KR101152761B1 - New beta-carotene producing strain and method for producing beta-carotene using the strain - Google Patents

Abstract

The present invention relates to a new variant strain of Dunaliella salina and to a method for producing beta-carotene using the same. Using the novel strain according to the present invention, it is possible to accumulate a large amount of beta-carotene by low energy consumption, so that beta-carotene can be efficiently produced at an industrial level.

Dunaliella, Beta-Carotene

Description

New beta-carotene producing strain and method for producing beta-carotene using the same {NEW BETA-CAROTENE PRODUCING STRAIN AND METHOD FOR PRODUCING BETA-CAROTENE USING THE STRAIN}

The present invention relates to a novel beta-carotene producing strain and a method for producing beta-carotene using the same. More specifically, the present invention relates to a novel variant strain of a novel beta-carotene producing strain, ie Dunaliella salina , which has high carotene production efficiency, and furthermore, to an efficient method for producing beta-carotene using the strain. It is about.

Dunaliella is a eukaryotes belonging to photosynthetic green algae, and has a high salt adaptation range capable of surviving salt concentrations ranging from about 1/10 of seawater to 5M close to saturated solution. Indicates. Two analytic classification of the position of Elah (Dunaliella) chloro pie and the other (Chlorophyta) Contact chloro Pai Kane (Chlrophyceae) Volvo Carl Les belonging to the river (Volvocales) two analytic Ella three (Dunaliellaceae) of the neck, cell volume from 50 to 100 It is a single-cell green algae up to. Their cells are surrounded by elastic thin mucosa and do not have the same rigid polysaccharide cell walls as normal plants (Ben-Amotz et al., Trends in Biotechnology , 8, 121-126, 1990).

Usually Dunaliella The microalgae of the genus are found in places with low nitrogen content, strong sunlight and high salt concentrations, and they are found to have beta-carotene in the cells from yellowish green to orange when exposed to such strong environmental stress conditions. do. These beta-carotenes have been reported to accumulate with oily granulocytes in the thylakoid interstitial spaces in the chloroplasts (Ben-Amotz et al., J. Phycol. 18: 529-537). It is supposed to protect.

Carotenoids are orange-based pigments (yellow to bright red) that are mainly present in the peels of green and yellow vegetables such as carrots, old pumpkins, green peppers, sesame leaves, lettuce, leeks, and spinach. Among them, beta-carotene is a precursor of almost all carotenoids, which are converted into vitamin A, an essential nutrient in the body. Vitamin A is a fat-soluble vitamin that, unlike water-soluble vitamins, is stored for a period of time in tissues such as the liver. Vitamin A itself may be toxic in large amounts, but beta-carotene is known to be a source of vitamin A, which is not toxic even when consumed in large amounts because it is converted into the body only as needed. Beta-carotene is not only widely used as a food additive due to its antioxidant effect, high nutritional value and unique color to prevent oxidation damage in animal tissues, but also as an anticancer agent and antioxidant for skin aging. . However, the amount of beta-carotene present in green and yellow vegetables and fruits is not so high that its production does not meet demand.

Despite the usefulness of beta-carotene, natural beta-carotene is not easy to mass production. A typical production method of beta-carotene is the direct extraction of beta-carotene from green yellow vegetables. However, this method is affected by seasonal and climatic fluctuations, takes a long time, and has a low yield.

Chemical synthesis of beta-carotene has also been attempted to achieve more stable production and higher yields, but the safety of chemical synthesis products has not yet been recognized, making them unsuitable for food use.

Due to insufficient and unstable production and problems related to the safety of human use, much research has been conducted on the use of Dunaliella , a green alga that synthesizes carotenoids (Packo et al., 2008). Dunaliella is used for beta-carotene production in some industrial applications, but due to the nature of the beta-carotene synthesis of these algae, satisfactory yields have not been achieved. have.

The present inventors have developed a novel mutant strain to develop a method for producing natural beta-carotene more effective than the production method using a conventionally used strain.

Accordingly, the present invention is to develop a novel mutant strain having excellent beta-carotene synthesis ability, to provide an effective beta-carotene manufacturing method using the same.

Thus, in one aspect, the present invention provides novel Dunaliella salina mutants.

The present inventors treated EMS, a mutagenic substance, in Dunaliella salina CCAP19 / 18, a strain registered in Culture Collection of Algae and Protozoa (CCAP), which has a relatively high beta-carotene content. Mutant strains with significantly higher beta-carotene content than the original strain CCAP19 / 18 were prepared and selected. Analysis of morphological and physiological characteristics such as growth and beta-carotene synthesis characteristics of the prepared and selected strains to confirm that it is a novel beta-carotene synthesis strain superior to the previously reported strains and Dunaliella salina ( Dunaliella salina ) It was named LS202 strain. The strain was deposited on December 15, 2008 by the KCTC in the Korea Research Institute of Bioscience and Biotechnology, 52 Eun-dong, Yuseong-gu, Daejeon, Korea, and was given a deposit number of KCTC 11442BP (hereinafter referred to as' Dunaellia Salina ( Dunaliella salina ) referred to as KCTC 11442BP '.

Regarding the morphological features of Dunaliella salina KCTC 11442BP according to the present invention, micrographs of the original species Dunaliella salina CCAP19 / 18 and the mutant strain according to the present invention are shown in FIG. Figure 4 shows the profile pigment profile of each of the Dunaliella strain, Dunaliella Bardawil ( D.bardawil ), Dunaliella Salina ( D.salina ) CCAP19 / 18, two under the same light intensity The three strains of D. salina LS202 were grown in different shapes, and the original species, D. salina CCAP19 / 18, was thin and mutant strains and dunaliella. Bardabil ( D. bardawil ) has a relatively round shape, especially the mutant strain was found to be dark orange with the accumulation of beta-carotene. Two flagellas were faintly observed on the thin side of the cell, but no flagella was observed in Dunalella bardawil (see FIG. 4b). All three strains show a similar growth pattern under the same culture conditions (medium containing 1.5 M NaCl, luminous intensity of 100 μM photons / m²s) during the logarithmic growth period from 2 days of inoculation to about 1 week, and then enter the stable phase. (See FIG. 3), the mutant strain was confirmed that the cell itself is large in size, instead of a small increase in the number of cells.

On the other hand, in relation to the molecular characteristics of Dunaliella salina KCTC 11442BP according to the present invention, the electrophoresis results of PCR products amplified using ITS primers in Figure 7, respectively, Dunaliella salina ( D.salina ) ITS 1 and 2 of CCAP19 / 18 and ITS 1 and 2 of variant strain LS202. In addition, as can be seen in the schematic diagram of FIG. 8, the mutant strain was the closest to the original species CCAP19 / 18, and it was confirmed that the strain was derived from CCAP19 / 18 rather than mistaken contamination of other strains.

In another aspect the invention provides Dunaliella salina KCTC 11442BP strain, characterized by producing beta-carotene.

Dunaliella salina KCTC 11442BP of the present invention has the advantage that the biomass increase is superior to the original strain as well as Dunaliella Bardabil (see Table 2), the brightness mainly used in conventional beta-carotene biosynthesis The beta-carotene accumulates abundantly at luminosity far below the level (see FIGS. 1 and 4).

In another embodiment the invention provides beta- in an amount of at least 2 mg, preferably at least 4 mg , more preferably at least 8 mg per 7 mg of chlorophyll a within 7 days, even under low luminosity in the range from 50 to 200 μmol photons / m 2 s. Dunaliella salina KCTC 11442BP strain is characterized by accumulating carotene.

In contrast, the original strain derived from the novel mutant strain according to the present invention hardly detects beta-carotene accumulation even after culturing for several days at a light intensity of about 400 μmol photons / m 2. Such characteristics of the mutant strain of the present invention are energy efficient compared to the conventional method of irradiating strong light for beta-carotene accumulation when Dunaliella is used for biosynthesis, thereby reducing production costs. In addition, even using the same amount of medium, it is possible to produce a large amount of biomass and high concentration of beta-carotene, thereby improving the yield.

Therefore, in another aspect the present invention provides a method for producing beta-carotene comprising culturing a novel Dunaliella salina KCTC 11442BP strain according to the present invention.

By using the strain of the present invention having the characteristics as described above, since beta-carotene is efficiently biosynthesized at low energy, that is, low brightness, beta-carotene can be economically produced.

Cultivation of the strain of the present invention for beta-carotene production can be done in artificial medium or natural seawater. Artificial medium for the growth and beta-carotene production of Dunaliella salina strain of the present invention is 0.1 to 5.0 M, preferably 0.5 to 4.5 M, Mg ²⁺ 0.05 to 500 mM, preferably 0.1 to 10 NaCl mM, 0.1 to 10 mM of K ^+, preferably from 0.1 to 20 mM, preferably of 0.3 mM to 5 mM, Ca ²⁺ is a buffer solution of pH 7.0 to 9.0 containing from 0.2 to 5 mM. The artificial medium also contains a source of iron such as FeCl ₃ from 0.5 to 50 mM, preferably from 4 to 10 mM, SO ₄ ^2- ions from 0.1 to 10 mM, preferably from 0.3 to 7 mM, NO ₃ ⁻ ions as a nitrogen source. It is further added 1 to 20 mM, preferably from 3 to 7 mM, or NH ₄ ⁺ ions in an amount of 0.5 to 2.5 mM, preferably 1 to 2 mM, the phosphate 0.1 to 50 mM, preferably 0.2 to 10 mM can do. As the carbon source, 25 mM NaHCO ₃ may be added or CO ₂ , CaCO _3, or the like may be supplied. In addition, trace elements such as H ₃ BO ₃ , MnCl ₂ , ZnCl ₂ , CuCl ₂ , Na ₂ MoO ₄ , NaVO ₃ , and CoCl ₂ may be added to promote growth of the strain. Specific examples of media compositions are listed in Table 1 in the Examples. For cultivation of the strain of the present invention and beta-carotene production can be used as a medium to concentrate the seawater by partially evaporating or to add the components to the same concentration range as the artificial medium in the seawater.

By inoculating the strain in the medium, the culture may be shaken at 20 to 35 ° C., preferably at 22 to 30 ° C. at a rate of about 150 rpm for 4 to 8 days, and during the incubation period, 50 to 200 μmol photon / m 2 s preferably It is preferable to adjust the pH to 7.0 to 9.0 by irradiating with light of about 100 μmol photon / m ₂ and adding CO ₂ , hydrochloric acid and nitric acid to the culture.

In order to maximize the production of beta-carotene, maximizing the growth of the strain at the beginning of cultivation and then promoting the production of beta-carotene by changing the composition and culture conditions of the medium, such as salt concentration, nitrogen source or roughness, in the late culture. Can be. Such concentration adjustment can be carried out by methods well known to those skilled in the art.

In the case of Dunalella strains, after incubating at low luminosity, such as 50 to 200 μmol photon / m 2s, artificially increasing the light intensity to 400 to 2000 μmol photon / m 2s to induce beta-carotene synthesis. The method is known, and cultured by changing the illumination as described above, it is possible to produce beta-carotene more than 10 times compared to the case of continuing to culture at low light.

On the other hand, the new strain of the present invention is capable of accumulating a large amount of beta-carotene even under significantly lower illuminance than Dunaliella strains. Thus, there is an advantage that a corresponding effect can be achieved without such additional energy input. However, in order to maximize the production of beta-carotene can be supplied with light beyond the optimal illumination for strain growth in culture. Dunaliella contains natural beta-carotene, and under the cell wall, a large amount of beta-carotene is synthesized to protect chlorophyll a, a chlorophyll important for photosynthesis when the culture environment deteriorates, such as strong light irradiation. It is known to form a beta-carotene membrane.

After the end of the culture, the microalgae may recover the cells through spontaneous precipitation of the culture or centrifugation. The intracellular beta-carotene was extracted by mixing 90% acetone in the recovered cells and centrifuged according to the method of the present invention to detect the beta-carotene at 445 nm using HPLC for supernatant. The content can be measured.

Alternatively, the cells may be treated with DMSO and beads, followed by extraction with methanol to obtain an extract. The extract may be saponinized, and beta-carotene is extracted with a heptane solvent to measure absorbance at 436 nm. In this case, the amount of beta-carotene can be calculated as follows.

Beta-carotene (%) = Abs ₄₃₆ / {196 x (mass (g) x dry mass (g))} x 25 ml x 1.25 x 100 x 0.84

Since the mutant strain of the present invention has high photosynthetic efficiency, the biomass increase is superior to other strains, and the mutant strain of the present invention exhibits a high biomass increase using light energy efficiently even at low luminous intensity. It is highly applicable in the industrial field. Recently, the use of microalgae for CO ₂ reduction has been actively studied due to the environmental problem caused by the increase of greenhouse gas (CO ₂ ) (Konstantine and Colman, Brian, 30L 745-752, 2007). In this system, the mutant strain can efficiently increase the biomass under a very low light intensity using CO ₂ as a carbon source, and the biomass thus produced can be used as a biofuel.

In particular, it has been found that beta-carotene binds to intracellular lipid granules (Lers et al., 1990). This means that the mutant, which accumulates high concentrations of beta-carotene, also accumulates excess lipid granules, which could be used as a source of biodiesel. Research into the use of plant-derived lipids as a source of biodiesel has also been widely conducted worldwide. The mutant strains could be the target of this new technology.

Using the novel strain according to the present invention, it is possible to accumulate a large amount of beta-carotene by low energy consumption, so that beta-carotene can be efficiently produced at an industrial level.

Specific embodiments of the present invention will be described through the following examples.

The terminology used herein is not to be construed as being limited to a general and dictionary meaning, and the concept of a specific term may be defined within an appropriate range in order to explain the present invention in the best manner. Various equivalent or modified embodiments that are within the scope of the invention disclosed herein are also apparent to those skilled in the art in view of the present specification or the prior art in the art. The embodiments described below are only described for illustrative purposes, and the scope of the present invention is limited only by the claims.

<Examples>

Example 1 Mutation Induction and Culture

Dunaliella salina CCAP 19/18 strain (hereinafter also referred to as `` progeny strain '') was distributed from Culture Collection of Algae and Protozoa (Liquid), and the liquid medium of the composition shown in Table 1 below Incubated in 'liquid medium'. Culture conditions were about 100μmol photons / ㎡s of light intensity, the temperature was 25-26 ℃, and shaken twice a day while stationary culture.

Incubate Dunaliella salina CCAP 19/18 until log phase, harvest the cells by centrifugation, resuspend them to a cell density of 2 × 10 ⁸ cells / mL, and use EMS as a mutant inducer. (Ethylmethane sulphonate) was treated to a final concentration of 0.2 μM. After incubation for 2 hours, the EMS used for the treatment was washed with liquid medium, centrifuged to harvest only cells, resuspended in liquid medium, cultured in dark for one day, and then the same composition as liquid medium. The plate was transferred to a solid medium to which only agar was added. After one week, colonies formed on a solid medium were separated, transferred to a liquid medium, and cultured in a liquid.

Example 2: Comparison of Cell Density and Growth of Isolated Mutant strains

In order to compare the cell growth rate and the final growth of the wild and mutant strains inoculated at a cell density of 5 × 10 ⁵ cells / mL in the medium of the components shown in Table 1, and then 20, 90, 200μmol photons / ㎡s respectively Incubated at light intensity. After the start of the culture, the culture solution was taken on each of the first, second, fourth, sixth, eighth, nineth, tenth, and fourteenth days, and the number of cells per milliliter was counted under a microscope using a hemocytometer (FIG. 3).

In addition, to compare their respective final biomass (biomass), 400 mL of the incubating suspension was centrifuged to harvest only the cells, followed by drying in a dry oven at 80 ° C. for 24 hours, and then comparing the respective dry weights. (See Table 2).

The mutant strain was excellent in beta-carotene content, but as can be seen in Figure 3, as the incubation period elapsed, the increase in cell number was small compared to wild strains. However, as can be seen in Figure 1, the mutant strain is not the objective comparison of biomass only by the number of cells because the size of each cell larger than the original strain. Therefore, dry weights were measured to compare their final biomass. As shown in Table 2 above, the final biomass in terms of dry weight because of the large volume of each cell despite the small number of cells can be confirmed that the mutant is significantly superior.

Example 3. Pigment Analysis of Isolated Mutant Wine

Colonies generated after mutant treatment were isolated and continued incubation of a single colony was performed by HPLC analysis using HPLC. Strains grown in the culture conditions described in Example 1 were harvested and the pigment was extracted using 80% acetone, and the supernatant was centrifuged again, filtered through a nylon filter, and then injected into HPLC for analysis.

In order to separate the pigment, the total flow rate of the solvent was 1.2 mL / min, and uniformly decreased Tris of pH 8.0 to 14% and acetonitrile to 84% to 0%, respectively, from 0 to 15 minutes. Ethyl acetate starts at 2% and increases by 68% and 32%, respectively, by the 15th minute. Then keep this solvent ratio for 3 minutes (15th to 18th minute), then return to the rate when starting each solvent for 1 minute (18th to 19th minute) and then remain for 6 minutes It was kept post-run. The pump used a LC-20A Prominence from Shimadzu, a column using Waters Spherisorb ™ S5 ODS1 4.6 × 250 mm, 5 μm Cartridge Column (Maple St. Milford, Massachusetts, USA) and the column temperature was maintained at 40 ° C. The detector analyzed the data using a photodiode array detector (SPD-M20A, Shimadzu) and carotenoid pigments including beta-carotene at 445 nm and chlorophyll a at 670 nm. , Denmark) was used as a standard to determine the concentration of beta-carotene and chlorophyll a purchased from the standard.

Figure 4 is a pigment profile of each strain grown at 200μmol photons / ㎡s for 5 days, showing a pigment analysis chromatogram analyzed using HPLC (High Performance Liquid Chromatography) according to the method described in Example 3. As can be seen in this chromatogram, the mutant strain accumulates much higher beta-carotene (peak numbers 8 and 9) than the original strain, and is superior to commercially available D. bardawil . You can check it.

Figure 5 is a graph quantitatively analyzing the pigment content of each strain grown under various light intensity (20, 100, 200, 400μmol photons / ㎡s) using the chromatogram. Graph (A) shows the ratio of beta-carotene per unit chlorophyll divided by the amount of beta-carotene (mg) in 1 mL of the culture suspension divided by the amount of chlorophyll a (mg). Graph (B) shows the amount of beta-carotene in one cell divided by the amount of beta-carotene in 1 mL of the culture suspension divided by the number of cells in the same amount of culture suspension. As can be seen in both graphs, the mutant strain was accumulating higher concentrations of beta-carotene than the other two strains.

Example 4 Production of Beta-Carotene in Isolated Variants

The amount of beta-carotene accumulated in the mutant strain isolated by visual observation and quantitative analysis by HPLC was compared with the amount accumulated in the original strain and Dunalella bardawil ( D. bardawil ).

As shown in FIG. 1, the mutant strains grown under the same luminous conditions (100 μmol photons / m 2 s) are larger in size than the original strain and turn orange due to the large amount of beta-carotene. In order to determine the degree of accumulation of beta-carotene according to the light intensity, the result of culturing for 7 days at different light intensity (20, 100, 200, 400μmol photons / ㎡s) was observed. As can be seen it has been found to have a good beta-carotene synthesis ability to be able to distinguish the color difference of the culture. When quantitatively analyzed by HPLC, it was proved to be much superior to Dunaliella Bardawil , which is already commercially available.

Variants of the present invention can easily be compared visually, at a relatively low luminous intensity of 100 μmol photons / m 2, that beta-carotene synthesis accumulates higher amounts of beta-carotene than D. bardawil , a known species. Could. This luminosity condition is much lower than the luminosity that promotes beta-carotene accumulation as reported previously, suggesting that there is an advantage that a desired product can be obtained with high energy and high efficiency.

Example 5. Photosynthetic efficiency of isolated mutant strains

Beta-carotene is a pigment present in the chloroplasts with lipid granules, and the photosynthetic efficiency was compared in anticipation that the presence of beta-carotene in the chloroplasts affects photosynthetic efficiency. To confirm the effect of beta-carotene content on mutant chloroplasts on photosynthetic efficiency, photosynthetic efficiency was compared by measuring oxygen production with increasing light intensity. Clark-type oxygen electrode from Hansatech Instrumnet Ltd. was used to measure the amount of oxygen generated. Oxygen generation amount is diluted to the amount of cells containing 2μM chlorophyll to 5mL and then measured the amount of oxygen generated while increasing the brightness as shown in the graph (Fig. 6) every two minutes, the results are shown in Figure 6 It was.

As can be seen in Figure 6 it was confirmed that the oxygen generation amount of the variant species of the present invention is higher than D. bardawil ( D. bardawil ) containing a significant level of beta-carotene, high oxygen production means high photosynthetic efficiency do.

Example 6: Amplification Experiment by ITC PCR

In order to identify the mutant strain and the systematic location of the original species, the internal transcribed spacer (ITS) portion was amplified by polymerase chain reaction (PCR). Dunaliella 's intranuclear DNA was extracted using the QIAGEN DNeasy Plant Mini Kit according to the protocol provided in the product. Intron's iTaq DNA polymerase, dNTP, and buffer were put together in the reaction solution, and DNA extracted from Dunaliella was used as a template, and the ITS portion was amplified using the primers of Table 3 below. For ITS amplification, the reaction solution was cycled 30 times at 94 ° C for 30 seconds, at 58 ° C for 30 seconds, and at 70 ° C for 30 seconds. As a result, an amplified DNA fragment having a size of about 300-400 bp was obtained (see FIG. 7).

Example 7 Molecular Genetic Positioning of Mutant strains Using Schematic Diagram

In order to analyze the ITS sequence, the PCR product of the ITS amplified by PCR was electrophoresed (Fig. 7), and then the amplified portion was cut out from the agarose gel, DNA was separated using QIAGEN's gel extraction kit, and then the base was placed on the bionics. Sequence analysis was commissioned. The analyzed sequences were aligned with the ITS of several strains of Dunaliella , registered with NCBI (http://www.ncbi.nlm.nih.gov/). First, the ITS sequences of each strain were compared using the clustalX program, and the compared sequences were analyzed using the MEGA4 program to draw a phylogenetic tree to analyze the systematic relationships. The schematic diagram of FIG. 8 was drawn using the maximum parsimony method, and was represented numerically with the confidence level through 500 repeated bootstrap tests (maximum 100). As shown in the phylogenetic tree of FIG. 8 obtained as a result, the mutant strain was the closest to the original species CCAP19 / 18, and it was confirmed that the strain was derived from CCAP19 / 18 rather than mistaken contamination of other strains.

1 is a photomicrograph of Dunaliella salina CCAP19 / 18, which is the original strain derived from the mutant strain of the present invention, and the mutant strain (KCTC 11442BP strain) of the present invention (magnification 1,000 ×). (A): Dunaliella salina CCAP 19/18; (B): Dunaliella salina KCTC 11442BP. Black lines in the picture indicate 10 μm.

FIG. 2 is a photograph showing the results of culturing three strains (wild strains, mutant strains, and D. bardawil ) under various luminous conditions (20, 100, 200, 400 μmol photons / ㎡s). . Third, fifth and seventh days after incubation from top to bottom, respectively; wt: original strain, LS202: KCTC 11442BP strain, D. bar: Dunaliella bardawil .

3 is a graph showing the Won Kyun own, two flying it Ella Salina (Dunaliella salina) CCAP19 / 18 and throwing away the two mutants of the present invention Ella Salina (Dunaliella salina) cells increased the number of KCTC 11442BP pattern. wt: original strain, m: mutant strain of the present invention, numbers indicate treated luminosity.

Figure 4a is a HPLC analysis graph showing the pigment profile of each Dunaliella strain. (A) wt, (B) LS202 (KCTC 11442BP), (C) D.bardawil

1: neoxanthin, 2: violaxanthin, 4: lutein, 5: zeaxanthin, 6: chlorophyll b 7: chlorophyll a, 8, 9: Two isomers of beta-carotene.

Figure 4b is a photograph showing the observation of each Dunaliella strain with an Olympus BX51 microscope (magnification 1,000X). Black lines in the picture indicate 10 μm.

5 is a graph comparing the beta-carotene content of the strains according to the present invention Dunaliella salina CCAP19 / 18, Bardawil ( D. bardawil ), respectively. After 7 days of incubation under the light conditions indicated in each graph, the pigment content was measured using HPLC.

(A) Beta-carotene content per mg of chlorophyll a according to brightness (mg); (B) Beta-carotene content per cell according to brightness (ng).

Figure 6 is a graph comparing photosynthetic efficiency of Dunaliella salina KCTC 11442BP mutant and D. bardawil ( D. bardawil ). M: mutant strain, b: D. bardawil .

Figure 7 shows the results of electrophoresis of the PCR product amplified using the ITS primer.

Lane 1: Two of throwing away Ella Salina (D. salina) CCAP19 / 18 ITS 1,

Lane 2: Dunaliella Salina (D. salina ) CITS19 / 18 ITS 2,

Lane 3: ITS 1 of mutant LS202,

Lane 4: ITS of Mutant LS202 2.

Figure 8 shows a schematic of Dunaliella salina strains. 8 is a schematic diagram of the following Dunaliella strains and CCAP19 / 18 (wt), and ITS1 and ITS2 sequences of mutant strains were combined and aligned using the clustalX program, and the CNI algorithm (Close-Neighbor) in the MEGA4 program. The results of 500 bootstrap tests using the Interchange algorithm are shown in the schematic diagram using the maximum parsimony method.

Claims (7)

Dunaliella salina KCTC 11442BP strain from Dunaliella salina CCAP19 / 18, having the ability to produce beta-carotene at low luminosity of 50 to 200 μmol photons / m 2 s. The Dunaliella salina KCTC 11442BP strain according to claim 1, characterized in that beta-carotene is produced at a low brightness of 50 to 200 μmol photons / m 2 s. The Dunaliella salina KCTC 11442BP according to claim 2, characterized in that beta-carotene accumulates in an amount of 2 mg or more per 1 mg of chlorophyll a when cultured for 7 days under a light intensity of 50 to 200 μmol photons / m 2. Strain. The pure culture of Dunaliella salina KCTC 11442BP strain derived from Dunaliella salina CCAP19 / 18 of claim 1. A method for producing beta-carotene comprising culturing Dunaliella salina KCTC 11442BP strain derived from Dunaliella salina CCAP19 / 18 at a low brightness of 50 to 200 μmol photons / m 2 s. delete The method of claim 5, wherein the culturing is a method for producing beta-carotene, characterized in that the salinity of the medium is made at 0.5M to 5M based on NaCl concentration.

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