WO2017039344A1 - Method for producing microalgae of which autophagy is inhibited and amounts of starch and lipids are increased - Google Patents

Abstract

The present invention relates to: a method for producing microalgae of which the amounts of starch and lipids are increased, comprising a step of inhibiting autophagy while culturing the microalgae under an autophagy inducing condition; microalgae produced by the method such that the amounts of intracellular starch and lipids are increased; biomass including the microalgae; a method for producing biodiesel by using the biomass; and biodiesel produced by the method. When using the method of the present invention, microalgae, of which the amounts of intracellular starch and lipids are increased, can be produced and the produced microalgae, of which the amounts of intracellular starch and lipids are increased, can be used as biomass for producing biodiesel, and thus the microalgae can be useful in the economical production of biodiesel.

Description

Method for producing microalgae with suppressed autophagy and increased lipid and starch content

The present invention relates to a method for producing microalgae in which autophagy is suppressed and lipid and starch content is increased, and more specifically, the present invention includes the step of inhibiting autophagy while culturing the microalgae in autophagy induction conditions. Method for producing microalgae with increased lipid and starch content, microalgae prepared by the above method with increased lipid and starch content in cells, biomass containing the microalgae, and biodiesel production using the biomass It relates to a method and a biodiesel produced by the method.

In the face of depletion of fossil energy and global warming all over the world in recent years, the development of new and renewable energy such as eco-friendly bio, hydrogen, solar, etc. Promoting supply and use. Although many efforts are being made to develop new and renewable energy in Korea, it is still in the research stage. Bioenergy is renewable and has the advantage of reducing global warming acceleration by fixing carbon dioxide. Currently, research on bioethanol, biobutanol, biodiesel, etc. has been actively conducted.

Among the bioenergy, biodiesel is being actively researched. Biodiesel is produced by conversion into methyl ester or ethyl ester form by the transesterification process of fatty acid contained in biomass, and has a flash point of 150 ℃, which is less than diesel. It does not stick well and is classified as a non-flammable liquid because it is more stable than gasoline (45 ° C.), which is highly volatile at low temperatures, and is more stable because it catches fire at high temperatures. In addition, unlike diesel, biodiesel is known to have low emissions of carcinogens and mutations when burned and non-toxic energy with other emissions significantly lower.

Such biodiesel is known to be biologically produced using microalgae. Microalgae have 25 times higher efficiency of solar energy than plants, so they are used for feed or fertilizer. Especially, Spirulina sp., Chlorella sp. And Dunaliella sp. Nostoc sp. Is also used as a health food. Since the microalgae have excellent solar energy utilization efficiency, the microalgae having excellent lipid content can be used as biomass for the production of biodiesel. Such microalgae with excellent lipid content are known as Botryococcus sp. And Schiochytrium sp. The microalgae do not have high lipid content in normal living conditions, but nutrient supply is stopped. When a certain time elapses in this state, lipids accumulate in the cell. The microalgae may be used as a biomass for the production of biodiesel by using such characteristics, but in order to increase the lipid content of the microalgae, the first step of culturing and growing the microalgae, and the expanded microalgae In order to stop the supply of nutrients and incubate for a certain period of time to increase intracellular lipid content, the second step requires excessive time, and thus has not yet been used for industrial production of biodiesel. Research into the development of microalgae with excellent lipid content for the production of biodiesel has been actively conducted.

For example, U.S. Patent Publication No. 2012-0329099 discloses a method for producing microalgae overexpressing isoamylase to produce starch and lipids in high yield, and Korean Patent Publication No. 2014-0010898 discloses Disclosed is a method of culturing microalgae under conditions deficient in sulfur while regulating, to accumulate starch from the microalgae. However, in the microalgae in which isoamylase is overexpressed, since the produced starch and lipids not only accumulate in the cell but are also discharged to the outside, the content of starch and lipids accumulated in the cell is not high, and sulfur is controlled while controlling the concentration of carbon dioxide. When the microalgae are cultured under the deficient condition, there is a risk that the microalgae are killed.

The present inventors have diligently researched to develop microalgae having a high lipid content, and when the microalgae were cultivated under a condition in which autophagy was induced, the inhibition of autophagy was not only increased, but also the lipid content in the cells was increased. Since the content is also increased, it has been confirmed that the microalgae with increased lipid and starch content can be used as biomass for the production of biodiesel, thus completing the present invention.

One object of the present invention is to provide a method for producing microalgae with increased lipid and starch content in cells.

Another object of the present invention is to provide microalgae with increased lipid and starch content in the cells produced by the above method.

Still another object of the present invention is to provide a biomass including microalgae with increased lipid and starch content in cells.

Still another object of the present invention is to provide a method for preparing biodiesel using the biomass.

Another object of the present invention is to provide a biodiesel produced by the above method.

By using the method of the present invention, it is possible to prepare microalgae with increased lipid and starch content in cells, and the prepared microalgae with increased lipid and starch content in cells can be used as biomass for the production of biodiesel. As such, it may be widely used for economic production of biodiesel.

Figure 1a Chlamydomonas genus microalgae autophagy from reinfardtii CC124) and autophagy inhibitor treatment, micrograph showing the results of comparing the production of fat globules, WT represents the microalgae of the control group induced autophagy, WT + R The microalgae treated with rapamycin after induction of predation are shown, WT + B represents microalgae treated with bafilomycin after induction of autophagy, and WT + W represents microalgae treated with watmanin after induction of autophagy.

Figure 1b shows rapamycin (R) or watts in wild type Chlamydomonas microalgae (WT) cultured in conditions that do not induce autophagy ((+) 48N) or conditions that induce autophagy ((-) 48N). It is a graph showing the result of comparing the content (top) of fat globules according to the treatment of manin (W) and the level (bottom) of the green fluorescence generated therefrom.

Figure 2 shows the results of comparing the production ability of fat globules when the wild type strain of Chlamydomonas microalgae and the mutant strains in which the expression of the VPS34 gene was suppressed were cultured in nitrogen supply medium (+ N) or nitrogen deficiency medium (-N). As a micrograph showing, WT indicates wild-type microalgae in which autophagy was induced, and T411 indicates mutant microalgae in which expression of the VPS34 gene was suppressed.

Figure 3a shows a wild-type microalgae (WT), transformed microalgae (Mock) or mutant microalgae (T411) introduced with an empty expression vector nitrogen supply medium ((+) N) or nitrogen deficiency medium ((-) N) After culturing under the same conditions at, the graph showing the results of counting the number of cells.

Figure 3b is a wild type microalgae (WT) or mutant microalgae (T411) incubated in the same conditions in nitrogen supply medium ((+) N) or nitrogen deficient medium ((-) N), and then compared the size of the cells A graph showing the results.

Figure 3c is a wild-type microalgae (WT) or mutant microalgae (T411) incubated in the same conditions in nitrogen supply medium ((+) N) or nitrogen deficiency medium ((-) N), then the aspect ratio of the cells (cell aspect It is a graph which shows the result of comparing ratio.

Figure 3d is a photograph showing the color of the culture after incubating the wild type microalgae (WT) or mutant microalgae (T411) under the same conditions in nitrogen supply medium ((+) N) or nitrogen deficiency medium ((-) N) to be.

Figure 3e is a wild type microalgae (WT) or mutant microalgae (T411) incubated in the same conditions in nitrogen supply medium ((+) N) or nitrogen deficient medium ((-) N), and then per unit dry weight of cells It is a graph which shows the result of comparing the ratio of triglyceride (TAG).

Figure 3f is a wild type microalgae (WT) or mutant microalgae (T411) incubated in the same conditions in nitrogen supply medium ((+) N) or nitrogen deficient medium ((-) N), and then the content of intracellular starch It is a graph showing the result of the comparison.

Figure 3g is a wild-type microalgae (WT) or mutant microalgae (T411) incubated in the same conditions in nitrogen supply medium ((+) N) or nitrogen deficiency medium ((-) N), the content of total protein in cells It is a graph showing the result of comparing.

Figure 3h shows a wild-type microalgae (WT), transformed microalgae (Mock) or mutant microalgae (T411) introduced with an empty expression vector nitrogen supply medium ((+) N) or nitrogen deficiency medium ((-) N) After culturing under the same conditions, the graph shows the result of comparing the activity of PI3P per unit weight of protein.

4A is an electron micrograph showing the results of analyzing the intracellular structure of the wild-type microalgae (WT) in nitrogen-deficient medium ((-) N) under the same conditions, and then performing TEM analysis, where A is self-extinguishing Represents an autophagosome analogous structure, L represents fat globules, S represents starch, and V represents vacuoles.

Figure 4b is a microscopic microalgae (T411) cultured in nitrogen deficient medium ((-) N) under the same conditions, and then carried out TEM analysis to analyze the intracellular structure is an electron micrograph showing.

Figure 5a is a diagram showing the results of analyzing the transcripts increased expression more than two times in mutant microalgae or wild-type microalgae cultured in nitrogen supply medium.

Figure 5b is a diagram showing the results of analyzing the transcripts increased expression more than two times in mutant microalgae or wild-type microalgae cultured in a nitrogen feed medium containing rapamycin.

Figure 5c is a diagram showing the results of analysis of transcripts with increased expression more than two times in mutant microalgae or wild-type microalgae cultured in nitrogen deficient medium.

Figure 5d is a diagram showing the results of analysis of transcripts with increased expression more than two-fold in wild-type microalgae cultured in nitrogen deficient medium, nitrogen feed medium and rapamycin-containing nitrogen feed medium.

Figure 5e is a diagram showing the results of analysis of transcripts with increased expression more than two-fold in mutant microalgae cultured in nitrogen deficient medium, nitrogen supply medium and rapamycin-containing nitrogen supply medium.

Figure 6a is a heat map analysis of the expression level of the genes involved in the autophagy regulation process according to the autophagy induction in the wild-type strain and VPS34 gene expression inhibitory strain.

Figure 6b is a heat map analysis of the expression level of the gene involved in the synthesis of triglycerides (TAG) according to autophagy induction in wild-type strains and VPS34 gene expression inhibitory strain.

The present inventors have been paying attention to the autophagy process while performing various studies to develop microalgae having a high lipid content for the production of biodiesel. Autophagy is the process of recycling part of intracellular organelles when cell survival is threatened under certain conditions, where cellular and intracellular organs degenerate into a double-membrane-bound structure called an autophagosome. The degenerated autodigestion vesicles are fused with lysosomes to form autolysosomes, and the degenerated material by the autolysosomes is hydrolyzed and reused. The autophagy process plays an important role in regulating cellular functions such as hunger survival, protection from infectious bacteria and regulation of neurodecay, which is an evolutionarily conserved process that appears in all eukaryotic cells from yeast to mammals. . Since the autolysosomes generated during the autophagy process contain lipids of various components, it was expected that the intracellular lipid content could be increased when the materials contained in the autolysosomes were not reused. To confirm this, the microalgae of the genus Chlamydomonas ( Chlamydomonas) reinfardtii CC124) was cultured in a nitrogen-deficient medium to induce autophagy, and treatment of autophagy inhibitors to the microalgae induced by autophagy promoted the formation of vacuoles, and the production of fat globules in the vacuoles was also remarkable. It was confirmed to increase. Since the autophagy inhibitors are known to exhibit their effects by inhibiting PI3K-related signaling in cells, a mutant microalgae in which the PI3K-related signaling is suppressed is produced, and the produced mutant microalgae are depleted in nitrogen supply medium or nitrogen deficient. As a result of culturing in the medium, it was confirmed that the production of lipid was suppressed when cultured in nitrogen supply medium, but the production of lipid was increased when cultured in nitrogen deficient medium. As a result of analyzing the characteristics of the mutated microalgae, cell proliferation is suppressed to produce a relatively small number of cells, but the size of the cells is increased, the shape of the cells is close to a circle, and photosynthesis is not performed smoothly. It was confirmed that the triglyceride (TAG) is produced at high levels, the intracellular starch content is increased, and the activity of PI3P is decreased. In addition, it was confirmed that autophagy-like structures did not exist structurally, and fat globules and starch exist in vacuoles.

As such, when the microalgae are cultivated under autophagy induction conditions, the content of lipids and starch in the cells is increased, and thus the microalgae cultivated by the above method may be used as biomass for the production of biodiesel. In addition, it can be used for economic production of biodiesel.

As one embodiment for achieving the above object, the present invention provides a method for producing microalgae with increased lipid and starch content, comprising the step of inhibiting autophagy while culturing the microalgae in autophagy induction conditions. .

The term "microalgae" of the present invention, also referred to as "phytoplankton", refers to a unicellular prokaryote that survives in an aquatic environment, reproduces with spores, and photosynthesizes with photosynthetic pigments.

In the present invention, the microalgae may be any kind of microalgae capable of accumulating lipids in cells, especially as long as the microalgae can accumulate lipids in cells when they are cultured under autophagy induction conditions. Examples of the microalgae include, but are not limited to, Chlorella sp. Microalgae, Scenedesmus sp. Microalgae, Parachlorella sp. Microalgae , which can accumulate lipids in cells. Microalgae of the genus Nanonochloropsis sp., Botryococcus sp. Microalgae, Ettlia sp. Microalgae, Chlamydomonas sp. As another example, the microalgae of Chlamydomonas can accumulate lipids in the cells when cultured under autophagy-inducing conditions and inhibits autophagy. As Chlamydomonas Lane Party (Chlamydomonas reinfardtii ) strain, and as another example, Chlamydomonas rain party CC124 ( Chlamydomonas reinfardtii CC124) strain.

The term "autophagy" of the present invention, also referred to as "autophagy", refers to a cellular function of disassembling and recycling the material or organelles that are not needed in the cell by enclosing the double membrane into the lysosome. The autophagy degenerates the cytoplasm and intracellular organs into a double-membrane-bound structure called an autophagosome, and the degenerated autodigestion fuses with a lysosome to form an autolysosome, The material degraded by the autolysosomes is hydrolyzed and reused.

The term "autophagy induction condition" of the present invention means a condition that induces the generation of autophagy processes from microalgae. Autophagy processes occur in conditions where cell survival is difficult, such as a lack of carbon sources, a lack of nitrogen sources, a lack of trace elements, a rapid change in external environmental pH, and a change in external environmental salt concentration.

In the present invention, the autophagy induction conditions may be interpreted as a culture condition that induces the autophagy from microalgae, but does not induce the death of the microalgae, specifically induce the autophagy process Among the possible conditions, it may be a deficiency condition of a nitrogen source that does not induce the death of the microalgae.

The term "autophagy suppression" of the present invention means inhibiting or delaying the progress of the autophagy process of the microalgae even under the conditions for inducing autophagy.

In the present invention, the method of inhibiting autophagy may be a method of inhibiting the intracellular signaling system involved in the microalgae autophagy process, for example, PI3K involved in the microalgae autophagy process. It may be a method of inhibiting a related signaling system, and as another example, the microalgae may be treated with an autophagy inhibitor that inhibits PI3K related signaling involved in the microalgae autophagy process, or It may be a method using a mutant microalgae in which the expression of the PI3K catalytic subunit type 3 protein involved in PI3K-related signaling involved is suppressed. Treat microalgae with or treat wortmannin, an autophagy inhibitor that inhibits PI3K-related signaling involved in the predation process Ryunae party may be a method using a mutant strain of microalgae The siRNA a process for the PI3K catalytic subunit claim VPS34 gene encoding a type III protein which is involved in the PI3K related signaling involved in the phagocytic process. For example, in the present invention, mutant microalgae treated with siRNA for the VPS34 gene having the following nucleotide sequence was used.

amiFor: 5'-ctagtCTGCGACATAAACTTGTATGAtctcgctgatcggcaccatgggggtggtggtg atcagcgctaTCATTCAAGTTTATGTCGCAGg-3 '(SEQ ID NO: 1)

amiRev: 5'-ctagcCTGCGACATAAACTTGAATGAtagcgctgatcaccaccacccccatggtgccg atcagcgagaTCATACAAGTTTATGTCGCAGa-3 '(SEQ ID NO: 2)

As used herein, the term "culture" refers to a series of actions for growing microorganisms under appropriately artificially controlled environmental conditions.

In the present invention, the culturing may be interpreted to mean a method of culturing microalgae, the culturing method may be performed using a method well known in the art. Specifically, the culturing may be performed continuously in a batch process or in a fed batch or repeated fed batch process.

In order to culture the microalgae, it is necessary to meet the survival requirements of specific strains in an appropriate manner while controlling the temperature, pH, etc. under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like. Carbon sources that can be used are mainly CO ₂ and carbonate, and mixed sugars of glucose and xylose may be used as the carbon source, and sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, cellulose, and soybean oil Oils such as sunflower oil, castor oil, coconut oil and the like, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid. These materials can be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation product, skim soy cake or its degradation product Can be. These nitrogen sources may be used alone or in combination. The medium may include, as personnel, monopotassium phosphate, dipotassium phosphate and corresponding sodium-containing salts. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used. For example, microalgae as a culture medium for the _{NaNO 3, K 2 HPO 4,} MgSO 4 .7H 2 O, CaCl 2 .2H 2 O, citric acid, Na ₂ EDTA, ammonium ferric citrate, green (Ammonium ferric citrate green) , Na ₂ CO _3, and trace metals solution _{(H 3 BO 3, MnCl 2} .4H 2 O, ZnSO 4 .7H 2 O, Na 2 MoO 4 .2H 2 O, CuSO 4 .5H 2 O, Co (NO 3) BG11 medium containing ₂ .6H ₂ O) can be used.

In addition, antifoaming agents such as fatty acid polyglycol esters can be used to inhibit bubble generation. Gas (eg, air) can be injected to maintain aerobic conditions. The temperature of the culture can usually be maintained at 20 ° C to 35 ° C, preferably 25 ° C to 30 ° C.

In addition, the nature of the microalgae to perform photosynthesis is preferable to provide light during the cultivation, the amount of light provided and the time provided can be appropriately adjusted by those skilled in the art as needed, luminous conditions of about 50 to 100μmole / ㎡ / s Can provide light.

According to one embodiment of the present invention, Chlamydomonas reinfardtii CC124 was cultured in a nitrogen deficient medium to induce autophagy, while treating watmanin (wortammanin) known as an autophagy inhibitor, Production of mutant microalgae with increased production and production of fat globules (FIG. 1), and inhibition of PI3K-related signaling, an intracellular signaling system affected by the autophagy inhibitors, and nitrogen production of the mutant microalgae As a result of culturing in a feed medium or nitrogen deficient medium, the production of lipid was suppressed when cultured in a nitrogen supply medium, but the production of lipid was increased when cultured in nitrogen deficient medium (Fig. 2), the strain strain fine Characterization of algae has shown that not only produces high levels of triglycerides (TAG) (FIG. 3E), but also high starch content. With the increased (Fig. 3f) it was confirmed.

As another embodiment for achieving the above object, the present invention is prepared using the above method, to provide a microalgae with increased lipid and starch content in the cells compared to the strains that do not inhibit autophagy.

When the microalgae prepared by the above method are cultured under the same autophagy induction conditions, the lipid and starch content in the cells is increased as compared to the wild-type strain which is not inhibited by autophagy. At this time, the lipid content is increased in the cell is not particularly limited, but may be a phospholipid as an example, in another example, phosphatidic acid (PA), phosphatidyl-ethanolamine (PE), PS (phosphatidylserine) belonging to phospholipids And, as another example, it may be MG (monoacylglycerol), a decomposition product of triglyceride (TAG), EA (eicosadienoic acid), which is a stress-induced unsaturated fatty acid. In particular, since phospholipids such as PA, PE, and PS are contained in mutant microalgae at a significantly higher level than wild-type microalgae without autophagy (FIG. 6), the phospholipids are microalgae prepared by the method of the present invention. Can be used as the main marker to distinguish it from microalgae prepared by conventional methods.

As another embodiment for achieving the above object, the present invention provides a biomass comprising microalgae with increased lipid and starch content in cells and a method for producing biodiesel using the biomass and the method Provides biodiesel.

As used herein, the term "biomass" refers to a variety of algae and plant resources produced by photosynthesis, such as trees, grasses, branches of crops, leaves, roots, fruits and the like.

In the present invention, the biomass may be interpreted as microalgae, cultures containing the microalgae, culture fractions, and the like, which are cultured in a medium under stress conditions and have increased lipid and starch contents in cells. It can be used as a raw material of biodiesel.

On the other hand, the method for producing a biodiesel of the present invention (a) obtaining a lipid component from the biomass; And (b) adding methanol to the obtained lipids and reacting under an alkali catalyst to obtain FAME (fatty acid methyl), which is biodiesel. At this time, in order to increase the yield of FAME may further comprise the step of removing the reaction by-product glycerol.

In the production method of the biodiesel of the present invention, in order to obtain a lipid component from the biomass, various known methods can be used. For example, the microalgae contained in the biomass may be dried and obtained by physically crushing the microalgae, or lipid components may be extracted from the microalgae by adding an organic solvent to the biomass. In this case, a non-polar solvent may be used as the solvent, and preferably hexane, dimethyl sulfoxide (DMSO), dimethyl carnonate (DMC), or the like may be used.

In addition, since the lipid component obtained from the biomass is derived from the microalgae with increased lipid content in the cells provided by the present invention, PA (phosphatidic acid), PE (phosphatidyl-ethanolamine), PS (phosphatidylserine), MG ( monoacylglycerol), EA (eicosadienoic acid) and the like may be included alone or in combination.

The term "bio-diesel" of the present invention broadly refers to the whole of pollution-free fuel made from vegetable oils, and narrowly to fatty acid methyl esters prepared using vegetable oils such as soybean oil as raw materials. ). The biodiesel may be prepared by various methods, and may be generally prepared by treating methanol with an alkaline catalyst in triglycerides.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example  1: microalgae District ball  Affecting generation Self-feeding  Effect of Inhibitors

Chlamydomonas of the genus Chlamydomonas reinfardtii CC124) was inoculated in TAP medium and cultured, and the medium was replaced with nitrogen-deficient medium to induce autophagy. (lipid droplet, LD) was stained with green fluorescence, treated with chlorophyll α, and the vacuole was stained with red fluorescence, and then observed by confocal microscopy (FIG. 1A). At this time, the microalgae cultured without the autophagy inhibitor was used as a control.

Figure 1a Chlamydomonas genus microalgae reinfardtii CC124) induces autophagy and treated with autophagy inhibitors, a micrograph showing the results of comparing the production of fat globules, WT represents the microalgae of the control group induced autophagy, WT + W The microalgae treated with watmanine after induction of predation is shown, and the DIC shows a result obtained by the differential interference contrast (DIC) method. As shown in Figure 1a, compared to the microalgae of the control group induced autophagy, when treated with autophagy inhibitors, it was confirmed that not only the formation of vacuoles but also the production of fat cells in the vacuoles was significantly increased.

In addition, rapamycin, an inhibitor of autophagosome production, is induced in the microalgae of Chlamydomonas under conditions that do not induce autophagy by culturing in normal medium or inducing autophagy by culturing in a nitrogen-deficient medium. Or after treatment with wartmannin (wortmannin) known as autophagy inhibitors, the same method was carried out to compare the content of fat globules and the level of green fluorescence resulting therefrom (Fig. 1b).

Figure 1b shows rapamycin (R) or watts in wild type Chlamydomonas microalgae (WT) cultured in conditions that do not induce autophagy ((+) 48N) or conditions that induce autophagy ((-) 48N). It is a graph showing the result of comparing the content (top) of fat globules according to the treatment of manin (W) and the level (bottom) of the green fluorescence generated therefrom. As shown in FIG. 1B, watmanin induced intracellular fat accumulation even when autophagy was not induced, but rapamycin did not induce intracellular fat accumulation.

Example  2: VPS34  Characterization of Mutant Microalgae with Inhibited Gene Expression

Since it is known that PI3K-related signaling is involved in autophagy, mutant microalgae in which expression of the PI3K catalytic subunit type 3 protein involved in PI3K-related signaling was suppressed were prepared and analyzed.

Example  2-1: VPS34  Production of Mutant Microalgae with Inhibited Gene Expression

Chlamydomonas of the genus Chlamydomonas mutant microalgae incorporating an expression vector comprising a polynucleotide encoding siRNA for the VPS34 gene in order to inhibit expression of the VPS34 gene encoding PI3K catalytic subunit type 3 protein involved in PI3K related signaling in reinfardtii CC124) T411 strain was prepared. The wild-type microalgae (W) and the mutant microalgae (T411) were incubated in nitrogen supply medium (+ N) or nitrogen deficiency medium (-N), respectively, and then fluorescently stained by the method of Example 1, and the results were obtained. Confirmed by confocal microscopy (FIG. 2).

Figure 2 shows the results of comparing the production ability of fat globules when the wild type strain of Chlamydomonas microalgae and the mutant strains in which the expression of the VPS34 gene was suppressed were cultured in nitrogen supply medium (+ N) or nitrogen deficiency medium (-N). As a micrograph showing, WT indicates wild-type microalgae in which autophagy was induced, and T411 indicates mutant microalgae in which expression of the VPS34 gene was suppressed. As shown in Figure 2, when cultured in a nitrogen supply medium, the production of lipids in both wild-type microalgae and mutant microalgae was inhibited, but when cultured in nitrogen-deficient medium, the production of lipids is increased in both wild-type microalgae and mutant microalgae It was confirmed that the amount of lipid production was significantly increased in mutant microalgae compared to wild-type microalgae.

Example  2-2: VPS34  Cellular Characterization of Mutant Microalgae with Inhibited Gene Expression

In order to analyze the characteristics of the mutant microalgae prepared in Example 2-1, a transformed microalgae (Mock) into which an empty expression vector containing no polynucleotide encoding siRNA for the wild type microalgae or VPS34 gene was introduced. And various cell characteristics (cell number, cell size, cell aspect ratio, color of culture, ratio of TAG per unit dry weight of cell, starch content, total protein content and activity of PI3P per unit protein) were compared (Fig. 3a to Fig. 3). 3h).

Figure 3a shows a wild-type microalgae (WT), transformed microalgae (Mock) or mutant microalgae (T411) introduced with an empty expression vector nitrogen supply medium ((+) N) or nitrogen deficiency medium ((-) N) After culturing under the same conditions at, the graph showing the results of counting the number of cells. As shown in Figure 3a, the mutant strain microalgae was inhibited cell proliferation, it was confirmed to produce a relatively small number of cells.

Figure 3b is a wild type microalgae (WT) or mutant microalgae (T411) incubated in the same conditions in nitrogen supply medium ((+) N) or nitrogen deficient medium ((-) N), and then compared the size of the cells A graph showing the results. As shown in Figure 3b, the mutant microalgae was confirmed to increase the size of the cell compared to wild-type microalgae.

Figure 3c is a wild-type microalgae (WT) or mutant microalgae (T411) incubated in the same conditions in nitrogen supply medium ((+) N) or nitrogen deficiency medium ((-) N), then the aspect ratio of the cells (cell aspect It is a graph which shows the result of comparing ratio. As shown in Figure 3c, the mutant strain microalgae showed a lower aspect ratio of the cell than the wild-type microalgae, it was confirmed that the morphology is closer to the round shape.

Figure 3d is a photograph showing the color of the culture after incubating the wild type microalgae (WT) or mutant microalgae (T411) under the same conditions in nitrogen supply medium ((+) N) or nitrogen deficiency medium ((-) N) to be. As shown in Figure 3d, the wild type microalgae and mutant microalgae culture when the culture in the nitrogen supply medium showed the same level of green, indicating that photosynthesis is performed smoothly, but when cultured in nitrogen-deficient medium, The green color of wild-type microalgae and mutant microalgae was reduced, and the green micro-algae of mutant microalgae showed more yellow color than wild-type microalgae. It was not known.

Figure 3e is a wild type microalgae (WT) or mutant microalgae (T411) incubated in the same conditions in nitrogen supply medium ((+) N) or nitrogen deficient medium ((-) N), and then per unit dry weight of cells It is a graph which shows the result of comparing the ratio of triglyceride (TAG). As shown in Figure 3e, mutant microalgae produced higher levels of TAG than wild-type microalgae, it was confirmed that this feature is more marked when cultured in nitrogen deficient medium.

Figure 3f is a wild type microalgae (WT) or mutant microalgae (T411) incubated in the same conditions in nitrogen supply medium ((+) N) or nitrogen deficient medium ((-) N), and then the content of intracellular starch It is a graph showing the result of the comparison. As shown in Figure 3f, mutant microalgae showed higher levels of intracellular starch compared to wild-type microalgae, it was confirmed that this feature is more marked when cultured in nitrogen deficient medium.

Figure 3g is a wild-type microalgae (WT) or mutant microalgae (T411) incubated in the same conditions in nitrogen supply medium ((+) N) or nitrogen deficiency medium ((-) N), the content of total protein in cells It is a graph showing the result of comparing. As shown in Figure 3g, the mutant microalgae showed a higher total protein content than the wild-type microalgae when cultured in a nitrogen supply medium, but the mutant microalgae and wild-type microalgae when cultured in a nitrogen-deficient medium. It was confirmed that the total protein content in the algae showed an equivalent level.

Figure 3h shows a wild-type microalgae (WT), transformed microalgae (Mock) or mutant microalgae (T411) introduced with an empty expression vector nitrogen supply medium ((+) N) or nitrogen deficiency medium ((-) N) After culturing under the same conditions, the graph shows the result of comparing the activity of PI3P per unit weight of protein. As shown in FIG. 3h, the PI3P activity was higher when cultured in nitrogen deficient medium than when cultured in nitrogen supply medium, and the activity of PI3P was higher in wild type microalgae than in mutant microalgae. It was confirmed.

Example  2-3: VPS34  Structural Characterization of Mutant Microalgae with Inhibited Gene Expression

To analyze the structural characteristics of the mutant microalgae prepared in Example 2-1, wild-type microalgae (WT) or mutant microalgae (T411) were cultured under the same conditions in nitrogen-deficient medium ((-) N). Intracellular structure was analyzed by transmission electron microscopy (TEM), and then compared (FIGS. 4A and 4B).

4A is an electron micrograph showing the results of analyzing the intracellular structure of the wild-type microalgae (WT) in nitrogen-deficient medium ((-) N) under the same conditions, and then performing TEM analysis, where A is self-extinguishing Represents an autophagosome analogous structure, L represents fat globules, S represents starch, and V represents vacuoles. In addition, Figure 4b is a microscopic microalgae (T411) cultured in nitrogen deficient medium ((-) N) under the same conditions, and then carried out TEM analysis to analyze the intracellular structure is an electron micrograph showing the results. As shown in Figures 4a and 4b, autodigestion-like structures were observed only in wild-type microalgae, not in mutant microalgae, and it was confirmed that fat globules and starch exist in vacuoles.

Example  2-4: Transcript  analysis

Mutant microalgae or wild-type microalgae prepared in Example 2-1; Culture in nitrogen feed or nitrogen deficient medium; And a combination of three conditions of treatment of rapamycin, an autophagosome production inhibitor, with each other to obtain respective cultures. Transcripts with increased expression levels were analyzed (FIGS. 5A-5E).

Figure 5a is a diagram showing the results of analyzing the transcripts increased expression more than two times in mutant microalgae or wild-type microalgae cultured in nitrogen supply medium. As shown in Figure 5a, 2,136 transcripts were increased more than 2 times in mutant microalgae cultured in nitrogen supply medium, 2,544 transcripts increased more than 2 times in wild-type microalgae cultured in nitrogen supply medium, It was confirmed that 13,061 transcripts did not increase expression more than two-fold.

Figure 5b is a diagram showing the results of analyzing the transcripts increased expression more than two times in mutant microalgae or wild-type microalgae cultured in a nitrogen feed medium containing rapamycin. As shown in Figure 5b, the mutant microalgae cultured in a nitrogen feed medium containing rapamycin increased expression more than 1,331 transcripts, 1,171 in wild-type microalgae cultured in a nitrogen feed medium containing rapamycin It was confirmed that the expression of transcripts increased more than two times, and that of 15,239 transcripts did not increase expression more than two times.

Figure 5c is a diagram showing the results of analysis of transcripts with increased expression more than two times in mutant microalgae or wild-type microalgae cultured in nitrogen deficient medium. As shown in Figure 5c, 3,274 transcripts were more than two-fold increased in mutant microalgae cultured in nitrogen-deficient medium, 3,104 transcripts were increased more than two-fold in wild-type microalgae cultured in nitrogen-deficient medium, 11,363 transcripts confirmed no increase in expression more than two-fold.

Figure 5d is a diagram showing the results of analysis of transcripts with increased expression more than two-fold in wild-type microalgae cultured in nitrogen-deficient medium, nitrogen supply medium and rapamycin-containing nitrogen medium, Figure 5e is a nitrogen deficient medium This diagram shows the results of analysis of transcripts with increased expression more than two-fold in mutant microalgae cultured in nitrogen feed medium containing nitrogen, nitrogen feed medium and rapamycin. As shown in Figure 5d and 5e, when cultured under the same conditions, compared to wild-type microalgae, mutant microalgae slightly increased the number of transcripts increased more than two times by the supply of nitrogen, when nitrogen is not supplied Although the number of transcripts with increased expression more than two times was significantly reduced, it was confirmed that even when nitrogen was supplied, the number of transcripts with increased expression more than two times was significantly reduced when rapamycin was treated.

Example  3: Heatmap (Heat map) analysis

Wild-type microalgae (WT) or mutant microalgae (T411) were cultured under the same conditions in nitrogen supply medium (+ N) or nitrogen deficiency medium (-N), and then the genes involved in autophagy regulation (AMPK, ATG1). , ATG12, ATG3, ATG4, ATG5, ATG7, ATG8, Cre02.g076900.v5.5, Cre10.g443550.v5.5 and Cre16.g669800.v5.5), PI3K subunits (VPS15, VPS30 and VPS34) and neutral Changes in expression levels of genes involved in fat (TAG) synthesis (DGAT1, DGK, DGTT1, DGTT3, DGTT4 and DGTT5) were confirmed by heat map analysis (FIGS. 6A and 6B).

Figure 6a is a heat map analysis of the expression level of the genes involved in the autophagy regulation process according to the autophagy induction in the wild-type strain and VPS34 gene expression inhibitory strain. As shown in Figure 6a, when the gene involved in the autophagy regulation process was cultured in nitrogen deficient medium (-N), it was confirmed that the expression level of the mutant microalgae (T411) as a whole rather than the wild-type microalgae (WT). In particular, it was confirmed that the expression levels of ATG1 and ATG3 genes acting at the beginning of autophagy regulation were reduced to very low levels. In addition, the expression levels of the PI3K subunits (VPS15, VPS30 and VPS34) also showed low levels.

From the above results, it was analyzed that when the mutant microalgae (T411) were cultured in a nitrogen deficient medium (-N), autophagy and autophagosome formation involved therein were inhibited as compared to wild-type strains.

Figure 6b is a heat map analysis of the expression level of the gene involved in the synthesis of triglycerides (TAG) according to autophagy induction in wild-type strains and VPS34 gene expression inhibitory strain. As shown in Figure 6b, the gene involved in the synthesis of triglycerides (TAG) is confirmed that when the strain is cultured in nitrogen deficient medium (-N), the expression level of mutant microalgae (T411) as a whole rather than wild-type microalgae (WT) is increased as a whole It was.

Taken together, the strains that inhibit the expression of PI3K, when autophagy is induced, increase the synthesis amount of triglycerides and inhibit the formation of autodigestive vesicles involved in the degradation of triglycerides. By suppressing, it was found that the lipid content was increased compared to the wild type strain.

Claims (12)

1. A method for producing microalgae with increased lipid and starch content, comprising the step of inhibiting autophagy while culturing the microalgae under autophagy induction conditions.
2. The method of claim 1,

   The microalgae is a microalgae of the genus Chlorella, microalgae of the genus Styreclonium or microalgae of the genus Chlamydomonas.
3. The method of claim 1,

   Wherein said autophagy induction condition is a lack of a nitrogen source.
4. The method of claim 1,

   Inhibition of autophagy is carried out by treating a autophagy inhibitor or by using a mutant microalgae in which PI3K related signaling is involved in the microalgae autophagy process.
5. The method of claim 4, wherein

   Wherein said autophagy inhibitor is wormmanin.
6. The method of claim 4, wherein

   The mutant microalgae is a microalgae in which the expression of the PI3K catalytic subunit type 3 protein involved in PI3K related signaling is suppressed.
7. The method of claim 6,

   Expression inhibition of the PI3K catalytic subunit type 3 (PI3K catalytic subunit type 3) protein is a method that is carried out by treating the microalgae siRNA for the VPS34 gene.
8. The method of claim 7, wherein

   The siRNA is composed of the nucleotide sequence of SEQ ID NO: 1 and 2.
9. A microalgae prepared by the method of any one of claims 1 to 8, wherein the microalgae have an increased content of lipids and starch in the cells compared to wild-type microalgae in which autophagy is not inhibited.
10. A biomass comprising microalgae with increased lipid and starch content of the cells of claim 9.
11. A method for preparing biodiesel using the biomass of claim 10.
12. Biodiesel prepared by the method of claim 11.

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