• ALGAE
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• v.37 no.4
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• pp.317-329
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• 2022

Carotenoids are effective antioxidants that are found in various photosynthetic organisms. Marine microalgae are an advantageous bioresource for carotenoid production because they do not compete with other crops for freshwater and arable land. This study reports a newly isolated Dunaliella strain from the Geumhong Saltpan on Yeongjong Island, West Sea, Korea. The new strain was isolated and classified as Dunaliella salina through phylogenetic analysis and was named the OH214 strain (Deposit ID: KCTC14434BP). The newly isolated strain can survive in a wide range of NaCl concentrations (0.3-5.0 M NaCl), but grows well in 0.6 to 1.5 M NaCl culture medium. Under high-light conditions (500 ± 10 μmol photons m^-2 s^-1), the cells accumulated three times more β-carotene than under low-light conditions (50 ± 5 μmol photons m^-2 s^-1). The cells accumulated 2.5-fold more β-carotene under nitrogen-deficient (1 mM KNO₃) conditions (3.24 ± 0.36 ㎍ 10⁶ cells^-1) than in nitrogen-sufficient conditions (>5 mM KNO₃). The lutein content under nitrogen-deficient conditions (1.73 ± 0.09 ㎍ 10⁶ cells^-1) was more than 24% higher than that under nitrogen-sufficient conditions. Under the optimized culture condition for carotenoid induction using natural seawater, D. salina OH214 strain produced 7.97 ± 0.09 mg g DCW^-1 of β-carotene and 4.65 ± 0.18 mg g DCW^-1 of lutein, respectively. We propose that this new microalga is a promising strain for the simultaneous production of β-carotene and lutein.

• Journal of Plant Biology
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• v.39 no.3
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• pp.173-177
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• 1996

A novel calcium binding protein (CaBP) was purified to electrophoretic homogeneity from Dunaliella salina. In the course of purification experiment, this CaBP was identified as a monomer and its molecular weight was about 21 kDand isoelectric point (pI) value was about 4.1 using isoelectrofocusing. This CaBP was able to bind Ca2+ even in the pressence of an excess MgCl2 and KCI both in solution. In the SDS-PAGE, the Ca2+-bound form was slower than the Ca2+-free form in the nondenaturing PAGE. This means that the CaBP undergoes conformational change in the Ca2+-bound condition. Furthermore, UV absorption spectrum and fluorescence intensity of this CaBP was investigated. UV absorption peak was appeared at about 258 nm and decreased somewhat in Ca2+-bound condition. In the measurement of fluorescence, maximum intensity was appeared at 303 nm and decreased in Ca2+-bound state, similarly as UV absorption spectrum. These show distinct changes upon Ca2+-binding, which indicate of structural and/or dynamic changes largely reminiscent of other members of the EF-hand Ca2+-binding protein family.

• ALGAE
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• v.26 no.1
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• pp.3-20
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• 2011

The physiology of the unicellular green alga Dunaliella salina in response to abiotic stress has been studied for several decades. Early D. salina research focused on its remarkable salinity tolerance and ability, upon exposure to various abiotic stresses, to accumulate high concentrations of $\beta$-carotene and other carotenoid pigments valued highly as nutraceuticals. The simple life cycle and growth requirements of D. salina make this organism one of the large-scale commercially exploited microalgae for natural carotenoids. Recent advances in genomics and proteomics now allow investigation of abiotic stress responses at the molecular level. Detailed knowledge of isoprenoid biosynthesis mechanisms and the development of molecular tools and techniques for D. salina will allow the improvement of physiological characteristics of algal strains and the use of transgenic algae in bioreactors. Here we review D. salina isoprenoid and carotenoid biosynthesis regulation, and also the biotechnological and genetic transformation procedures developed for this alga that set the stage for its future use as a production system.

• Fisheries and Aquatic Sciences
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• v.19 no.7
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• pp.31.1-31.6
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• 2016

Background: The objective of this study was to develop an efficient selectable marker for transgenic Dunaliella salina. Results: Tests of the sensitivity of D. salina to the antibiotic chloramphenicol and the herbicide Basta$^{(R)}$ showed that cells ($1.0{\times}10^6cells/ml$) treated with 1000 or $1500{\mu}g/ml$ chloramphenicol died in 8 or 6 days, respectively, whereas D. salina cells ($1.0{\times}10^6cells/ml$) treated with 5, 10, 20, or $40{\mu}g/ml$ Basta$^{(R)}$ died in 2 days. Therefore, D. salina is more sensitive to Basta$^{(R)}$ than to chloramphenicol. To examine the possibility of using the phosphinothricin N-acetyltransferase (pat) gene as a selectable marker gene, we introduced the pat genes into D. salina with particle bombardment system under the condition of helium pressure of 900 psi from a distance of 3 cm. PCR analysis confirmed that the gene was stably inserted into the cells and that the cells survived in $5{\mu}g/ml$ Basta$^{(R)}$, the medium used to select the transformed cells. Conclusions: The findings of this study suggest that the pat gene can be used as an efficient selectable marker when producing transgenic D. salina.

• Journal of Plant Biology
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• v.38 no.3
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• pp.243-250
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• 1995

Involvement of calcium in signal transduction of salt stress was investigated in 1.7 M NaCl adapted Dunaliella salina, extremely halotolerant, unicellular green alga. When hyperosmotic (3.4 M NaCl) or Hypoosmotic (0.8 M NaCl) stress was treated, extracellular calcium was influxed in or intracellular calcium effluxed from D. salina, respectively, and these fluxes were proportional to the degree of stress. This might indicate indirectly that the change of calcium level occurred within the cells. In addition, the change of calcium flux was ahead of glycerol synthesis which has been known as the physiological response to salt stress. Osmoregulation was affected byextracellular calcium concentration, and increase of glycerol content as an osmoticum was inhibited about 50% by treatment of TFP and W-7 known as calmodulin specific inhibitors. Furthermore, in the case of the hyperosmotic stressed cells, the amount of 21 kD and 39 kD protein appeared to be calcium binding protein were increased. Among these, the 39 kD protein was detected only in the hyperosmotic stressed cells. The results obtained in the present work suggest that the possibility of calcium as a second messenger in the transduction of salt stress signal exists in the osmoregulation system of D. salina.

• Journal of Microbiology
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• v.43 no.4
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• pp.361-365
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• 2005

In this study, we chronicle the establishment of a novel transformation system for the unicellular marine green alga, Dunaliella salina. We introduced the CaMV35S promoter-GUS construct into D. salina with a PDS1000/He micro-particle bombardment system. Forty eight h after transformation, via histochemical staining, we observed the transient expression of GUS in D. salina cells which had been bombarded under rupture-disc pressures of 450 psi and 900 psi. We observed no GUS activity in either the negative or the blank controls. Our findings indicated that the micro-particle bombardment method constituted a feasible approach to the genetic transformation of D. salina. We also conducted tests of the cells' sensitivity to seven antibiotics and one herbicide, and our results suggested that 20 ${\mu}g$/ ml of Basta could inhibit cell growth completely. The bar gene, which encodes for phosphinothricin acetyltransferase and confers herbicide tolerance, was introduced into the cells via the above established method. The results of PCR and PCR-Southern blot analyses indicated that the gene was successfully integrated into the genome of the transformants.

• Journal of Microbiology and Biotechnology
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• v.29 no.9
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• pp.1453-1459
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• 2019

Zeaxanthin is an important pigment in the photo-protection mechanism of microalgae. However, zeaxanthin epoxidase, an enzyme involved in the accumulation and conversion of zeaxanthin, has not been extensively studied in microalgae. In this work, we report the expression pattern of zeaxanthin epoxidase in Dunaliella tertiolecta (DtZEP) at different light and diverse salinity conditions. To confirm the responsiveness to light conditions, the ZEP expression pattern was investigated in photoperiodic (16 h of light and 8 h of dark) and continuous (24 h of light and 0 h of dark) light conditions. mRNA expression levels in photoperiodic conditions fluctuated along with the light/dark cycle, whereas those in continuous light remained unchanged. In varying salinity conditions, the highest mRNA and protein levels were detected in cells cultured in 1.5 M NaCl, and ZEP expression levels in cells shifted from 0.6 M NaCl to 1.5 M NaCl increased gradually. These results show that mRNA expression of DtZEP responds rapidly to the light/dark cycle or increased salinity, whereas changes in protein synthesis do not occur within a short period. Taken together, we show that DtZEP gene expression responds rapidly to light irradiation and hyperosmotic stress. In addition, ZEP expression patterns in light or salinity conditions are similar to those of higher plants, even though the habitat of D. tertiolecta is different.

• Korean Journal of Microbiology
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• v.35 no.4
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• pp.289-295
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• 1999

Halotolerant microalga Dunaliella bardawil was reported to massively accumulate the ${\beta}$-carotene, which protects cells from excess light intensity. Maximum specific growth rate of 0.168/hr was achieved when cells were cultivated at 1 N NaCl, pH 8.0, light intensity 80 ${\mu}E/m^{2}/s$, agitation 70rpm. For the effectiv accumulation of ${\beta}$-carotene, ozone ro hydrogen peroxide was added to media which was irradiated with white fuorescent lamps with moderate light intensity of 250 ${\mu}E/m^{2}/s$. As a result, maximum volumetric content of ${\beta}$-carotene was 324 ${\mu}$g/㎖. The ${\beta}$-carotene extraction efficiency of vegetable oils was in the order of olive oil, sesame oil, rice brain oil, corn oil, and soy bean oil. Sonication and warming was effective in ${\mu}$-carotene extraction and finally 96.9% of ${\beta}$ could be extracted using olive oil.

• Journal of the Korean Society of Marine Environment & Safety
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• v.26 no.5
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• pp.514-522
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• 2020

To understand the ecotoxicological differences between representative Skeletonema sp. and Dunaliella tertiolecta, both producers as international standard test species for marine ecotoxicity testing, we compared each standard test method, and comparatively analyzed the suitability of the species for environmental assessment and their sensitivity to various test substances. Although most of the test conditions were the same in each method, there were differences in limitation of pH changing and the initial inoculation density in the validation criteria, which is supposed to originate from the low growth rate of D. tertiolecta. In terms of suitability, both species showed consistency in test performance by repeatedly meeting the validation criteria required by the standard test methods. The salinity ranges available for testing were 20 and 10 psu for Skeletonema sp. and D. tertioelecta, respectively. Finally, regarding sensitivity, the toxicity sensitivity of Skeletonema sp. was relatively higher than that of D. tertiolecta for the reference toxicant, actual polluted water discharged (ballast water), and other chemicals. This implies that using at least two species of microalgae from different classification groups could help increase the reliability and objectivity of test results in the performance of marine ecotoxicity tests using producers.

• Applied Chemistry for Engineering
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• v.28 no.3
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• pp.306-312
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• 2017

Changes in the cell growth and lipid accumulation of marine microalga Dunaliella tertiolecta were investigated in response to the combination of different stress factors including the variation of iron supply as a primary stress factor and different options in light irradiation and $CO_2$ supply as a secondary stress factor. High or limited Fe conditions could act as a stress for lipid synthesis. As a secondary stress factor, non-$CO_2$ condition was good for lipid accumulation, but the overall cell growth was sacrificed significantly after a long-time cultivation. Dark condition as a secondary stress factor also favored lipid accumulation and the extent of cell density reduction at the early period in the dark was small compared to other stress conditions. The two-stage cultivation strategy was necessary to maximize lipid production because tendencies of the cell growth and lipid content were not identical under the chosen stress condition. The first stage was for preparing a high cell density under the normal growth-favoring condition and the second stage was the stress condition to induce lipid accumulation in a short time. The short-term (12 h) incubation under the 5X Fe (3.25 mg/L) and dark conditions resulted in the best lipid productivity of 1.44 g/L/d providing 2 g/L inoculum at the second stage.