Inhibitors of sterol metabolism for their use to accumulate triglycerides in microalgae, and methods thereof

Abstract

The invention relates to a method for accumulating triacylglycerols in microalgae by inhibiting the sterol metabolism, by incubating the microalgae with an inhibitor of sterol metabolism. The invention also relates to a method for producing fatty acids, biofuels, pharmaceutical or cosmetic compositions, and also food supplements, comprising a triacylglycerols accumulation step in microalgae according to the invention. Finally, the invention concerns the use of an inhibitor of sterol metabolism to accumulate triglycerides in microorganisms, and preferably microalgae.

Claims

1. A method for triggering triacylglycerols accumulation in microalgae by inhibiting the sterol metabolism, wherein the method comprises a step of incubating the microalgae with an inhibitor of sterol metabolism, said inhibitor being a compound of formula (I) or a salt thereof: ##STR00012## wherein: R.sub.41 and R.sub.42, identical or different, represent a hydrogen atom, alkyl, alkenyl, alkynl, or hydroxyl, COR.sub.4a or COOR.sub.4a group, in which R.sub.4a represents a hydrogen atom, a linear or branched alkyl, aryl, heteroaryl group, optionally substituted with one or more groups independently selected from alkyl or cycloalkyl groups, or R.sub.41 and R.sub.42 form together an oxygen atom attached by a double bond; R.sub.43 represents a hydrogen atom, or an alkyl group; and R.sub.44, R.sub.45 and R.sub.46, identical or different, represent a hydrogen atom, an alkyl, alkoxy, hydroxyl group, or an oxygen atom attached by a double bond, said alkyl group being optionally substituted with one or more halogen atoms, and including optionally in its chain one or more sulfoxide functions, wherein the concentration of the inhibitor of sterol metabolism ranges from 1 M to 1M, and wherein the microalgae is selected from the group consisting of diatom microalgae species Phaeodactylum tricornutum and the Chromalveolata micro-algae species Nannochloropsis gaditana.

2. A method according to claim 1, wherein the incubation step is implemented in a nitrogen medium.

3. A method according to claim 1, wherein the inhibitor of sterol metabolism is selected from the group consisting of Ethinylestradiol and Estrone.

4. A method for producing fatty acids comprising a triggering of triacylglycerols accumulation step in microalgae as defined according to claim 1.

5. A method for producing biofuels comprising the following steps; (i) a triggering of triacylglycerols accumulation step in microalgae as defined according to claim 1, followed by (ii) an extraction step of the triacylglycerols accumulated in microalgae during step (i), and (iii) a trans-esterification step of the triacylglycerols recovered during step (ii).

6. The method according to claim 1, wherein the concentration of the inhibitor of sterol metabolism ranges from 5 M to 1M.

7. The method according to claim 1, wherein the concentration of the inhibitor of sterol metabolism ranges from 5 to 20 M.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In addition to the above provisions, the invention also comprises other provisions which will emerge from the remainder of the description which follows, and also to the appended drawings in which:

(2) FIG. 1 gives a schematic view of the sterol biosynthetic pathway via mevalonic acid, and represents the action of Mevastatin and Simvastatin (Liu et al., Mol. Biol. Rep. (2010) 37:1391-1395), Estrone (Merola and Arnold, Science, Vol. 144, 301-302 (1964), Butenafine and Terbinafine (Belter et al., Biol. Chem., Vol. 392, 1053-1075 (2011)),

(3) FIG. 2 represents pictures of a Phaeodactylum cell visualized by confocal microscopy after a 72 h cultivation in Nitrogen-rich N(+) or in Nitrogen-starved N() medium On the left side (A and C), cells are shown in phase contrast (phase). On the right side (B and D), cells are shown after excitation of Nile red at 488 nm and emission at 519 nm,

(4) FIG. 3a-h illustrates the dose response for Ethinylestradiol, Mevastatin, Simvastatin and Estrone, and

(5) FIG. 4 illustrates the dose response for Ethinylestradiol, Mevastatin and Simvastatin with an evaluation of the TAG content by staining with Nile Red and an evaluation of cells by counting in an aliquote fraction using a Malassez cell,

(6) FIG. 5 shows the effect of Butenafine on Nile Red accumulation in Nannochloropsis gaditana grown respectively in ESAW 1N1P media and ESAW 10N10P media. Nile Red Fluorescence was normalized by calculating the relative fluorescence units per million cells, and

(7) FIG. 6 shows the effect of Ethinylestradiol on Nile Red accumulation in Nannochloropsis gaditana grown respectively in ESAW 1N1P media and ESAW 10N10P media. Nile Red Fluorescence was normalized by calculating the relative fluorescence units per million cells.

DETAILED DESCRIPTION

Examples

1) Materials & Methods

(8) 1) 1-Phaeodactylum tricornutum Strain and Growth Conditions

(9) Phaeodactylum tricornutum (Pt1) Bohlin Strain 8.6 CCMP2561 (Culture Collection of Marine Phytoplankton, now known as NCMA: National Center for Marine Algae and Microbiota) was used in experiments.

(10) Pt1 was grown at 20 C. in 250 mL flask using enriched artificial seawater (ESAW) medium, prepared following the recommendations of the Canadian Center for the Culture of Microorganisms.

(11) To prepare the ESAW medium, four separated solutions are prepared, two solutions of salts (solutions 1 and 2), one solution of nutrients, and one solution of vitamins. Salts are added in order to distilled deionized water (DDW). When the salts in solutions 1 and 2 are completely dissolved, the solutions 1 and 2 are mixed together. The total volume is diluted with DDW.

(12) TABLE-US-00003 TABLE 3 Compositions of the ESAW salts solutions 1 and 2 Molecular weight Amount to weight Concentration (g .Math. mol.sup.1) (g/L solution) (mM) Solution 1: Anhydrous salts NaCl 58.44 20.756 362.7 Na.sub.2SO.sub.4 142.04 3.477 25.0 KCl 74.56 0.587 8.03 NaHCO.sub.3 84 0.17 2.067 KBr 119.01 0.0845 0.725 H.sub.3BO.sub.3 61.83 0.022 0.372 NaF 41.99 0.0027 0.0657 Solution 2: Hydrated salts MgCl.sub.26H.sub.2O 203.33 9.395 47.18 CaCl.sub.22H.sub.2O 147.03 1.316 9.134 SrCl.sub.26H.sub.2O 266.64 0.0214 0.082

(13) TABLE-US-00004 TABLE 4 Nutrient Enrichment Stocks Stock concentration Final concentration Solutions (g .Math. L.sup.1) (M) 1 NaNO.sub.3 46.67 549.1 2* Na.sub.2 glycerophosphate 6.67 21.8 3 Na.sub.2SiO.sub.39H.sub.2O 15.00 105.6 4** Na.sub.2EDTA2H.sub.2O 3.64 9.81 Fe(NH.sub.4).sub.2(SO.sub.4).sub.26H.sub.2O*** 2.34 5.97 FeCl.sub.36H.sub.2O 0.16 0.592 5 MnSO.sub.44H.sub.2O 0.54 2.42 ZnSO.sub.47H.sub.2O 0.073 0.254 CoSO.sub.47H.sub.2O 0.016 0.0569 Na.sub.2MoO.sub.42H.sub.2O 0.126 0.520 Na.sub.2EDTA2H.sub.2O 1.89 5.05 6 H.sub.3BO.sub.3 3.80 61.46 7 NaSeO.sub.3 0.00173 0.001 *Na.sub.2 glycerophosphate can be replaced with an equimolar stock of Na.sub.2HPO.sub.4. **Na.sub.2EDTA2H.sub.2O is added before the trace metals Fe(NH.sub.4).sub.2(SO.sub.4).sub.26H.sub.2O and FeCl.sub.36H.sub.2O. ***Fe(NH.sub.4).sub.2(SO.sub.4).sub.26H.sub.2O can be replaced with an equimolar stock of FeCl.sub.3. Solution 5 is adjusted to pH = 6 with 2 g of Na.sub.2CO.sub.3. Solution 4 can be heated to dissolve the iron.

(14) TABLE-US-00005 TABLE 5 Vitamin Stocks Stock concentration Final concentration Vitamin Stock (g .Math. L.sup.1) (mM) Thiamine 0.1 2.97 10.sup.1 Vitamin B12 0.002 1.47 10.sup.3 Biotin 0.001 4.09 10.sup.3

(15) To prepare the ESAW medium, the solutions are filtered through 0.45 m membrane filter with a glass fiber prefilter. A flask is acid-washed in 10% HCl and rinsed in distilled water before first use. To 1 L of filtered salt solution, 1 mL of Nutrient Enrichment Stock solutions 1, 2, 4, 5, 6 and 7, 2 mL of Nutrient Enrichment Stock solution 3, and 2 mL of the Vitamin Stock are added (Tables 4 and 5). To reduce precipitation during autoclaving, 1.44 mL of 1N HCl and 0.12 g of sodium bicarbonate are added. The obtained ESAW medium is then sterilized by autoclaving.

(16) Cells were grown on a 12:12 light (450 Einstein-1 sec.sup.1)/dark cycle (an Einstein defined the energy in one mole (6.02210.sup.23) of photons). Cells were sub-cultured every week by inoculate fresh media with of previous culture. Nitrogen-rich N(+) medium contained no source of nitrogen. Nitrogen-starved N(), medium contained 0.05 g/L NaNO.sub.3. To monitor cell growth, a genetically modified strain containing a Histone H4 protein fused to the yellow fluorescent protein was used (Siaut et al., Gene 406 (2007) 23-35).

(17) 1) 2-Principle of Nile Red Staining of Oil Droplets

(18) Accumulation of oil droplets can be monitored by Nile Red (Sigma Aldrich) fluorescent staining (Excitation wavelength at 485 nm; emission at 525 nm) as described by Ren et al. (Biotechnology for Biofuels 2013, 6:143), Cells were diluted and adjusted to a cell density that was linearly correlated with Nile Red fluorescence. Nile Red solution (40 L of 2.5 g.Math.mL.sup.1 stock concentration, in 100% DMSO) was added to 160 L cell suspension. Specific fluorescence was determined by dividing Nile Red fluorescence intensity by the number of cells. Oil bodies stained with Nile Red were then visualized using a Zeiss AxioScope.A1 microscope (FITC filter; Excitation wavelength at 488 nm; emission at 519 nm).

(19) Lipid droplets can be visualized. In FIG. 2, a Phaeodactylum cell was visualized by confocal microscopy after a 72 h cultivation in Nitrogen-rich N(+) medium or in Nitrogen-starved N() medium. On the left side, cells are shown in phase contrast (phase). On the right side, cells are shown after excitation of Nile red at 488 nm and emission at 519 nm. Lipid droplets can be clearly visualized.

(20) This principle was use to measure the presence of oil in Phaeodactylum tricornutum simply by using a spectrofluorometer (in these conditions, to lower the detection of other fluorophores within the cell, such as chlorophylls, excitation was at 530 nm and emission at 580 nm).

(21) 1) 3-Alternative Method for Oil Level Detection

(22) Alternatively, oil is extracted using solvents or another extraction method, separated and purified by thin layer chromatography and methanolyzed to produce fatty acid methyl esters and quantified by gas chromatography coupled to a ionization flame detector or a mass spectrometer.

(23) TAG were extracted from 200 mg of freeze-dried Phaeodactylum tricornutum cells in order to prevent lipid degradation. Briefly, cells were frozen in liquid nitrogen immediately after harvest. The freeze-dried cell pellet was resuspended in 4 mL of boiling ethanol for 5 minutes followed by the addition of 2 mL of methanol and 8 mL of chloroform at room temperature. The mixture was then saturated with argon and stirred for 1 h at room temperature. After filtration through glass wool, cell remains were rinsed with 3 mL of chloroform/methanol (2:1, v/v). In order to initiate biphase formation, 5 mL of NaCl 1% was then added to the filtrate. The chloroform phase was dried under argon before re-solubilization of the lipid extract in pure chloroform. To isolate TAG, lipids were run on silica gel thin layer chromatography (TLC) plates (Merck) with hexane/diethylether/acetic acid (70:30:1, v/v). Lipids were then visualized under UV light after pulverization of 8-anilino-1-naphthalenesulfonic acid at 2% in methanol. They were then scraped off from the TLC plates for further analyses. For acyl profiling and quantification of TAG, fatty acids were methylated using 3 mL of 2.5% H.sub.2SO.sub.4 in methanol during 1 h at 100 C. (including standard amounts of 21:0). The reaction was stopped by the addition of 3 mL of water and 3 mL of hexane. The hexane phase was analyzed by gas liquid chromatography (Perkin Elmer) on a BPX70 (SGE) column. Methylated fatty acids were identified by comparison of their retention times with those of standards and quantified by surface peak method using 21:0 for calibration. Extraction and quantification were done at least 3 times.

(24) 1) 4-Principle of Cell Normalization

(25) The number of cells in a sample can be evaluated using a fluorescent reporter, like the strain by Siaut et al. (Gene 406 (2007) 23-35) containing a genetic construction with Histone H4 protein fused to the Yellow Fluorescent Protein (YFP) (FIG. 5A of Siaut et al., Gene 406 (2007) 23-35).

(26) For cell counting, we can either use this strain called ptYFP and measure the fluorescence emitted by the YFP at 530 nm after excitation at 515 nm, or use any strain and estimate cell numbers by counting with a Malassez grid (supplier: Mareinfeld).

(27) 1) 5-Incubation of Phaeodactylum tricornutum with Inhibitors of the Sterol Metabolism and Detection of Oil Accumulation Triggered by the Treatment

(28) On the first day, we prepared 48 well plates by adding 4 mm glass beads sterilized with Ultra-Violet exposure in each well.

(29) We prepared fresh suspensions of microalgae in exponential growth phase. For cell normalization based on YFP fluorescence, cells of Phaeodactylum tricornutum containing a YFP reporter (ptYFP) cultured in N(+) ESAW medium were centrifuged at 3,500 rpm, 5 min. For cell normalization based on counting using a Malassez grid, cells of the Pt1 strain of Phaeodactylum tricornutum cultured in N(+) ESAW medium were centrifuged at 3,500 rpm, 5 min. The supernatant was discarded and the pellet suspended in N() ESAW. The microalgae were then centrifuged at 3,500 rpm, 5 min. The supernatant was discarded and the pellet suspended in N() ESAW. Cells were then diluted to 110.sup.6 cells/mL in N() ESAW. Samples were then separated into two batches. One was supplemented with 1 L/mL of 46.7 g/L NaNO.sub.3 stock to obtain a suspension of cells in N(+) ESAW medium. Another was left without NaNO.sub.3 to obtain a N() ESAW culture as a control for high lipid accumulation. In a 48-well clear NUNC plate, 450 L/well of 110.sup.6 cells/mL were dispensed.

(30) For a dose-response analysis, each well of the 48-well plate was then subjected to an appropriate incubation with 0, 1, 10 or 100 M of inhibitor of sterol metabolism using 50 L of the following: 50 L of a 10 time concentrated solution of inhibitor of sterol metabolism (5% DMSO:95% N(+) ESAW) or 50 L of Nitrogen-rich medium without any inhibitor of sterol metabolism (5% DMSO:95% N(+) ESAW) or Nitrogen-starved medium without any inhibitor of sterol metabolism (5% DMSO:95% N() ESAW) as a positive control.

(31) For an analysis of the effect of a single dose, an incubation was performed with 0, and a chosen concentration of inhibitor of sterol metabolism (50 M).

(32) In all cases, the edge of the plate was sealed using a parafilm. Plates were incubated for 48 h in an incubator with top lighting, 20 C., 100 rpm, 12 h/12 h light/dark.

(33) After an incubation of 48 hours, fluorescence was measured at the following excitation/emission wavelengths, 530/580 nm (to evaluate a baseline fluorescence prior Nile Red addition) and 515/530 nm (to evaluate YFP fluorescence). Following this first measure, 40 L of Nile Red (2.5 g/mL stock concentration, in 100% DMSO) are added. Plates are mixed and incubated 20 minutes at room temperature, protected from light. Nile Red fluorescence is then measured using a spectrofluorometer (excitation 530 nm/emission 580 nm).

(34) 1) 6-Incubation of Nannochloropsis gaditana with Inhibitors of the Sterol Metabolism and Detection of Oil Accumulation Triggered by the Treatment

(35) Experiments were performed in two different conditions of Nannochloropsis gaditana: cells were either cultured in ESAW containing 47 mg.Math.L.sup.1 NaNO.sub.3 and 3 mg.Math.L.sup.1 NaH.sub.2PO.sub.4 (medium 1N1P), or 470 mg/L NaNO.sub.3 and 30 mg.Math.L.sup.1NaH.sub.2PO.sub.4) (medium 10N10P). Cells in an exponential growth phase were collected via centrifugation at 3500 rpm for 10 minutes. The supernatant was discarded and the cells were resuspended in the same volume of ESAW medium (either 1N1P or 10N10P). The cultures were centrifuged again at 3500 rpm, for 10 minutes, and the supernatant was discarded. The pellet was resuspended in ESAW (either 1N1P or 10N10P) to obtain a concentration of 210.sup.6 cells/mL. Cell counts were performed using a Malassez counting chamber, allowing 10 minutes for the cells to settle before counting.

(36) Twenty milliliters of 210.sup.6 cells/mL of Nannochloropsis gaditana in ESAW (10N10P) and ESAW (1N1P) were dispensed into sterile glass conical flasks. Stocks of inhibitor of sterol metabolism were prepared in DMSO. Inhibitors of sterol metabolism were added to the 20 mL Nannochloropsis gaditana samples at final concentrations of 10 M, 30 M, or 100 M. The maximum final concentration of DMSO in the samples was 1% (v/v), All cultures were incubated for seven days at 100 rpm, 12 h/12 h light/dark cycle, 50 E.Math.m.sup.2.Math.s.sup.1, 20 C.

(37) Each day, an aliquot was taken from each flask in order to perform a Nile Red stain and cell counts. 160 L per sample was added to black 96 well plates, and allowed to settle for 10 minutes. In order to detect any background noise, fluorescence was measured at excitation and emission of 530 nm and 580 nm, respectively. 40 L of 2.5 g.Math.mL.sup.1 Nile Red in DMSO was added to each well, and mixed thoroughly. After 20 minutes of incubation, Nile Red fluorescence was measured at excitation and emission of 530 nm and 580 nm, respectively.

(38) Nile Red Fluorescence was normalized by calculating the relative fluorescence units per million cells. Results were expressed as a percentage of Nile Red fluorescence of Nannochloropsis gaditana cultured in complete medium (either ESAW (10N10P) or ESAW (1N1P)).

(39) 1) 7-Inhibitors of Sterol Metabolism

(40) Inhibitors of sterol metabolism were obtained from the Prestwick library for their ability to trigger the accumulation of lipid droplets within the cells of Phaeodactylum tricornutum, and then purchased from Sigma-Aldrich,

(41) TABLE-US-00006 TABLE 6 Inhibitors of sterol metabolism selected from the Prestwick library Chemical name Structure Molecular Formula Mevastatin C.sub.23H.sub.34O.sub.5 Butenafine C.sub.23H.sub.27N Simvastatin C.sub.25H.sub.38O.sub.5 Estrone 0 C.sub.18H.sub.22O.sub.2 Ethinylestradiol C.sub.20H.sub.24O.sub.2

2) Results

(42) Phaeodactylum tricornutum was incubated for 48 h in presence of 10 M of Mevastatin, Butenafine, Simvastatin, Estrone, Ethinylestradiol and Terbinafine.

(43) In all cases the presence of oil per cell increased by a factor of at least 1.5, based on Nile Red staining.

(44) FIG. 3a-h illustrates the dose response for Ethinylestradiol, Mevastatin, Simvastatin and Estrone with an evaluation of the TAG content by staining with Nile Red, and an evaluation of cells by monitoring YFP fluorescence from an expressed Histone H4 reporter gene. On the left, FIG. 3a-h shows the Nile Red levels in percent of untreated cells and the number of cells estimated by the YFP fluorescence expressed in percent of untreated cells. The right panels show the increase of oil content per cell, by the ratio of Nile Red fluorescence/YFP fluorescence, expressed in percent of untreated cells. The oil content per cell increases with drug concentration and consequently level of sterol metabolism inhibition and ranges from 120 to 400% when compared to untreated cells.

(45) FIG. 4 illustrates the dose response for Ethinylestradiol (A), Mevastatin (B) and Simvastatin (C) with an evaluation of the TAG content by staining with Nile Red and an evaluation of cells by counting in an aliquote fraction using a Malassez cell. In histograms of FIG. 4, the white bars indicate the evaluation of cell numbers in percent of untreated cells, and the black bars indicate the Nile Red per 10.sup.6 cell, in percent of untreated cells. The oil content per cell, and consequently level of sterol metabolism inhibition, increases with incubation of 50 M of inhibitor, and ranges from 120 to 350% when compared to untreated cells.

(46) Nannochloropsis gaditana was incubated in presence of Butenafine and

(47) Ethinylestradiol.

(48) FIGS. 5 and 6 illustrate the effect of Butenafine and Ethinylestradiol on Nile Red accumulation in Nannochloropsis gaditana grown respectively in ESAW 1N1P media and ESAW 10N10P media. Nile Red Fluorescence was normalized by calculating the relative fluorescence units per million cells.

(49) Both Butenafine and Ethinylestradiol trigger the accumulation of oil in Nannochloropsis gaditana, in different media and with a time course that can be observed at least for 7 days.