into algae, 12 C 5+ The algae belonging to the genus Chlamydomonas are preferred. 12 C 5+ When irradiating with an ion beam, the range of the dose is preferably 10 to 250 Gy, more preferably 50 to 100 Gy. After ion beam irradiation, recovery culture is performed for several days, and the resulting cell population is used as a mutant library, which can be screened as described below. The recovery culture is performed by leaving the cells stationary for 3 days or more under conditions such as neutral white fluorescent light with appropriate light intensity.

[0034] Therefore, in one embodiment of the present disclosure, examples of microalgae that can be used in the method of the present disclosure include the mutant strain KOR1 derived from Chlamydomonas sp. JSC4 strain, and strains derived from KOR1.

[0035] In another embodiment, for example, when preparing a mutant having enhanced enzyme activity of starch branching enzyme (BE), gene introduction can be performed. "Introduction" of a gene refers to introducing an exogenous or endogenous gene, preferably a functional gene, into, for example, a chromosomal genome, etc., by an appropriate introduction technique. A gene can be introduced using a vector such as a phage or a plasmid, and natural transformation, conjugation, protoplast-PEG, electroporation, etc. can also be used for gene introduction. In addition, by utilizing a target gene recombination method known in the art, an exogenous functional gene can be introduced by replacing it with an endogenous functional gene. Note that an exogenous functional gene is a gene that does not originally exist in the chromosomal genome of the organism, and can be a gene derived from another organism or a synthetic gene produced by PCR or the like. Gene introduction also includes conversion to a desired gene by genome editing of an existing genome.

[0036] In one embodiment, when preparing a mutant having enhanced enzyme activity of starch branching enzyme (BE), a method of introducing a mutation into a gene encoding the target enzyme, a method of newly introducing a base sequence derived from the same or different species, a method of modifying a gene or base sequence involved in the control of the target enzyme gene, etc. can be mentioned. A method of introducing a mutation into an enzyme gene can be a site-specific mutagenesis method. Specific examples of a method of introducing a site-specific mutation include a method using an SOE-PCR reaction, an ODA method, and a Kunkel method. In addition, a commercially available kit such as a Site-Directed Mutagenesis System Mutan-SuperExpress Km Kit (Takara Bio Inc.), a Transformer TM Site-Directed Mutagenesis Kit (Clonetech), and a KOD-Plus-Mutagenesis Kit (Toyobo Co., Ltd.) can be used. In addition, a target gene can be obtained by randomly mutating a gene and then evaluating the enzyme activity and analyzing the gene by an appropriate method.

[0037] (2) Screening Primary screening of variants can be performed using a fluorescence activated cell sorter (FACS). Intracellular oil droplets of cultured cells are stained with fluorescent dyes such as BODIPY and Nile Red, and cells with strong fluorescence intensity are separated by FACS. The intensity of BODIPY fluorescence, fluorescence derived from staining of intracellular oil droplets such as Nile Red fluorescence, and chlorophyll autofluorescence (used as an index of cell size) of individual cells is analyzed by FACS, and cells with high fluorescence derived from staining of intracellular oil droplets such as BODIPY fluorescence and Nile Red fluorescence per chlorophyll autofluorescence (for example, the top 1 to 0.5%) can be separated. In addition, it is also possible to concentrate the target cells by repeating the above-mentioned culture and separation by FACS multiple times.

[0038] The cells were seeded on agar medium and exposed to light with a light intensity of 50 μmol photons / m 2 The cells are cultured under neutral white fluorescent light for about 100 s until colonies are formed. The candidate strains obtained in the primary screening are cultured in a microwell plate, and the cells are allowed to accumulate fats and oils, which are then analyzed by gas chromatography-mass spectrometry (GC-MS) to perform secondary screening of high fat and oil accumulation strains. In the secondary screening, the amount of biomass, fats and oils, carbohydrates, etc. are measured according to the method described below, and the fat and oil content and carbohydrate content are calculated to select the mutant strain of the present disclosure.

[0039] The microalgae obtained by mutation breeding are suspended in a medium described below in an incubator such as a flask under conditions of 0.04% to 10% CO2, preferably 0.5% to 5% CO2, and more preferably 1% to 3% CO2, at 15°C to 40°C, preferably 20°C to 35°C, and more preferably 25°C to 30°C, and pre-cultured for about 2 to 10 days, preferably 2 to 7 days, and more preferably 2 to 5 days under light conditions that allow photosynthesis. When the microalgae grow smoothly and the number of cells is sufficient, expansion culture is performed, and main culture is performed for 5 to 20 days, preferably 7 to 16 days, and more preferably 10 to 14 days under light conditions that allow photosynthesis as described above.

[0040] Regarding the light conditions, conditions that allow the final recovery of a large amount of oil and fat are preferable. Continuous cultivation under conditions that allow photosynthesis is possible, or 50 to 2000 μmol photons / m 2 The cells may be cultured under a day-night cycle of about 12 hours of light and 12 hours of darkness using natural white fluorescent lights with a light output of about 100 μmol photons / m 2 ·Sec~2,000μmol photons / m 2 seconds range, typically 80 μmol photons / m 2 ·Sec~1,000μmol photons / m 2 sec, typically 100 μmol photons / m 2 ·sec~500μmol photons / m 2 ·Second range is more preferable.

[0041] As the culture method used in the present disclosure, a stationary culture method can be used, but considering the productivity of the algae body of the microalgae and the productivity of the oil and fat components, culture by a shaking culture method or a deep aeration stirring culture method is preferable. The shaking culture may be a reciprocating shake or a rotary shake.

[0042] The medium used for the above culture can be appropriately set depending on the type of microalgae used, and when using the genus Chlamydomonas, for example, there is no particular limitation as long as the medium can grow microalgae belonging to the genus Chlamydomonas. For example, examples of the basal medium include Modified Bold 6N (MB6N) medium, TAP medium, HSM medium, BG-11 medium, BBM medium, etc., and MB6N medium is more preferable because it can produce oil and fat components with high efficiency.

[0043] A characteristic of the culture used in the method of the present disclosure is that it is cultured under conditions where the concentration of the nitrogen nutrient source in the medium is low. Culture under conditions where the nitrogen nutrient source concentration is low may be culture under nitrogen deficiency conditions due to nitrogen consumption accompanying growth, or culture by transplanting algae bodies into a medium with a low concentration of the nitrogen nutrient source. Cultivation under conditions where the nitrogen nutrient source concentration is low makes it possible to produce oil and fat components with high efficiency. In the method of the present disclosure, the concentration of the nitrogen nutrient source contained in the medium is determined by calculating the concentration of nitrate ions contained in the medium as the optical density (OD) at a wavelength of 220 nm. 220 ) as an indicator, an ion sensor, or a method for measuring absorbance using a color-developing reagent.

[0044] Thus, in one embodiment of the method of the present disclosure, a step of depriving the nitrogen nutrient source can be further included, which can initiate the production of oils and fats.

[0045] Biomass volume In one embodiment, in the method of the present disclosure, the amount of biomass can be calculated using the dry weight of the cells as an index. The measurement of the amount of biomass can be performed by a method known to those skilled in the art, and the method is not limited, but can be performed, for example, as follows. That is, a required amount of microalgae cells obtained by the above-mentioned culture is collected 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 weight of the empty microtube is subtracted to obtain the dry weight (mg) of the collected dried algae. Furthermore, the amount of biomass (g / L) contained in the culture solution can be calculated by dividing this by the amount of culture solution used for the measurement. After weighing, the dried algae are used for the measurement of fats and oils described below.

[0046] fats and oils In one embodiment, the microalgae used in the method of the present disclosure are characterized by a high oil content, for example, the oil content under a day-night cycle condition of 12 hours light / 12 hours dark is usually 20% by weight or more, preferably 30% by weight or more, and more preferably 40% by weight or more. The microalgae used in the method of the present disclosure are also characterized by a high oil production rate, and the oil production rate under a day-night cycle condition of 12 hours light / 12 hours dark is 120 g / m 3 / day or more, 150 g / m 3 / day or more is preferable, and 180 g / m 3 / day or more is more preferable. In this specification, the term "oil production rate under a day-night cycle condition of 12 h light / 12 h dark" refers to the maximum oil production rate achieved in the main culture for 14 days. In addition, the term "oil content under a day-night cycle condition of 12 h light / 12 h dark" refers to the maximum oil content per dry algae body achieved in the main culture for 14 days.

[0047] In the method of the present disclosure, the measurement of fats and oils can be performed by a method known to those skilled in the art, and the method is not limited, but can be performed, for example, as follows. For the measurement of fats and oils, the dried algae prepared in the biomass measurement experiment is used. The dried algae are weighed in a microtube dedicated to crushing and subjected to the measurement. 0.5 mm diameter glass beads are added to the microtube and the cells are crushed by a multi-beads shocker device. The fats and oils in the cells are methylated using a fatty acid methylation kit (manufactured by Nacalai, etc.), and the fatty acid methyl esters thus produced are quantified by gas chromatography-mass spectrometry (GC-MS), and the fats and oil content (wt%) per dried algae and the fats and oil (oil) production amount (mg / L) per culture solution can be calculated. The fats and oil (oil) production amount (mg / L) per culture solution is calculated by multiplying the fats and oil content by the amount of biomass. In addition, the culture solution can be sampled over time to check the increase or decrease over time in the lipid content (weight %) per dry algae body and the lipid (oil) production amount (mg / L) per culture solution. Furthermore, by measuring the amount of lipid obtained per day from a unit culture solution (L), the lipid (oil) production rate (mg / L / day) can be calculated. The lipid production rate can be calculated by dividing the lipid production amount (mg / L) by the number of days required for cultivation, starting from day 0 of cultivation.

[0048] The oils and fats accumulated by the microalgae used in the method of the present disclosure include triglycerides composed of fatty acids such as palmitic acid, linoleic acid, stearic acid, linolenic acid, and oleic acid, and methyl ester compounds of these fatty acids have high combustion efficiency and are useful as biodiesel fuel, etc.

[0049] <Oil and fat production> The microalgae obtained as described above can be cultured in the absence of salt stress, such as in freshwater conditions, to produce fats and oils. In one embodiment, the culture in the absence of salt stress can be carried out by imparting high salt stress resistance to the microalgae through another genetic modification, and fats and oils can also be produced by culturing in brackish water or seawater conditions. In such cases, the culture conditions can be a combination of the above-described pre-culture and / or main culture conditions and a medium containing high concentrations of seawater salt, artificial seawater, sodium chloride, etc.

[0050] As a method for extracting oil and fat components from cultured microalgae, a conventional oil and fat extraction method can be used, in particular, a general extraction method using an organic solvent such as a chloroform / methanol system, as represented by the Folch method or the Bligh-Dyer method, can be used, but is not limited to these.

[0051] In one embodiment, for the cultivation of microalgae in the production of oils and fats, for example, the microalgae can be cultured while being expanded stepwise from a scale of several hundred mL, and finally sown in an outdoor raceway pond. In the outdoor raceway pond cultivation, the medium to be used is not particularly limited as long as it is low-cost, but it can be prepared by adding necessary nutrient sources to freshwater (industrial water) or seawater, and the nitrogen nutrient source can be adjusted to a concentration that causes deficiency within a few days after the start of cultivation. During the cultivation period, the algae concentration and the nitrogen nutrient source concentration are monitored, and after the deficiency of the nitrogen nutrient source is confirmed, cultivation is continued for another 4 to 10 days. During the cultivation period, 1% to 100% CO2 can be continuously supplied, or the pH of the culture solution can be monitored and CO2 can be supplied only when the pH becomes equal to or higher than a certain level.

[0052] In another aspect of the present disclosure, there is provided a composition for producing oils and fats, the composition comprising a microalga having an enhanced rate of carbohydrate degradation in the absence of salt stress. Such a composition can have any of the features described elsewhere herein.

[0053] In another aspect of the present disclosure, a system for producing oils and fats is provided, the system comprising: 1) a microalgae having an enhanced carbohydrate degradation rate in the absence of salt stress; 2) a culture medium; and 3) a culture facility. Such a system may comprise any of the features described elsewhere herein.

[0054] (General technology) The molecular biological, biochemical and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999).These methods are described in Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Special Edition of Experimental Medicine: "Gene Introduction & Expression Analysis Experimental Methods" Yodosha, 1997, etc., and the relevant parts (possibly in their entirety) of these are incorporated herein by reference.

[0055] Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, gene synthesis and fragment synthesis services such as those of GeneArt, GenScript, Integrated DNA Technologies (IDT), etc. can be used. In addition, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (I996). Bioconjugate Techniques, Academic Press, etc., the relevant portions of which are incorporated herein by reference.

[0056] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when it is specified that "within the range of" "two values", the range includes the two values ​​themselves. All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth.

[0057] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims. EXAMPLES

[0058] (Example 1: Preparation of modified bodies) The starch debranching enzyme gene knockout strain was created using Chlamydomonas sp. JSC4 as the parent strain. Mutations were introduced by irradiating JSC4 cell populations with ion beams at the Takasaki Ion accelerators for Advanced Radiation Application (TIARA) facility of the National Institutes for Quantum and Radiological Science and Technology.

[0059] Specifically, an appropriate amount of algae was taken from the agar medium and pre-cultured for 3 days under the following conditions: Optical density at a wavelength of 750 nm (OD 750 The OD was then increased to 0.04, and the culture was continued for another 2 days under the following conditions. 750 The antibody was diluted with TAP medium so that the concentration was 0.5, and 100 μL of the diluted solution was applied to the TAP agar medium. 12 C 5+ The algae cells on the agar medium were irradiated with 50 Gy of ion beam. After irradiation, the light intensity was 50 μmol photons / m 2 The cells were then allowed to stand under a neutral white fluorescent lamp at 375 nm for 3 days or more for recovery culture, and the resulting cells were used as the JSC4 mutant library in the following experiments.

[0060] (Culture conditions) Use a two-tier flask Medium: 70mL mTAP CO2: 2% CO2 Light: 100μmol photons / m 2 / sFluorescent lamp Temperature: 30℃ Mixing: 100 rpm

[0061] (2) Primary screening Primary screening of mutants under day and night conditions was performed using a fluorescence activated cell sorter (FACS). Intracellular oil droplets of cells cultured under day and night conditions were stained with the fluorescent dye BODIPY, and cells with strong fluorescence intensity were sorted by FACS.

[0062] An appropriate amount of algal cells was taken from the agar medium for the mutant library prepared above, and pre-cultured for 3 days under the following conditions. OD 750 The cells were subcultured so that the β-amyloid ratio was 0.04, and then cultured for an additional 7 days under the conditions described below. After culture, the cells were harvested and diluted to 5 × 10 6 The cells were suspended in PBS at a concentration of 50 cells / mL. The fluorescent dye BODIPY was added to this at 50 μM and left in the dark for 5 minutes to fluorescently stain the intracellular oil droplets. The intensity of BODIPY and chlorophyll autofluorescence (used for standardization as an index of cell size) of each cell was analyzed by FACS (SONY SH800), and cells with high BODIPY fluorescence per chlorophyll autofluorescence (top 1-0.5%) were sorted. The filter set used for fluorescence detection is shown below. After sorting, the cells were plated on TAP agar medium and exposed to a light intensity of 50 μmol photons / m 2 The cells were cultured under neutral white fluorescent light at 3500 x 1000 / s until colonies were formed.

[0063] (Culture conditions) Use a two-tier flask Culture medium: 70mL MB6N + 2% sea salt CO2: 2% CO2 Light: 250 μmol photons / m 2 / s Fluorescent lamp (day-night cycle conditions: 12h light + 12h dark) Temperature: 30℃ Mixing: 100 rpm

[0064] (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)

[0065] (3) Secondary screening A secondary screening of high oil accumulating strains under day and night conditions was carried out by culturing the candidate strains in microwell plates and accumulating oil from the cells and analyzing it by gas chromatography-mass spectrometry (GC-MS).

[0066] An appropriate amount of the algal cells of the candidate mutant strain obtained in the primary screening was taken from the agar medium and pre-cultured for 4 days under the following conditions. OD 750 The cells were subcultured so that the ratio of the oil content to the total oil content was 0.1, and the main culture was continued for another 8 days under the conditions below. On the fourth day of the main culture, all cells were collected from each well and resuspended in MB0N + 2% sea salt to continue the culture. After the culture, 2 mL of culture medium was collected from each well, and KOR1 was obtained as a mutant strain that highly accumulates oil.

[0067] (Culture conditions) 12-well plates were used. Culture medium: Preculture: 3mL MB6N + 2% sea salt Main culture 1:3mL MB6N+2% sea salt Main culture 2:3mL MB0N+2% sea salt CO2: 2% CO2 Light: 100μmol photons / m 2 / s White LED (day-night cycle conditions: 12h light + 12h dark) Temperature: 30℃ Mixing: 100 rpm

[0068] (Example 2: Evaluation of fat and oil content) Chlamydomonas sp. KOR1, a disruptant of the starch debranching enzyme gene ISA1, was cultured in the freshwater medium MB6N (0% sea salt) and in a medium in which sea salt was added to MB6N (2% or 4% sea salt). Specifically, an appropriate amount of algae was taken from the agar plate medium and pre-cultured for 3 days under the following conditions. The optical density at a wavelength of 750 nm (OD 750 ) was 0.04, and main culture was carried out for an additional 7 days under the following conditions.

[0069] The culture conditions and the composition of MB6N medium are as follows. Culture conditions (two-tier flask, Biotron incubator) Media: (1) 70mL MB6N (2) 70mL MB6N + 2% sea salt (3) 70mL MB6N + 4% sea salt CO2: 50mL 2% CO2 adjusted solution (changed after sampling on the 2nd and 4th days of culture) Light: 250μmol photons / m 2 / s Daylight white fluorescent lamp Temperature: 30℃ Mixing: 100 rpm

[0070] MB6N medium composition (per 1L) NaNO3: 750mg K2HPO4: 38.3mg MgSO4·2H2O: 75 mg KH2PO4: 88mg NaCl: 25 mg CaCl2·2H2O: 25.2 mg FeCl3·6H2O: 1.77 mg ZnSO4·7H2O: 0.0732 mg CoSO4·7H2O: 0.016 mg MnSO4·5H2O: 0.584 mg Na2MoO4·2H2O: 0.00148 mg Na2SeO3: 0.001728mg NiCl2·6H2O: 0.001488 mg

[0071] After culturing under the above culture conditions, biomass measurement, nitrate measurement, and lipid content of the cells were each performed by the following methods.

[0072] Biomass Measurement The required amount of cells was collected in a weighed microtube, washed once with distilled water, and then freeze-dried overnight. After drying, the microtube was weighed again, and the weight of the empty microtube was subtracted to obtain the dry weight (mg) of the collected cells. This was then divided by the volume of culture solution used for the measurement to obtain the amount of biomass (g / L) contained in the culture solution.

[0073] Nitrate Measurement Since NaNO3 absorbs light with a wavelength of 220 nm well, the amount of NaNO3 contained in the medium was measured using the optical density (OD) at a wavelength of 220 nm. 220 The OD was used as an indicator. The cells were removed by centrifugation to prepare the culture supernatant. The culture supernatant was appropriately diluted with distilled water and the OD 220 A calibration curve was created using MB6N containing known concentrations of NaNO3, and the NaNO3 concentration in the culture supernatant was calculated based on this.

[0074] Oil and fat measurement Approximately 3 mg of dried algal cells obtained from the biomass measurement were weighed into a microtube designed for crushing and subjected to the measurement. 0.5 mm diameter glass beads were added to the microtube and the cells were crushed using a multi-beads shocker device. The oils and fats in the cells were methylated using a fatty acid methylation kit (Nacalai), and the fatty acid methyl esters produced by this were quantified by gas chromatography-mass spectrometry (GC-MS).

[0075] The results are shown in Figures 1 to 3. As can be seen from Figure 3, KOR1 showed the same level of fat and oil content even in 0% seawater salt as in 2% and 4% seawater salt. Since the starch debranching enzyme gene is disrupted in KOR1, it is thought that the carbohydrate decomposition rate is enhanced, resulting in a high fat and oil content even in the absence of salt stress.

[0076] (Example 3: Creation of modified organisms by gene transfer) The preparation of the mutant by gene transfer is carried out by introducing DNA into Chlamydomonas sp. JSC4 cells by electroporation.

[0077] Specifically, an appropriate amount of algae is taken from the agar medium and pre-cultured for 2 days under the following conditions: Optical density at a wavelength of 750 nm (OD 750 ) to 0.04, and the cell density was increased to 0.8 × 10 6 cell / mL ~ 2.5 × 10 6 Continue culturing until the cell density reaches 1.0×10 cells / mL. After culturing, centrifuge the cells to separate them. 8 Suspend the cells in TAP + 40 mM sucrose at 10 cells / mL and place on ice. Add the DNA to be introduced at 10 ng / μL and perform electroporation. After static culture overnight under low light conditions, inoculate the cells on TAP agar medium (containing 10 μg / mL paromomycin) and culture until colonies are formed.

[0078] (Culture conditions) Use a 200mL Erlenmeyer flask Medium: 70mL TAP Light: 50 μmol photons / m 2 / sWhite LED Temperature: 25℃ Mixing: 100 rpm

[0079] To enhance enzyme activity, the genes of highly active starch branching enzymes endogenous to Chlamydomonas sp. or from other organisms, and amylase and starch phosphorylase genes are prepared by gene synthesis or other methods. Candidates for highly active enzymes from other organisms include the starch branching enzyme from Solanum tuberosum and starch phosphorylase from Zea mays. One or more of these genes are placed downstream of a highly active promoter (Hsp70A promoter, RBCS2 promoter, etc.) and cloned into a general plasmid vector together with a selection marker (paromomycin resistance gene aphVIII, etc.). The plasmid is linearized by restriction enzyme treatment, or the DNA sequence containing these factors amplified by PCR is introduced into cells by electroporation.

[0080] (Example 4: Example of using a variant that improves salt stress tolerance) Creation of mutants with improved salt stress tolerance Using the same procedure as in Example 1, a mutant library is prepared by irradiating a Chlamydomonas sp. strain in which carbohydrate decomposition has been promoted with ion beam irradiation. As a first screening step, this mutant library is subcultured once a week using MB6N medium containing a concentration of seawater salt (5% or more) at which Chlamydomonas sp. cannot normally grow. If an improvement in growth is observed during this process, the seawater salt concentration of the medium is gradually increased. Once a cell population that grows well even in a high seawater salt concentration (7% or more) is obtained, it is cloned by seeding it on an agar medium. As a second screening step, it is confirmed again that the isolated clones grow well even in a medium with a high seawater salt concentration (7% or more).

[0081] Experimental example using mutants with improved salt stress tolerance The mutant with enhanced carbohydrate decomposition and salt stress resistance was cultured and evaluated in MB6N medium containing 3% or more seawater salt in the same manner as in Example 2. This mutant is expected to achieve both high cell proliferation and high lipid accumulation even in a seawater salt concentration of 3% or more.

[0082] (Example 5: Example of depriving nitrogen nutrient source) Since the amount of nitrogen nutrient that can be consumed varies depending on the strain and culture conditions, culture is performed by changing the medium to one with a lower amount of nitrogen nutrient, such as MB3N medium (375 mg NaNO3), depending on the strain and culture conditions.

[0083] Example 6: Biofuel Production As a method for mass-cultivating microalgae, open-system cultivation using outdoor raceway ponds or closed-system cultivation using bioreactors are used to cultivate mutants with enhanced carbohydrate decomposition, which were prepared using the same procedures as in Example 1. Microalgae are harvested from this culture medium using centrifugation or other methods, and oil is extracted from the microalgae cells and methyl esterified for use as biodiesel fuel.

[0084] (Note) Although the present disclosure has been illustrated using the preferred embodiments thereof, it is understood that the present disclosure should be interpreted in scope only by the claims. It is understood that the patents, patent applications, and other documents cited in this specification are incorporated by reference into this specification in the same manner as if the contents themselves were specifically set forth herein. [Industrial Applicability]

[0085] According to the present disclosure, it is possible to produce oils and fats using microalgae at low production costs. In addition, it is expected that the culture in outdoor open ponds will be stabilized, and the method can be used for biofuel production, which is expected to be applied in industrial fields such as industrial products and the food industry.

Claims

Claim 1 A method for producing oil and fat, comprising the step of culturing microalgae with an enhanced rate of carbohydrate decomposition in the absence of salt stress. Claim 2 The method according to claim 1, wherein the enhancement of the rate of carbohydrate decomposition is achieved by genetic modification of the microalgae. Claim 3 The method according to claim 2, wherein the genetic modification includes improvement of the degradability of carbohydrates produced by the microalgae and / or improvement of the carbohydrate-degrading ability of the microalgae. Claim 4 The method according to any one of claims 1 to 3, wherein the carbohydrates include starch and glycogen. Claim 5 The method according to claim 2 or 3, wherein the genetic modification includes suppression or deficiency of the enzymatic activity of debranching enzyme (DBE) and / or enhancement of the enzymatic activities of branching enzyme (BE), amylase, and / or starch phosphorylase. Claim 6 The method according to claim 5, wherein the enhancement of the enzymatic activity is achieved by an increase in the gene copy number, utilization of a highly expressing promoter, and / or introduction of a highly active enzyme gene derived from another biological species. Claim 7 The method according to any one of claims 1 to 3, wherein the production of the oil and fat is carried out under freshwater conditions. Claim 8 The method according to any one of claims 1 to 3, wherein the microalgae include a mutant strain KOR1 derived from Chlamydomonas sp. JSC4 strain and strains derived from KOR1. Claim 9 The method according to any one of claims 1 to 3, further comprising the step of depriving of a nitrogen nutrient source. Claim 10 The method according to claim 9, wherein the production of the oil and fat is initiated by the deprivation of the nitrogen nutrient source. Claim 11 The method according to any one of claims 1 to 3, wherein the microalgae further include a modification for improving salt stress tolerance and include the step of culturing under brackish water conditions or seawater conditions. Claim 12 A composition for producing oil and fat, the composition including microalgae with an enhanced rate of carbohydrate decomposition in the absence of salt stress. Claim 13 The composition according to claim 12, wherein the enhancement of the rate of carbohydrate decomposition is achieved by genetic modification of the microalgae. Claim 14 The composition according to claim 13, wherein the genetic modification includes improvement of the degradability of carbohydrates produced by the microalgae and / or improvement of the carbohydrate-degrading ability of the microalgae. Claim 15 The composition according to any one of claims 12 to 14, wherein the carbohydrate comprises starch and glycogen.

16. The composition according to claim 12 or 14, wherein the genetic modification comprises suppression or deficiency of the enzymatic activity of starch debranching enzyme (DBE) and / or enhancement of the enzymatic activity of starch branching enzyme (BE), amylase, and / or starch phosphorylase.

17. The composition according to claim 16, wherein the enhancement of the enzymatic activity is achieved by an increase in the gene copy number, utilization of a highly expressing promoter, and / or introduction of a highly active enzyme gene derived from another biological species.

18. The composition according to any one of claims 12 to 14, wherein the production of the oil is carried out under freshwater conditions.

19. The composition according to any one of claims 12 to 14, wherein the microalgae comprises a mutant strain KOR1 derived from Chlamydomonas sp. JSC4 strain and strains derived from KOR1.

20. The composition according to any one of claims 12 to 14, wherein the microalgae is cultured under conditions lacking a nitrogen nutrient source.

21. The composition according to claim 20, wherein the production of the oil is initiated by the lack of the nitrogen nutrient source.

22. The composition according to any one of claims 12 to 14, wherein the microalgae further comprises a modification for improving salt stress tolerance and is cultured under brackish water conditions or seawater conditions.

23. A system for producing oil, comprising: 1) microalgae with an enhanced rate of carbohydrate decomposition in the absence of salt stress; 2) a culture medium; 3) culture equipment A system.

24. The system according to claim 23, wherein the enhancement of the rate of carbohydrate decomposition is achieved by genetic modification of the microalgae.

25. The system according to claim 24, wherein the genetic modification comprises improvement of the degradability of the carbohydrate produced by the microalgae and / or improvement of the carbohydrate-degrading ability of the microalgae.

26. The system according to any one of claims 23 to 25, wherein the carbohydrate comprises starch and glycogen.

27. The system according to claim 24 or 25, wherein the genetic modification comprises suppression or deficiency of the enzymatic activity of starch debranching enzyme (DBE) and / or enhancement of the enzymatic activity of starch branching enzyme (BE), amylase, and / or starch phosphorylase.

28. The system according to claim 27, wherein the enhancement of the enzyme activity is achieved by an increase in the gene copy number, the use of a highly expressing promoter, and / or the introduction of a highly active enzyme gene derived from another species.

29. The system according to any one of claims 23 to 25, wherein the production of the oil is carried out under freshwater conditions.

30. The system according to any one of claims 23 to 25, wherein the microalgae include a mutant strain KOR1 derived from Chlamydomonas sp. JSC4 strain and a strain derived from KOR1.

31. The system according to any one of claims 23 to 25, wherein the microalgae are cultured under conditions lacking a nitrogen nutrient source.

32. The system according to claim 31, wherein the production of the oil is initiated by the lack of the nitrogen nutrient source.

33. The system according to any one of claims 23 to 25, wherein the microalgae further include a modification for improving salt stress tolerance and are cultured under brackish water conditions or seawater conditions.

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