This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
D. salina is one of the recognized natural sources to produce β-carotene, and an useful model for studying the role of inhibitors and enhancers of carotenogenesis. However there is little information in D. salina regarding whether the isoprenoid substrate can be influenced by stress factors (carotenogenic) or selective inhibitors which in turn may further contribute to elucidate the early steps of carotenogenesis and biosynthesis of β-carotene. In this study, Dunaliella salina (BC02) isolated from La Salina BC Mexico, was subjected to the method of isoprenoids-β-carotene interference in order to promote the interruption or accumulation of the programmed biosynthesis of carotenoids. When Carotenogenic and non-carotenogenic cells of D. salina BC02 were grown under photoautotrophic growth conditions in the presence of 200 µM fosmidomycin, carotenogenesis and the synthesis of β-carotene were interrupted after two days in cultured D. salina cells. This result is an indirect consequence of the inhibition of the synthesis of isoprenoids and activity of the recombinant DXR enzyme thereby preventing the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol (MEP) and consequently interrupts the early steps of carotenogenesis in D. salina. The effect at the level of proteins and RNA was not evident. Mevinolin treated D. salina cells exhibited carotenogenesis and β-carotene levels very similar to those of control cell cultures indicating that mevinolin not pursued any indirect action in the biosynthesis of isoprenoids and had no effect at the level of the HMG-CoA reductase, the key enzyme of the Ac/MVA pathway.
bioactive compoundsIntroduction
Carotenoids, whose bioactive role has become the object of considerable scientific and public attention in the last years, are a rich source of antioxidants used commercially in cosmetics and food supplements for both animals and humans. Recently, the consumer demand for natural β -carotene has increased and epidemiological studies suggest anti-cancer activity of this pro-vitamin A pigment. β-carotene is massively accumulated (up to 10% on dry weight basis) within globules in the inter-thylakoid spaces of the chloroplast of the halophilic microalgae Dunaliella salina [1]. D. salina is considered the best commercial source of natural β-carotene in the world [2]. The molecule of β-carotene is made up of eight isoprene units, which are cyclised at each end. The configuration of each double-bond can occur in different geometrical forms in D. salina [3]: all trans (1) and 9-cis isomers (2) .
Several studies have shown that in Dunaliella, β-carotene functions as a means of storing carbon and quenching oxygen singlets. In addition, β-carotene globules have been implicated in photoprotection or absorption in the periphery of the chloroplast [4].
Preliminary investigations showed that the regulation of carotenoid biosynthesis in D. salina in almost all instances was related to deficiency in nitrate, sulfate, and phosphate in the culture media as well as high light intensity and high sodium chloride concentration [5–7].
Isoprenoids are synthesized by condensation of their isopentenyl diphosphate pools (IPP), considered the universal precursor of five-carbon building block in the biosynthesis of all carotenoids and β-carotene in plants and algae [8]. Two pathways for these precursors are known: the mevalonate pathway occurring in eucaryotes, Archeobacteria and cytosol of higher plants and the recently discovered non-mevalonate pathway (also known as 1-deoxy-D-xylulose 5-phosphate (DXP) pathway or 2-C-methyl-D-erythritol (MEP) [9, 10].
The studies concerning the early steps of carotenogenesis followed by Dunaliella salina are scarce and there is less information regarding whether the isoprenoid substrate can be influenced by carotenogenic conditions or selective inhibitors, viz, mevinolin and fosmidomycin. This approach can be useful to improving production level by manipulating precursor enhancers, to control downstream isoprenoid and carotenoids and further advance in the biosynthesis of β-carotene and carotenogenesis in D. salina. The present research was aimed at evaluating the isoprenoid-β-carotene interference approach by the action of inhibitors in the programmed synthesis of the early steps of carotenogenesis and β-carotene accumulation in an indigenous strain of Dunaliella salina isolated from La Salina, Ensenada, B.C. (Mexico).
Materials and MethodsDunaliella salina strain and Carotenogenesis
Cells of D. salina isolated from La Salina B.C. (32° 05’; 118° 40’) in the Northwest coast of México were used in this study. The isolated strain was correctly assigned by [11], lately corroborated with molecular biological techniques [12] and maintained under the acronym Dunaliella salina BCO2 in the strains collection of the Department of marine Biotechnology. Cells were maintained in a growth médium (control) containing, unless otherwise stated, 1 M NaCl, 5 mM MgSO4, 0.3 mM CaCl2, 5 mM NaNO3, and 0.2 mM KH2PO4 at pH 7.5–8, and a mixture of micronutrients, as previously described [13]. D. salina cells were collected by centrifugation, then they were transferred to carotenogenic media containing 2.5 mM NaNO3 and 2 M NaCl respectively. Bacteria free cultures were developed by successive generational growth under continuous illumination with cool-white fluorescent lamps at 382 μE/m2/s, and maintained at 20 ± 2 ºC. Three experimental replicate were set-up for each treatment.
Inhibitors of Carotenogenesis
The two carotenogenesis inhibitors were used: mevinolin, an inhibitor of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the mevalonate pathway and fosmidomycin, an inhibitor of 1-deoxy-D-xylulose-5-phosphate reductoisomerase that suppresses the biosynthesis of isoprenoids and accumulation of carotenoids (β-carotene) respectively in the non-mevalonate pathway. Mevinolin was purchased from Sigma (USA), which was previously converted to the water-soluble sodium salt as described in [14]. Fosmidomycin was purchased from Molecular Probes (USA) and was dissolved in culture medium prior to application to the Dunaliella cultures. The active concentration of inhibitors mevinolin (1μM) and fosmidomycin (200 μM) was determined previously in algal cultures at a cell density of 106 cells/ml. Aliquots were taken from the stock solutions in order to obtain the active concentration for each inhibitor, viz, control medium added with 1 μM mevinolin. In the case of fosmidomycin we added to the control medium the following concentrations of fosmidomycin: 50, 100, 150 and 200 μM, unless otherwise stated.
Growth and pigments
Cell grown was determined by counting immobilized cells (by Lugol) with a hemocytometer. For total chlorophyll and carotenoids determination, the centrifuged algae obtained (3000 X g) was resuspended in 90% acetone in water and stirred by vortex. After filtration, chlorophyll was determined by reading at 665 nm the absorption spectra of the extracts recorded in a Hewlett-Packard Diode-Array spectrophotometer. The absorbances of the extract at 450 nm were used assuming that 1 (%) (w/w) of β-carotene have an average extinction coefficient of 2600 cm–1. Pigments separation was performed by using a Hewlet Packard (Agilent) series 1100 HPLC (Agilent technologies Palo Alto, CA) with a C18 reverse phase 300SB-C18 Zorbax column (5 µ, 150 mm). An isocratic step was eluted with 85:10:5 acetonitrile, methanol and dichloromethane (v/v/v) respectively at a flow rate of 1 mL/min [15]. Standard of β-carotene and Chlorophyll were ran under the same conditions.
Protein measurements
Protein content of Dunaliella was determined according to [16]. The algae were collected by centrifugation at 3500 X g for 15 minutes at 10ºC. Thereafter the samples pellets were resuspended in 5 ml NaOH 1N and incubated in a water bath at 100 ºC for 10 minutes followed by a second alkaline extraction for a full protein extraction of the cell concentrates [17]. Protein measurement were carried-out reading at 595 nm and the measurements compared with a calibration curve prepared by using bovine serum albumin following procedures of Bio-Rad protein-assay kit.
Cellular RNA
Total RNA in Dunaliella salina BC02 was extracted according to the protocol of [18]. The algae was collected by centrifugation at 13000 g during 15 min. Thereafter, the algal pellet was powdered with a mortar and pestle and added 300 μl of RNA lysis solution (Aquapure RNA Bio-Rad 732-6370) followed by vigorous mixing. All manipulations were carried-out at low temperature conditions. The protein-DNA recovered was placed on ice bath (5 mins) and centrifuged at low temperature ,13000 g. The aqueous (upper) phase was then transferred into a new tube, re-extracted with 300 μl de isopropanol, and centrifuged (13000 g, 15 min, 20°C) and the supernatant was carefully discarded. The pellet was washed with 70% etanol, centrifuged briefly, and the supernatant was discarded. The pellet was then dried at room temperatura and resuspended in 50 μl RNA hidratation solution (Bio-Rad 732-6370). To determine the RNA concentration, the OD260 was measured. A solution of RNA whose OD260=1 contains approximately 40 μg.ml-1 [19].
Results and Discussion
Preliminary investigations were carried-out in order to determine the optimal concentration of each inhibitor. Application of fosmidomycin, at a range of concentrations from 50 to 200 μM was assessed in cultured D. salina BCO2 cells. At concentrations below 150 µM, fosmidomycin had no influence on the biosynthesis of β-carotene or on their number of D. salina cells (Table 1), in contrast, when D. salina was exposed to 200 μM fosmidomycin caused growth inhibition and arrested the carotenoids accumulation that was apparent at day 7 of cultivation (Figure 1C). Mevinolin, did not exerted an inhibitory effect on carotenoids accumulation and growth (<5% of control) at a concentration of 1µM, and at 48 h of cultivation as shown in Table 1 and Figure 1B. Once selected the appropriate concentration of each inhibitor, we proceeded to assess its effect on the synthesis of carotenoids, chlorophyll, β-carotene, protein and RNA in D. salina grown under carotenogenic and non-carotenogenic conditions. When D. salina cells were grown under conditions which enhances carotenoids accumulation and in presence of the highest concentration of fosmidomycin (200 μM, the highest response among the assayed concentrations), a decrease in the relative content in carotenoids was registered after four days of culture (Figure 2). This result represented, less than 75% of the content in the cells cultured under non-carotenogenic conditions (control). On the contrary, when D. salina cells were cultured under carotenogenic conditions and exposed to 1 μM mevinolin, this inhibitor did not exerted a significant change in the relative content of carotenoids during the exposure time (four days) when compared to the cells cultured under non-carotenogenic conditions (control) (Figure 2).
Analysing the ratio β-carotene to Chlorophyll ratio (Figure 3) during the exposure time to the inhibitors, highest ratios were registered under carotenogenic conditions exposed to mevinolin when compared to the non-carotenogenic cells exposed to fosmidomycin.
Mevinolin treated D. salina cells continue to grow and synthesizing carotenoids, as observed in figure 4, cells turned orange-yellow and a decrease in cell density was observed and the β-carotene levels were similar to those of control cell cultures. Figure 4 shows green cells as indicative of photosynthetic growth and blocked synthesis of carotenoids and β-carotene in D. salina by fosmidomycin. The fact that the treatment with fosmidomycin did not completely suppress the system of renewal of its pools of isoprenoids and respective products from carotenogenesis in Dunaliella salina, viz, β-carotene indicates the effective inhibition of the essential regulatory step, DOXP reductoisomerase (generated under photosynthetic conditions) of the DOXP/MEP pathway by fomidomycin which is a structural analogue to 2-C-methylerythrose 4-phosphate, the intermediate in the enzymic reaction of DXR [20]. The results evidenced the absence of HMGR, as well as a suppression of the C5-units of biosynthesis, resulting in a shortage of the bulk isoprenoids like carotenoids, β-carotene, and chlorophylls (by phytol reduction, side chain of chlorophylls) at the level of chloroplast, the exclusive site of carotenogenesis in D. salina.
All experiments were performed in order to favor growth and to try to enhance the incorporation of carbon (from carbon dioxide) from air during photosynthesis. The photosynthetic autotrophic conditions promoted in cultures of Dunaliella leads to synthesis of pentose phosphate cycle substrates in the Calvin cycle from pyruvate pools, which conversion stimulate the apparent synthesis of the first precursors of the IPPP/DMAPP and lately incorporated into the chloroplast isoprenoids and carotenoids (Figure 5).
Carbon dioxide via photosynthesis has been reported as the main carbon source for isoprenoid biosynthesis via the mevalonate-independent methylerythritol 4-phosphate route in the marine diatoms Phaeodactylum tricornutum and Nitzschia ovalis [22].
The action of fomidomycin slightly decreased the content of protein in D. salina, under non-carotenogenic conditions (Figure 6). At 1.0 μM mevinolin, there was no decrease in the content of protein. A similar trend was observed in RNA content during carotenogenesis. In principle, cellular RNA content was constant during carotenogenic conditions when D. salina was exposed to the assayed concentrations in mevinolin and fosmidomycin. Results in other Chlorophyte [23] have shown that total carotenoids were related to total proteins, suggesting that the enzyme (s) responsible for carotenoid synthesis must account for only a small part of total protein. This mechanism could indicate the importance of nitrogen to achieve adequate level of RNA and protein formation required for carotenoid biosynthesis during carotenogenesis.
Limited information exist on the important early steps of isorpenoid biosynthesis leading to carotenoid formation in Dunaliella salina. [10, 24] reported the biosynthesis of plastidic isoprenoids in heterotrophic chlorophyte and non carotenogenic microalgae. Dunaliella salina, an obligate photoautotrophic microalga that cannot use dissolved organic compounds is one of the most important microalgae in biotechology considering its hyperproducing properties of β-carotene.
The role of carotenoid accumulation in D. salina dependent as it is on photosynthesis for its continued growth and survival, and their production requires redirection of a substantial amount of the cells' carbon resources into synthesis of isoprenoids. The accumulation of carotenoids in D. salina (β-carotene represents approx. 95% in dry weight) is considered as a general response to stress conditions that presumably is of advantage to its survival in extreme natural conditions in which this alga grows [1].
Thus, the initiation of algal carotenogenesis exclusively occurs in the chloroplast as a result of the photoactivation of the carotenogenic enzymes [3]. We were unable to demonstrate a cytosolic branch of IPP biosynthesis. Our results suggest there is a low probability in Dunaliella salina cells of crosstalk between the chloroplast DXP/MEP and the cytosolic MVA pathways, as was previously reported in plants and certain algae [25, 20], since massive accumulation of carotenoids and β-carotene occurred only in the interthylakoid spaces of the chloroplast in this Chlorophyte.
The high difference in carotenoids accumulated in D. salina exposed to fosmidomycin under carotenogenic and non-carotenogenic conditions can exclude the possible effect of the the stored isoprenoids and carotenoids during the short experimental period used here. Among the posible factors to be considered in the partial percentage of inhibition of carotenoids biosynthesis by fosmidomycin (lower than 100 μM), we speculate and associate our result to a partial lack of uptake of fosmidomycin into cells by D. salina or inactivation of the action of this inhibitor throghout the culture period. In this work we have shown that isoprenoids-mediated carotenoids interference can be effectively applied by using carotenogenesis inducers, as well as inhibitors, viz, fosmidomycin or mevinolin. Even when more significantly, the changes in pigments involved in photosynthesis (chlorophylls and carotenoids) are relatively bigger than those in RNA and protein in D. salina isolated from the hipersaline lagoon La salina from Baja California Mexico.
This research was supported in part by The council of science and technology from Mexico (CONACYT) under the research project “Biotechnological potential of carotenogenic microalgae from the Peninsula of Baja California Mexico” granted to J.P.M. We wish to thank M. Sc. Eduardo Morales for assistance in the HPLC analysis. Special thanks are due to the anonymous reviewers for valuable suggestions and comments.
References and NotesBen-AmotzAEffect of irradiance and nutrient deficiency on the chemical composition of Dunaliella bardawilJ. Plant. Physiol198713147948710.1016/S0176-1617(87)80290-0BorowitzkaMMicroalgae as source of pharmaceuticals and other biologically active compoundsJ. Appl. Phycol1995731510.1007/BF00003544OrsetSCHYoungAJExposure of low irradiance favors the synthesis of 9-cis β, β-carotene in Dunaliella salina (Teod.)Plant Physiol200012260961710.1104/pp.122.2.60910677453Ben-AmotzAShaishAβ-carotene biosynthesisDunaliella: Physiology, Biochemistry and BiotechnologyAvronMBen-AmotzACRC Press, Boca RatonFlorida, USA1992205216BorowitzkaMBorowitzkaLKesslyDEffects of salinity increase on carotenoid accumulation in the green alga Dunaliella salinaJ. Appl. Phycol1990211111910.1007/BF00023372Paniagua-MichelJDujardinESironvalCGrowth of Dunaliella bardawil under carotenogenic conditionsJ. Mar. Biotech19952101104Ben-AmotzAAvronMOn the factors which determine massive beta-carotene accumulation in the halotolerant alga Dunaliella bardawilPlant Physiol19837259359710.1104/pp.72.3.59316663050RohmerMThe discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plantsNat. Prod. Rep19991656557410.1039/a709175c10584331LangeBMRujanTMartinWCroteauRIsoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomesProc. Nat. Acad. Sci. USA200097131721317710.1073/pnas.24045479711078528SchwenderJGemundenCLichtenthalerHChlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway for the biosynthesis of isoprenoidsPlanta200121241642310.1007/s00425000040911289606Gutierrez-MillanLEDesoxyribonucleic acid, protein and pigments during induced carotenogenesis in a new strain of Dunaliella salina (Chlorophyta isolated from a hipersaline coastal lagoon of Baja CaliforniaMSc Thesis, CICESE Mexico, 1996181Olmos-SotoJPaniagua-MichelJContreras-FloresRTrujillo-ValleMLMolecular identification of β-carotene hyper-producer strains of Dunaliella from saline environments using species-specific oligonucleotidesBiotechol. Letters20022436536910.1023/A:1014516920887Ben-AmotzAAvronMOn the Factors which Determine Massive β-Carotene Accumulation in the Halotolerant Alga Dunaliella bardawilPlant Physiol19837259359710.1104/pp.72.3.59316663050HagenCGrünewaldKFosmidomycin as an inhibitor of the non-mevalonate terpenoid pathway depresses synthesis of secondary carotenoids in flagellates of the green alga Haematococcus pluvialisJ Appl. Bot20007437140KhachikFBeecherGRGoliMBSeparation, identification, and quantification of carotenoids in fruits, vegetables and human plasma by high performance liquid chromatographyPure Appl. Chem199163718010.1351/pac199163010071BradfordMMA rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalyt. Biochem19767224829410.1016/0003-2697(76)90527-3942051BergesJFisherAHarrisonPA comparison of Lowry, Bradford and Smith protein assays using different protein standards and protein isolated from the marine diatom Thalassiosira pseudonanaMar. Biol199311518719310.1007/BF00346334SambrookJFritshEFManiatisTMolecular cloning: a laboratory manualCold Spring Harbor laboratory PressNew York; NY, USA1989BajguzAAsamiTEffects of brassinazole, an inhibitor of brassinosteroid biosynthesis, on light and dark grown Chlorella vulgaris.Planta200421886987710.1007/s00425-003-1170-914655015LichtenthalerHKThe 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plantsAnnu Rev Plant Physiol Plant Mol Biol199950476510.1146/annurev.arplant.50.1.4715012203LangeMKetchumEBCroteauRBIsoprenoid biosynthesisPlant Physiol200112730531410.1104/pp.127.1.30511553758CvejicJRohmerMCO2 as main carbon source for isoprenoid biosynthesis via the mevalonate-independent methylerythritol 4-phosphate route in the marine diatoms Phaeodactylum tricornutum and Nitzschia ovalisPhytochem200053212810.1016/S0031-9422(99)00465-3SirevRLevineRPTranscription and translation of carotenoid synthesis in Chlamydomonas reinhardtiiPlanta1973111738410.1007/BF00386737DischASchwenderJMüllerCLichtenthalerHRohmerMDistribution of the mevalonate and glyceraldehyde phosphate/pyruvate pathways for isoprenoid biosynthesis in unicellular algae and the cyanobacterium Synechocystis PCC 6714Biochem J19983333813889657979LauleOFürholzAChangHZhuTWangXHeifetzPGruissemWLangeBCrosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis inArabidopsis thaliana Proc Nat Acad Sci USA20031006866687110.1073/pnas.1031755100Figures and Table
HPLC chromatogram of Dunaliella salina cultured in the control growth media (A) after 7 days of culture and in presence of 200 μM fosmidomycin (B) and 1 µM mevinolin (C). The peaks correspond to the following pigments: 1, violaxanthin; 2, neoxanthin; 3, lutein; 4, zeaxanthin; 5, Chlorophyll a; 6, all-trans β-carotene.
Relative changes in the total carotenoid content during carotenogenesis (line triangle) in D. salina expose to fosmidomycin (line square) and mevinolin (line diamond). Data are means ± SD (each point represent n=3).
β-Carotene-to-chlorophyll ratio in Dunaliella salina exposed to metabolic inhibitors under carotenogenic conditions (Bars) and non-carotenogenic control (line). Values are means ± SD (each point represent n=3).
Culture flasks containing D. salina cells cultured under carotenogenic conditions and exposed to mevinolin (uninterrupted synthesis of β-carotene, left) and to fosmidomycin (blocked synthesis of β-carotene, right) respectively.
Supposed simplified pathway of the early steps of carotenogenesis and β-carotene accumulation in Dunaliella salina (modified after [21]).
Protein (dark bar) and RNA content (gray bar) in D. salina during carotenogenesis and exposed to inhibitors: 1, Control (C); 2, C+ 1.0 μM mevinolin; 3-6=, C+ 50, 100, 150 and 200 μM fosmidomycin respectively.
Effect of the concentrations of mevinolin (Mev) and fosmidomycin (Fos) in growth, Chlorophyll and total carotenoids of D. salina under carotenogenic growth phase. Mean values indicate standard deviation (n = 3).