TY - JOUR
AU - Hapala, Ivan
AB - Abstract Squalene is a valuable natural substance with several biotechnological applications. In the yeast Saccharomyces cerevisiae, it is produced in the isoprenoid pathway as the first precursor dedicated to ergosterol biosynthesis. The aim of this study was to explore the potential of squalene epoxidase encoded by the ERG1 gene as the target for manipulating squalene levels in yeast. Highest squalene levels (over 1000 μg squalene per 109 cells) were induced by specific point mutations in ERG1 gene that reduced activity of squalene epoxidase and caused hypersensitivity to terbinafine. This accumulation of squalene in erg1 mutants did not significantly disturb their growth. Treatment with squalene epoxidase inhibitor terbinafine revealed a limit in squalene accumulation at 700 μg squalene per 109 cells which was associated with pronounced growth defects. Inhibition of squalene epoxidase activity by anaerobiosis or heme deficiency resulted in relatively low squalene levels. These levels were significantly increased by ergosterol depletion in anaerobic cells which indicated feedback inhibition of squalene production by ergosterol. Accumulation of squalene in erg1 mutants and terbinafine-treated cells were associated with increased cellular content and aggregation of lipid droplets. Our results prove that targeted genetic manipulation of the ERG1 gene is a promising tool for increasing squalene production in yeast. squalene epoxidase, terbinafine, erg1 mutations, squalene accumulation, Saccharomyces cerevisiae Introduction Squalene is a linear polyunsaturated triterpene occurring naturally in almost all organisms from unicellular prokaryotic to multicellular eukaryotic plants and animals. The most important role of squalene is the involvement in the makeup of membranes as the precursor of membrane constituents, for example sterols in eukaryotes (Bloch, 1965) or hopanoids in eubacteria (Ourisson et al., 1979), or directly as a membrane component in archaea (Tornabene et al., 1979). In addition to these membrane-linked functions, squalene fulfills also other specific roles in various cell types and tissues, for example maintenance of bacteriorhodopsin function (Hendler & Dracheva, 2001), protection of human skin as lubricant and antioxidant (De Luca & Valacchi, 2010), or stabilization of nonbilayer structures in retinal rod outer segment membranes (Fliesler et al., 1997). Specific physicochemical and antioxidant properties of squalene imply also its applications in food industry, pharmacology, and cosmetics (for review, see Spanova & Daum, 2011). Extensive industrial use of squalene is hampered by limited resources and its relatively high price. Shark liver as the traditional source of squalene is currently being substituted by plant sources such as olive or amaranth oil (He & Corke, 2003; García-González et al., 2008). Another promising approach in the search for new squalene resources is the use of microorganisms as squalene producers on industrial scale (Ghimire et al., 2009; Nakazawa et al., 2012). Among these, yeasts represent an interesting alternative due to easy genetic and physiological manipulation of squalene content. Similarly to the majority of eukaryotes, the yeast Saccharomyces cerevisiae synthesizes squalene in the isoprenoid (mevalonate) pathway as the first precursor dedicated to the synthesis of fungal native sterol – ergosterol (see Daum et al., 1998, for review). Under normal conditions, yeast cells rapidly utilize squalene for the formation of sterol planar ring. Consequently, squalene levels in growing or resting aerobic cultures are usually very low. On the other hand, significant amounts of squalene can be found in yeast grown under specific conditions such as hypoxia or in specific mutants. Squalene levels in the yeast S. cerevisiae depend generally on three factors: the rate of squalene production in the mevalonate pathway, the rate of squalene utilization in the postsqualene part of ergosterol biosynthesis, and finally, on the capacity of yeast cells to store accumulated squalene in lipid droplets as specific storage compartments for neutral lipids. The flux in the yeast presqualene pathway is regulated at two major control sites – hydroxymethylglutaryl-CoA (HMG-CoA) reductase and squalene synthase (Daum et al.,1998). Yeast S. cerevisiae has two isoforms of HMG-CoA reductase, Hmg1p and Hmg2p, that are encoded by the genes HMG1 and HMG2 (Basson et al., 1986). The regulation of HMG-CoA reductase activity in yeast is rather complex and involves the differential control of transcription of HMG1 and HMG2 genes by oxygen and heme via the Hap1/Rox1 system, translational control of Hmg1p via feedback inhibition by an isoprenoid signal as well as stimulation of Hmg2p degradation by geranylgeranyl pyrophosphate and an oxysterol signal (for review, see Burg & Espenshade, 2011). So far, HMG-CoA reductase has been the preferred target in attempts to increase squalene levels in yeast. Donald et al. (1997) overexpressed the catalytic domain of Hmg1p under a strong constitutive promoter in a multicopy plasmid which lead to severalfold higher squalene levels and increased ergosterol content. Similar results were obtained by expression of truncated cytosolic form of Hmg1p that was not subject to feedback inhibition (Polakowski et al., 1998) or by expression of a stable form of the hypoxic isoenzyme Hmg2p under the control of inducible galactose promoter (Mantzoridou & Tsimidou, 2010). All three approaches resulted in significant increase in squalene level that was associated with reduced growth rate in some cases (Donald et al., 1997; Asadollahi et al., 2010). Less information is available about the regulation of squalene synthase encoded by the ERG9 gene. Several studies have shown that squalene synthase activity is subject to feedback inhibition by ergosterol (Bard & Downing, 1981; M'baya et al., 1989). According to the results of Kennedy et al. (1999), transcription of the ERG9 gene is positively regulated by an isoprenoid precursor (probably mevalonate) and repressed by feedback inhibition by ergosterol. Probably the tight regulation by ergosterol contributed to the limited effectiveness of squalene synthase in increasing cellular squalene content in S. cerevisiae. Squalene levels in yeast depend also on the rate of its conversion to ergosterol as the final product of sterol biosynthesis. Reduction in the activity of the postsqualene part of ergosterol biosynthesis may thus be an alternative approach for increasing squalene content in yeast. As the first step of squalene metabolism (epoxidation to oxidosqualene) is dependent on molecular oxygen (Jahnke & Klein, 1983), squalene accumulates in cells grown under anaerobic conditions (David & Kirshop, 1973). Suitability of squalene epoxidase encoded by the gene ERG1 as the target for manipulation of squalene levels in yeast was demonstrated by the accumulation of squalene in cells treated by the specific squalene epoxidase inhibitor – the antimycotic terbinafine (Ryder, 1992; Klobučníková et al., 2003; Naziri et al., 2011; Ta et al., 2012). Oxidosqualene cyclase and lanosterol demethylase encoded by the ERG7 and ERG11 genes, respectively, represent other possible targets for manipulation of squalene content. Defective activity of Erg7p results in the accumulation of oxidosqualenes and dioxidosqualene while squalene levels are affected in a limited extent only (Mo et al., 2003). Lanosterol demethylase (Erg11p) is the first heme-dependent enzyme in ergosterol biosynthesis, and elevated levels of squalene were correspondingly observed in heme-deficient cells (Astin & Haslam, 1977; Spanova et al., 2010), as well as in cells treated with azoles as lanosterol demethylase inhibitors (Sanati et al., 1997). However, lanosterol was the major ergosterol precursor accumulating in these cells. Disruption of the activities of enzymes involved in late steps in ergosterol synthesis pathway showed even lower efficiency in increasing cellular squalene as these enzymes are able to use several ergosterol precursors as substrates, and the blocks can be overcome by alternative routes. The affected yeast cells accumulate usually multiple sterol species that are able to fulfill partially ergosterol functions in the membranes (Fryberg et al., 1973). Squalene accumulation may be limited also by the capacity of yeast cells to store lipids. Similar to triacylglycerols (TAG) and steryl esters (SE), yeast cells sequester squalene in lipid droplets (Leber et al., 1994; Spanova et al., 2010). Yeast lipid droplets represent dynamic organelles responding by their lipid and protein composition to the cellular metabolic state (Grillitsch et al., 2011). Their biogenesis is intimately linked to the synthesis of TAG and SE. Sandager et al. (2002) showed that the quadruple mutant with simultaneously deleted genes encoding acyltransferases involved in the formation of TAG (Dga1p, Lro1p) or SE (Are1p, Are2p) did not form lipid droplets but was still viable. Using a series of triple mutants expressing only one of these acyltransferases, Czabany et al. (2008) proved that either TAG or SE alone was sufficient for lipid droplet formation and that the nature of stored lipid affected the internal structure of lipid droplets. This effect of lipids on lipid droplet structure was confirmed also for squalene accumulated in hem1 mutants (Spanova et al., 2012). The size and number of lipid droplets in S. cerevisiae is affected by the homeostasis of neutral lipids, as indicated by mutants defective in TAG lipases accumulating high levels of TAG (Kurat et al., 2006) or by cells accumulating squalene (Ta et al., 2012). However, even under conditions of high accumulation of neutral lipids, cellular content of lipid droplets in S. cerevisiae does not reach the levels seen in some oleaginous yeast (e.g. Yarrowia lipolytica). This indicates that some species-specific limits exist for the formation of lipid droplets although the mechanisms of the control of this process are largely unknown. HMG-CoA reductase activity has been so far the preferred target for manipulating squalene levels in yeast and much less attention has been paid to the postsqualene part of ergosterol synthesis pathway. In this study, we focused on the squalene epoxidase (Erg1p) as the target for experimental manipulation of squalene content in the yeast S. cerevisiae. Using partial inhibition of squalene epoxidase activity with the specific inhibitor terbinafine and specific erg1 mutations, we explored the role of this enzyme in squalene accumulation with the aim to disclose possible limits and ‘bottlenecks’ that may affect accumulation of squalene in this important industrial microorganism. As squalene accumulated in yeast cells is stored in lipid droplets, we were also interested how increased squalene levels affect the cellular content and morphology of these organelles. Materials and methods Chemicals Terbinafine hydrochloride, lipid standards (ergosterol, cholesterol, lanosterol, triolein, cholesteryl oleate, oleic acid), and δ-aminolevulinic acid (δ-ALA) were obtained from Sigma-Aldrich. Growth media components (yeast autolysate, peptone, agar) were from Becton Dickinson/Difco. Thin-layer chromatography (TLC) plates and organic solvents (HPLC grade) were from Merck (Germany). Nile Red was from Molecular Probes. Other standard chemicals of the highest purity available were purchased from various suppliers. Yeast strains Saccharomyces cerevisiae strain BY4741 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) was obtained from Euroscarf collection. Its hem1 derivative (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 hem1::LEU2) was prepared in our laboratory by disruption of the HEM1 gene with LEU2 disruption cassette (Kohut et al., 2011). Saccharomyces cerevisiae strains of the KLN1 series were generously provided by F. Turnowski (Graz, Austria). Strain KLN1-ERG1 [MATα leu2 ura3 trp1 ERG1::URA3 (pNS1)] has disrupted chromosomal copy of ERG1 gene and is transformed with the centromeric plasmid pRS315 containing 2.3 kb PstI fragment with the wild-type ERG1 gene (Leber et al., 2003). Strain KLN1-erg1L37P [MATα leu2 ura3 trp1 ERG1::URA3 (pAB1)] has disrupted chromosomal copy of ERG1 gene and is transformed with the centromeric plasmid pRS315 containing 2.3 kb PstI fragment with the allelic erg1L37P gene bearing a single base substitution generated by random mutagenic PCR (Ruckenstuhl et al., 2007). Strain KLN1-erg1Q443UAG [MATα leu2 ura3 trp1 ERG1::URA3 (pCN1)] has disrupted chromosomal copy of ERG1 gene and is transformed with the centromeric plasmid pRS315 containing 2.3 kb PstI fragment with the allelic erg1Q443UAG gene bearing a single base substitution generated by random mutagenic PCR (F. Turnowski, unpublished data). Cultivation conditions Cells were grown aerobically in liquid YPD media (1% yeast extract, 2% peptone, and 2% glucose) in Erlenmeyer flasks with cotton stoppers containing the medium up to 1/10 of flask volume. The cultivation was performed in rotatory shaker at temperatures indicated in individual experiments. Where indicated, growth supplements (50 mg L−1 δ-ALA, 20 mg L−1 sterols, 0.06% Tween80 as the source of unsaturated fatty acids) were added to the media after autoclaving. Anaerobic growth was performed in YPD media (supplemented with sterols and Tween80 as described above) that were prepared as for cultivation of anaerobic methanogenic bacteria (Smigan et al., 1994). Shortly, YPD media supplemented with resazurin (redox indicator with color shift at −42 mV; final concentration 0.005%) were placed in thick-walled flasks closed by special gas-tight rubber stoppers. Media were degassed by short boiling and then extensively flushed with catalytically deoxygenated nitrogen. Traces of oxygen remaining in the medium were removed by the addition of Na2S (final concentration 6 μg mL−1) and l-cysteine (final concentration 8 μg mL−1). After autoclaving, sterols and Tween80 (source of oleic acid) were added to the media by gas-tight Hamilton syringe to concentrations indicated above. Prior to experimental cultivations for lipid analysis, yeast cultures were adapted to anaerobiosis by precultivation of inocula in anaerobic medium for 48 h. Terbinafine susceptibility assay Sensitivity to terbinafine was determined by drop tests on agar plates. Cells grown in YPD for 20 h at 28 °C were washed, and the cell density was adjusted to 106 cells mL−1. Five microliter drops of fourfold serial dilutions (corresponding to 104–101 cells) were spotted on YPD agar plates containing various concentrations of terbinafine. Growth was evaluated after 2 days of incubation at 28 °C. Lipid extraction and TLC analysis of neutral lipids Total lipids for TLC analysis were extracted by a modified procedure of Bligh & Dyer (1959). Washed cells were disrupted by vortexing with glass beads (diameter 0.4 mm, 6 × 1 min, with cooling on ice), and lipids were extracted by hot methanol (30 min at 65 °C) followed by 2-h incubation in chloroform–methanol–H2O (1 : 2 : 0.8) at room temperature. The organic phase containing lipids was withdrawn and evaporated under N2 stream. Dry lipid residue was dissolved in chloroform–methanol (2 : 1), and aliquots corresponding to 4 × 108 cells were applied to silica gel TLC plates by semi-automatic sample applicator (CAMAG Linomat 5, Switzerland). Neutral lipids were separated by ascending two-step TLC as described by Spanova et al. (2010) (first step: petroleum ether–diethyl ether–acetic acid, 70 : 30 : 2; second step: petroleum ether–diethyl ether, 49 : 1). Individual lipid spots were visualized by charring the plates with the solution consisting of 0.63 g MnCl2·4H2O, 60 mL of water, 60 mL of methanol, and 4 mL of concentrated sulfuric acid. Relative amounts of individual lipids were determined by densitometry at 600 nm (CAMAG TLC Scanner 3, Switzerland). Individual peaks were identified by lipid standards run on the same plate. Nonsaponifiable lipid extraction and quantitative high-performance liquid chromatography analysis of sterols Nonsaponifiable lipids for high-performance liquid chromatography (HPLC) analysis were isolated by the modified procedure of Breivik & Owades (1957). Shortly, cells broken by homogenization with glass beads were incubated in 3 mL of 60% KOH (w/v) in 50% methanol (v/v) for 2 h at 70 °C. Nonsaponifiable lipids were extracted twice with 3 mL of n-hexane, and combined extract were dried under N2. Lipid residue was dissolved in acetone and analyzed by reversed-phase HPLC on Agilent 1100 instrument equipped with Eclipse XDB-C8 column (Agilent Technologies), diode array detector (Agilent Technologies), and Corona Charged Aerosol Detector (ESA Inc.). Sterols were eluted at 30 °C with 95% methanol at the flow rate of 1 mL min−1. The identity of peaks was determined from the retention times of standards (ergosterol, cholesterol, lanosterol, and squalene) and verified by their characteristic UV spectra. The quantity of sterols was calculated from the output of the Corona CAD detector using calibration curves constructed for individual lipid standards. Fluorescence microscopy Lipid droplets in yeast cells were visualized by confocal microscopy after Nile Red staining (Wolinski & Kohlwein, 2008). Inocula of yeast strains were grown overnight in 5 mL YPD medium at 28 °C. Experimental cultures were inoculated into 10 mL YPD media to the density of 2 × 106 cells mL−1 and grown 24 h at 28 °C with vigorous shaking. Cells from 1 mL of medium were collected by centrifugation (1000 g, 1 min), washed 2× in 50 mM Tris–HCl, pH 7.5, and suspended in 1 mL 50 mM Tris–HCl, pH 7.5. 0.5 μL Nile Red (stock solution 2 mg mL−1 in DMSO) was added to cell suspension (f.c. 1 μg mL−1). Suspension was gently mixed and incubated in the dark for 20 min at room temperature. After 20 min, suspension was centrifuged 2 min at 1000 g and cell pellet was suspended in the residual liquid. Eight microliter of cell suspension was mounted on microscopic slide and observed in Olympus confocal laser scanning microscope FV1000D supplemented with immersion objective 100× and excitation/emission optics for fluorescein isothiocyanate. Images were processed with fv10-asw software (version 3.0). The content and morphology of lipid droplets in control and squalene accumulating cells was evaluated by visual inspection of five independent images for each experimental condition. Twenty cells per image were selected from bright field images, and the number of single lipid droplets and lipid droplet aggregates was estimated for these cells in fluorescence images. Statistical evaluation Statistical significance of differences in squalene levels determined by quantitative HPLC was evaluated by Student's t-test or by anova one-way analysis of variance using the SigmaPlot software (Systat Software, San Jose, CA). Results and discussion Effect of anaerobiosis and heme deficiency on squalene levels in yeast cells Biosynthesis of ergosterol in S. cerevisiae is dependent on the availability of molecular oxygen and heme. Molecular oxygen is required for the first step of the pathway dedicated to sterol synthesis – transformation of squalene to 2,3-oxidosqualene by squalene epoxidase (Jahnke & Klein, 1983). Heme (synthesized itself in an oxygen-dependent manner) is required for several steps in ergosterol synthesis pathway, the first being the demethylation of lanosterol (Loper, 1992). Growth of anaerobic and/or heme-deficient S. cerevisiae cells is thus dependent on supplementation with external sterols. Both conditions (oxygen limitation and heme deficiency) have been described to increase squalene levels in growing yeast cells (David & Kirshop, 1973; Astin & Haslam, 1977; Mantzouridou et al., 2009; Spanova et al., 2010). Our first experiments were aimed at exploring accumulation of squalene in anaerobic and heme-deficient cells for comparison with a more targeted and controlled approach using partial inhibition of squalene epoxidase by terbinafine or mutations in the ERG1 gene. In contrast to most previous studies, we grew the yeast under strict anaerobic conditions (as used for cultivation of the strictly anaerobic methanoarchaea). In addition, we were looking for conditions that could increase squalene levels in anaerobic cells. The results of the quantitative analysis of nonsaponifiable lipids (sterols and squalene) shown in Fig. 1a revealed that although anaerobiosis significantly stimulated accumulation of squalene, its levels were still relatively low (31.13 ± 0.91 μg per 109 cells). As the activity of presqualene pathway was reported to be subject to feedback regulation by ergosterol or its derivative(s), we tested also the effect of ergosterol depletion on squalene levels in anaerobic cells. Ergosterol levels were manipulated in two ways – by general sterol depletion and by substitution of ergosterol by cholesterol. Growing anaerobic cells without external sterols (Tween80-grown cells) lead to total sterol depletion, and the cells grew only for 3–4 generations overt 24 h (from 2 × 106 to 2.5–5 × 107 cells mL−1). This residual growth was enabled by utilization of ergosterol stored in steryl ester fraction, and it ceased when the levels of free ergosterol in membranes fell below the limit consistent with cell growth. Alternatively, growing anaerobic cells in medium supplemented with cholesterol and Tween80 resulted in a complete substitution of ergosterol by cholesterol. In contrast to sterol-depleted cells grown on Tween80, cholesterol-grown cells showed normal growth identical to ergosterol-grown cells. Both conditions (substitution of ergosterol by cholesterol and general sterol depletion) resulted in additional three to fourfold increase in squalene levels (to 87.58 ± 10.56 and 112.81 ± 23.27 μg per 109 cells for cholesterol- and Tween80-grown cells, respectively, Fig. 1a). This significant increase in squalene content is probably caused by release from feedback regulation of the mevalonate pathway by free ergosterol in S. cerevisiae. Quantitative HPLC analysis of the nonsaponifiable lipids showed that anaerobic cells grown in medium supplemented with cholesterol accumulated significantly more cholesterol compared with the levels of ergosterol in ergosterol-grown anaerobic cells (Fig. 1a). This difference can be explained by high efficiency of cholesterol esterification in anaerobic cells, as indicated by TLC analysis of neutral lipids (Fig. 1b). Following hydrolysis during saponification, this esterified cholesterol contributed to high levels of cholesterol detected by HPLC. High esterification of external cholesterol was reported also in earlier studies (Taylor & Parks, 1981; Valachovič et al., 2001), and it might reflect the role of sterol esterification in sequestration of unnatural sterols from the membranes (Valachovič et al., 2002). 1 View largeDownload slide Accumulation of squalene in anaerobically grown cells. Strain BY4741 was grown in aerobic (+O2) or anaerobic (−O2) conditions for 24 and 48 h, respectively (as described in Materials and methods) with following supplements in YPD media for anaerobic growth: Erg/Tw – 20 μg mL−1 ergosterol and 0.06% Tween80; Chol/Tw – 20 μg mL−1 cholesterol + 0.06% Tween80; Tw – 0.06% Tween80 (as the source of oleic acid). Lipids were extracted and analyzed by HPLC or TLC as described in Materials and methods. (a) Quantification of sterols as nonsaponifiable lipids by HPLC. Results are averages of three independent experiments ± SEM. Statistical significance of differences in squalene levels was compared by one-way analysis of variance. *P < 0.05 compared with aerobic control; §P < 0.05 compared with anaerobic cells grown on ergosterol + Tween80. (b) Typical TLC chromatography scans of neutral lipids in chloroform/methanol extracts. ERG, ergosterol; CHOL, cholesterol; LAN, lanosterol; SQ, squalene; FA, free fatty acids; TAG, triacylglycerols; SE, steryl esters. 1 View largeDownload slide Accumulation of squalene in anaerobically grown cells. Strain BY4741 was grown in aerobic (+O2) or anaerobic (−O2) conditions for 24 and 48 h, respectively (as described in Materials and methods) with following supplements in YPD media for anaerobic growth: Erg/Tw – 20 μg mL−1 ergosterol and 0.06% Tween80; Chol/Tw – 20 μg mL−1 cholesterol + 0.06% Tween80; Tw – 0.06% Tween80 (as the source of oleic acid). Lipids were extracted and analyzed by HPLC or TLC as described in Materials and methods. (a) Quantification of sterols as nonsaponifiable lipids by HPLC. Results are averages of three independent experiments ± SEM. Statistical significance of differences in squalene levels was compared by one-way analysis of variance. *P < 0.05 compared with aerobic control; §P < 0.05 compared with anaerobic cells grown on ergosterol + Tween80. (b) Typical TLC chromatography scans of neutral lipids in chloroform/methanol extracts. ERG, ergosterol; CHOL, cholesterol; LAN, lanosterol; SQ, squalene; FA, free fatty acids; TAG, triacylglycerols; SE, steryl esters. The effect of heme depletion on squalene levels was studied in the same genetic background (BY4741) bearing hem1Δ mutation that caused deficiency in δ-ALA synthase. Similar to anaerobic cells, hem1Δ mutant cannot synthesize ergosterol and desaturate fatty acid and it is dependent on the supply of ergosterol and unsaturated fatty acid. Addition of δ-ALA to the growth media reconstitutes heme synthesis, and cells become competent for ergosterol biosynthesis and fatty acid unsaturation. Quantification of nonsaponifiable lipids in hem1Δ mutant cells grown under conditions of heme deficiency or anaerobiosis (Fig. 2) brought very similar results to the anaerobiosis in wild-type cells – squalene did accumulate, but at a relatively low level. This indicates that despite different mechanisms of the inhibition of postsqualene pathway in anaerobic cells (direct inhibition of squalene epoxidase by oxygen depletion) and heme deficiency (inhibition of lanosterol demethylase), the final effect on squalene accumulation is similar. 2 View largeDownload slide Accumulation of squalene in heme-deficient cells. Strain BY4741hem1Δ was grown in aerobic (+O2) or anaerobic (−O2) conditions for 24 and 48 h, respectively (as described in Materials and methods), with following supplements in YPD media: δALA – 50 μg mL−1 δ-aminolevulinic acid; Erg/Tw – 20 μg mL−1 ergosterol and 0.06% Tween80. Lipids were extracted, and sterols in nonsaponifiable lipid fraction were quantified by HPLC as described in Materials and methods. Results shown are averages of three independent experiments ± SEM. Statistical significance of differences in squalene levels was compared by t-test. *P < 0.05 compared with aerobic cells supplemented with δALA. ERG, ergosterol; LAN, lanosterol; SQ, squalene. 2 View largeDownload slide Accumulation of squalene in heme-deficient cells. Strain BY4741hem1Δ was grown in aerobic (+O2) or anaerobic (−O2) conditions for 24 and 48 h, respectively (as described in Materials and methods), with following supplements in YPD media: δALA – 50 μg mL−1 δ-aminolevulinic acid; Erg/Tw – 20 μg mL−1 ergosterol and 0.06% Tween80. Lipids were extracted, and sterols in nonsaponifiable lipid fraction were quantified by HPLC as described in Materials and methods. Results shown are averages of three independent experiments ± SEM. Statistical significance of differences in squalene levels was compared by t-test. *P < 0.05 compared with aerobic cells supplemented with δALA. ERG, ergosterol; LAN, lanosterol; SQ, squalene. Squalene accumulation in cells with reduced squalene epoxidase activity: effect of terbinafine Epoxidation of squalene, the first step of squalene metabolism, is a logical target for modification of squalene levels in yeast. Squalene epoxidase (Erg1p) is inhibited by the allylamine antimycotics, for example terbinafine, and this treatment was shown to induce squalene accumulation (Ryder, 1992; Klobučníková et al., 2003; Naziri et al., 2011; Ta et al., 2012). In our experiments, we wanted to find out whether partial inhibition of squalene epoxidase activity by increasing concentrations of terbinafine will reveal a maximum level of squalene consistent with normal vitality of yeast cells. The used terbinafine concentrations ranged from low subinhibitory concentrations (2.5–7.5 μg mL−1) with negligible effect on growth up to higher concentrations causing severe growth defects (40–50 μg mL−1; Fig. 3). As shown in Fig. 4a, increasing concentrations of terbinafine caused gradual increase in cellular squalene levels and reduction in free ergosterol levels. At terbinafine concentration of 7.5 μg mL−1, the increase in squalene levels became statistically significant, while the growth of treated cells was only slightly affected (average final cell density 2.5 × 108 vs. 1.3 × 108 cells mL−1 for 0 and 7.5 μg mL−1 terbinafine, respectively). From 30 μg mL−1 terbinafine, a plateau in squalene accumulation at about 600–700 μg squalene per 109 cells was reached and squalene levels did not change by increasing terbinafine concentration up to 50 μg mL−1 (the highest concentration tested). It must be emphasized that growth of these cells with high squalene accumulation was significantly compromised (growth yield reduced to 4.5 × 107 and 1.6 × 107 cells mL−1at 30 and 50 μg mL−1, respectively). TLC densitometric analysis of neutral lipids (Fig. 4b) revealed that even the lowest tested concentrations of terbinafine caused a drop in steryl ester levels. This indicated that already at terbinafine concentrations partially inhibiting squalene epoxidase activity cells experienced deficit in sterols. The limiting amounts of ergosterol synthesized in these cells were utilized for membrane biogenesis and not for the formation of SE as the storage form of sterols. As the formation of TAG depends primarily on the availability of fatty acids, TAG levels should not be affected in terbinafine-treated cells. Reduced TAG peak on TLC densitograms at high terbinafine concentrations (30 μg mL−1 terbinafine in Fig. 4b) may be caused by different charring intensity as this sample was separated on a different plate. However, as we could not perform a thorough quantitative analysis on these TLC densitograms, we cannot exclude that this change reflects some general adjustment of lipid composition of lipid droplets at these extreme conditions. 3 View largeDownload slide Susceptibility of the strain BY4741 to terbinafine. Susceptibility of the strain BY4741 to squalene synthase inhibitor terbinafine was estimated by drop tests on YPD agar plates as described in Materials and methods. Briefly, cells were grown overnight in YPD medium, washed, and serially diluted in sterile water. Five microliter aliquots of diluted cell suspensions containing 104–103–102–101 cells were spotted on YPD agar plates with indicated concentrations of terbinafine and incubated at 28 °C for 2 days. 3 View largeDownload slide Susceptibility of the strain BY4741 to terbinafine. Susceptibility of the strain BY4741 to squalene synthase inhibitor terbinafine was estimated by drop tests on YPD agar plates as described in Materials and methods. Briefly, cells were grown overnight in YPD medium, washed, and serially diluted in sterile water. Five microliter aliquots of diluted cell suspensions containing 104–103–102–101 cells were spotted on YPD agar plates with indicated concentrations of terbinafine and incubated at 28 °C for 2 days. 4 View largeDownload slide Effect of squalene epoxidase inhibition by terbinafine on squalene accumulation. Strain BY4741 was grown aerobically for 24 h in YPD media containing various concentrations of terbinafine (0–50 μg mL−1). Lipids were extracted and analyzed by HPLC or TLC as described in Materials and methods. (a) Quantification of sterols as nonsaponifiable lipids by HPLC. Results shown are averages of 3–5 independent experiments ± SEM. Statistical significance of differences in squalene levels was compared by one-way analysis of variance. *P < 0.05 compared with untreated control. (b) Typical TLC chromatography scans of neutral lipids in chloroform/methanol extracts of BY4741 cells treated by 0, 5, 10, and 30 μg mL−1 terbinafine. ERG, ergosterol; LAN, lanosterol; FA, free fatty acids; TAG, triacylglycerols; SE, steryl esters; SQ, squalene. 4 View largeDownload slide Effect of squalene epoxidase inhibition by terbinafine on squalene accumulation. Strain BY4741 was grown aerobically for 24 h in YPD media containing various concentrations of terbinafine (0–50 μg mL−1). Lipids were extracted and analyzed by HPLC or TLC as described in Materials and methods. (a) Quantification of sterols as nonsaponifiable lipids by HPLC. Results shown are averages of 3–5 independent experiments ± SEM. Statistical significance of differences in squalene levels was compared by one-way analysis of variance. *P < 0.05 compared with untreated control. (b) Typical TLC chromatography scans of neutral lipids in chloroform/methanol extracts of BY4741 cells treated by 0, 5, 10, and 30 μg mL−1 terbinafine. ERG, ergosterol; LAN, lanosterol; FA, free fatty acids; TAG, triacylglycerols; SE, steryl esters; SQ, squalene. Absence of oxygen and the application of terbinafine should have similar effect on yeast cells with respect to the content of squalene and ergosterol. Under both conditions, cells should experience reduction in squalene epoxidase activity demonstrated in lowering ergosterol levels and accumulation of squalene. However, if we compare amount of squalene in anaerobic cells (Fig. 1a) and in terbinafine-treated cells (Fig. 4a), we see striking differences: while anaerobic cells reached 80–100 μg squalene per 109 cells only under conditions of severe ergosterol depletion, severalfold higher squalene levels (over 300 μg per 109 cells) were observed in cells treated with 10 μg mL−1 terbinafine with relatively normal ergosterol levels. Adaptation to anaerobiosis is associated with a general reprogramming of cellular metabolism (for review, see Snoek & Steensma, 2007) that may include a complex attenuation of lipid metabolism. On the other hand, exposure to subinhibitory concentrations of terbinafine is an instant targeted intervention and affected cells respond in a simple way that includes general upregulation of ergosterol metabolic pathway. Such targeted partial inhibition of squalene epoxidase is apparently the more efficient way for increasing squalene levels in S. cerevisiae than anaerobiosis or heme deficiency. Squalene accumulation in cells with reduced squalene epoxidase activity: effect of mutations in the ERG1 gene Our previous experiments with terbinafine validated squalene epoxidase as a suitable target to increase squalene levels in the yeast S. cerevisiae. The use of mutations in the ERG1 gene encoding squalene epoxidase is an alternative and biotechnologically more feasible approach for the modification of squalene epoxidase activity. In our previous work on mutational mapping of the ERG1 gene (Leber et al., 2003; Ruckenstuhl et al., 2007), we utilized the KLN1 strain with disrupted chromosomal copy of the ERG1 gene that was transformed with a series of centromeric plasmids harboring mutated variants of the ERG1 gene under the control of the native promoter. The erg1 mutations selected in these experiments as hypersensitive to terbinafine had reduced activity of squalene epoxidase in vitro and showed increased radiolabeling of squalene by 14C-acetate (Ruckenstuhl et al., 2007). These characteristics of terbinafine-hypersensitive mutants are interesting with respect to the production of squalene. We have chosen two strains bearing different types of erg1 mutation for further analysis. The erg1L37P strain carried a single missense mutation causing the substitution of Leu for Pro at the position 37 of the Erg1p. This substitution was located in the FADI domain of squalene epoxidase and resulted in weak enzymatic activity and instability of the Erg1p (Ruckenstuhl et al., 2007). The other strain erg1Q443UAG carried a single nonsense mutation creating a stop codon at the position 443. Mutant cells synthesized an inactive truncated Erg1p; however, their survival was enabled by partial read-through of the stop codon and formation of a small fraction of functional full-length Erg1p (F. Turnowski, unpublished data). As a control, we used the KLN1 strain with disrupted chromosomal ERG1 gene that harbored the wild-type ERG1 gene in the same centromeric plasmid. Both erg1 mutants chosen for detailed analysis were extremely sensitive to terbinafine (Fig. 5). Quantification of nonsaponifiable lipids extracted from cells grown under standard conditions (24 h of aerobic growth at 28 °C) revealed that both erg1 mutations caused high accumulation of squalene (Fig. 6a). The levels of squalene in the erg1Q443UAG mutant (600 μg per 109 cells) were similar to the levels observed in the BY4741 strain treated with the highest concentrations of terbinafine. In contrast to terbinafine-treated cells, both erg1 mutants showed undisturbed growth under the conditions of high squalene accumulation. Both mutants retained relatively high ergosterol levels, indicating that they were adapted to reduced squalene epoxidase activity. This was confirmed also by TLC analysis of neutral lipids (Fig. 6b), showing normal levels of free ergosterol and residual but significant levels of esterified sterols. 5 View largeDownload slide Hypersensitivity of erg1 mutants to terbinafine. Strains ERG1,erg1L37P, and erg1Q443UAG (derived from the strain KLN1 with disrupted chromosomal copy of ERG1 gene, see Materials and methods) were tested for sensitivity to terbinafine by drop test as described in Materials and methods. Briefly, cells were grown overnight in YPD medium, washed, and serially diluted in water. Five microliter aliquots of diluted cell suspensions containing 104–103–102–101 cells were spotted on YPD agar plates with indicated concentrations of terbinafine and incubated at 28 °C for 2 days. 5 View largeDownload slide Hypersensitivity of erg1 mutants to terbinafine. Strains ERG1,erg1L37P, and erg1Q443UAG (derived from the strain KLN1 with disrupted chromosomal copy of ERG1 gene, see Materials and methods) were tested for sensitivity to terbinafine by drop test as described in Materials and methods. Briefly, cells were grown overnight in YPD medium, washed, and serially diluted in water. Five microliter aliquots of diluted cell suspensions containing 104–103–102–101 cells were spotted on YPD agar plates with indicated concentrations of terbinafine and incubated at 28 °C for 2 days. 6 View largeDownload slide Effect of mutations in the ERG1 gene on squalene accumulation. Strains ERG1,erg1L37P, and erg1Q443UAG (derived from the strain KLN1 with disrupted chromosomal copy of ERG1 gene, see Materials and methods) were grown aerobically for 24 h in YPD media. Lipids were extracted and analyzed by HPLC or TLC as described in Materials and methods. (a) Quantification of sterols as nonsaponifiable lipids by HPLC. Results shown are averages of six independent experiments ± SEM. Statistical significance of differences in squalene levels was compared by one-way analysis of variance. *P < 0.05 compared with control ERG1 wt cells. (b) Typical TLC chromatography scans of neutral lipids in chloroform/methanol extracts. ERG, ergosterol; LAN, lanosterol; FA, free fatty acids; TAG, triacylglycerols; SE, steryl esters; SQ, squalene. 6 View largeDownload slide Effect of mutations in the ERG1 gene on squalene accumulation. Strains ERG1,erg1L37P, and erg1Q443UAG (derived from the strain KLN1 with disrupted chromosomal copy of ERG1 gene, see Materials and methods) were grown aerobically for 24 h in YPD media. Lipids were extracted and analyzed by HPLC or TLC as described in Materials and methods. (a) Quantification of sterols as nonsaponifiable lipids by HPLC. Results shown are averages of six independent experiments ± SEM. Statistical significance of differences in squalene levels was compared by one-way analysis of variance. *P < 0.05 compared with control ERG1 wt cells. (b) Typical TLC chromatography scans of neutral lipids in chloroform/methanol extracts. ERG, ergosterol; LAN, lanosterol; FA, free fatty acids; TAG, triacylglycerols; SE, steryl esters; SQ, squalene. Squalene content in yeast cells can be modified by altering cultivation conditions such as media composition or temperature. Although high temperature is known to reduce sterol biosynthesis in yeast, Shimizu & Katsuki (1975) observed almost twofold increase in squalene levels in S. cerevisiae cultivated at 40 °C. Similarly, Henderson et al. (2013) reported an increase in squalene content in industrial S. cerevisiae strains grown at 35 °C. On the other hand, Loertscher et al. (2006) and Tronchoni et al. (2012) observed significant accumulation of squalene in S. cerevisiae cultivated at low temperatures. Although the effect of cultivation temperature on squalene accumulation seems to be nonsystematic and strain specific, we tested the effect of low (22 °C) and high (35 °C) temperatures on production of squalene in our erg1 mutants and in the control ERG1 strain. Surprisingly, the response in squalene levels to changes in cultivation temperature was specific for each ERG1 allele despite the same genetic background. Yeast strain expressing the wild-type allele of ERG1 gene had significantly reduced squalene levels at 35 °C and slightly reduced squalene levels at 22 °C compared with standard cultivation at 28 °C (Fig. 7a). A reduction in squalene levels at 22 °C was observed also in the mutant erg1L37P expressing the erg1 allele with a single amino acid change in the FAD binding domain. Cultivation at 35 °C, however, caused a striking fourfold increase in squalene accumulation in this strain (Fig. 7b). Finally, the strain erg1Q443UAG expressing the truncated Erg1p showed no significant response to high cultivation temperature (35 °C), while increased squalene levels were observed at 22 °C (Fig. 7c). It is interesting that erg1L37P and erg1Q443UAG mutant strains showed squalene accumulation at the temperatures where their ability to grow was significantly compromised (number of cell doublings over 24 h of cultivation at corresponding temperatures was reduced by about 30% for each strain). This coincidence of extreme accumulation of squalene and growth defects may indicate that our erg1 mutants can tolerate only limited (although strikingly high) levels of squalene. 7 View largeDownload slide Effect of cultivation temperature on squalene accumulation in erg1 mutants. Strains ERG1 (a), erg1L37P (b), and erg1Q443UAG (c) were grown aerobically for 24 h in YEPD media at three different temperatures (22, 28, and 35 °C). Lipids were extracted, analyzed by HPLC, and quantified as described in Materials and methods. Results shown are averages of four independent experiments ± SEM. Statistical significance of differences in squalene levels in each strain was compared by one-way analysis of variance. *P < 0.05 for comparison with cultivation at 28 °C. ERG, ergosterol; SQ, squalene. 7 View largeDownload slide Effect of cultivation temperature on squalene accumulation in erg1 mutants. Strains ERG1 (a), erg1L37P (b), and erg1Q443UAG (c) were grown aerobically for 24 h in YEPD media at three different temperatures (22, 28, and 35 °C). Lipids were extracted, analyzed by HPLC, and quantified as described in Materials and methods. Results shown are averages of four independent experiments ± SEM. Statistical significance of differences in squalene levels in each strain was compared by one-way analysis of variance. *P < 0.05 for comparison with cultivation at 28 °C. ERG, ergosterol; SQ, squalene. Our results thus revealed that under standard conditions the effect of two terbinafine-hypersensitive erg1 mutations on cellular squalene was comparable to terbinafine treatment in the strain BY4741. We also showed that squalene levels in erg1 mutant strains could be further increased by growing the cells at low (erg1Q443UAG) or high (erg1L37P) temperature. Increased accumulation of squalene at different temperatures in erg1L37P and erg1Q443UAG mutants can be related to specific temperature sensitivity of mutated forms of Erg1p. Both erg1 mutants appear to be well adapted to low squalene epoxidase activity as they are able to accumulate high amounts of squalene without significantly compromised growth. This proves that genetic manipulation of squalene epoxidase is a promising approach for biotechnological production of squalene in yeast. Squalene effect on lipid droplet morphology For a long time, yeast lipid droplets were considered to be a relatively inert storage compartment for neutral lipids (TAG and SE). This view has changed in recent years, and lipid droplets are currently recognized as metabolically dynamic organelles involved in many aspects of cell physiology, particularly in processes linked to lipid metabolism (Grillitsch et al., 2011). Formation and maintenance of lipid droplets are associated with neutral lipid homeostasis (Sandager et al., 2002; Kurat et al., 2006; Czabany et al., 2008). Several recent reports linked squalene accumulation directly to lipid droplets: Spanova et al. (2010) showed that squalene produced in hem1 mutant is predominantly localized in lipid droplets. In a subsequent study based on a differential calorimetry of hem1 lipid droplets, Spanova et al. (2012) have found that squalene is intermixed with TAG in the central part of lipid droplets. Ta et al. (2012) reported that squalene accumulation in yeast (and animal) cells is accompanied by increased cellular content and aggregation of lipid droplets. We were therefore interested how the very high accumulation of squalene induced under our experimental conditions will be reflected in the formation and morphology of lipid droplets. The most common way to study cellular content and/or morphology of lipid droplets is fluorescence microscopy of cells stained with Nile Red, a fluorescent dye with high affinity to neutral lipids (Greenspan et al., 1985). Under appropriate staining conditions, this dye stains preferentially lipid droplets in yeast cells (Wolinski & Kohlwein, 2008). As shown in the Fig. 8, Nile Red staining of the strain BY4741 grown in the absence of terbinafine revealed relatively low number (average 1.9 per cell) of isolated globular lipid droplets in most cells. Treatment with 10 μg mL−1 terbinafine showed a striking effect on the cellular content and morphology of lipid droplets. The level of fluorescent staining by Nile Red was much higher in these cells, indicating increased content of lipid droplets. The average number of single round-shaped lipid droplets was reduced to 1.2 per cell; however, the structures stained by Nile Red had predominantly large size and irregular shape which indicated aggregation of lipid droplets. Only a small fraction (about 10%) of terbinafine-treated cells was free of these lipid droplet aggregates compared with 83% for untreated control cells. The changes in lipid droplet content and morphology observed in terbinafine-treated cells were consistent with the observation of Ta et al. (2012) who reported accumulation of lipid droplet aggregates in yeast cells after terbinafine treatment and correlated this effect with increased levels of squalene. Although we could not compare squalene content directly (Ta et al. quoted only a relative 3.5- to 4-fold increase of squalene over the untreated control compared with 30-fold increase under our experimental conditions), the effect of terbinafine treatment on lipid droplet morphology was similar in both studies. 8 View largeDownload slide Effect of terbinafine treatment on lipid droplet morphology and content. Strain BY4741 was grown aerobically for 24 h in YEPD media containing 0 or 10 μg mL−1 of terbinafine. Cells were stained with Nile Red and their images were taken by confocal microscopy and evaluated as described in Materials and methods. Bar: 10 μm. 8 View largeDownload slide Effect of terbinafine treatment on lipid droplet morphology and content. Strain BY4741 was grown aerobically for 24 h in YEPD media containing 0 or 10 μg mL−1 of terbinafine. Cells were stained with Nile Red and their images were taken by confocal microscopy and evaluated as described in Materials and methods. Bar: 10 μm. A slightly different pattern was observed in the erg1 mutants (Fig. 9). Control strain expressing the wild-type copy of ERG1 gene contained on average 3.4 relatively small single lipid droplets per cell. Although these ERG1 cells contained high amount of squalene (133 μg per 109 cells, see Fig. 6a), small lipid droplet aggregates were present in a minor fraction of cells (36%). Similarly, erg1L37P mutant that accumulated significantly higher amounts of squalene than BY4741 cells treated with 10 μg mL−1 terbinafine (450 vs. 319 μg per 109 cells, respectively) showed significantly lower level of Nile Red staining than terbinafine-treated cells. Particularly, the aggregated lipid droplets were present only in about one-half of these cells (57%, compared with 90% in cells treated with 10 μg mL−1 terbinafine). The strain erg1Q443UAG accumulating high amounts of squalene (over 600 μg per 109 cells) showed substantial increase in single LDs (average 5.7 per cell) and although LD aggregates were present in 74% cells, they were much smaller than the aggregates observed in cells treated with 10 μg mL−1 terbinafine. Our results thus indicate that although squalene accumulation stimulates formation of lipid droplets, other factors that squalene content may be involved in the phenomenon of lipid droplet aggregation as described by Ta et al. (2012). 9 View largeDownload slide Lipid droplet content and morphology in erg1 mutants with defective squalene epoxidase. Strains ERG1,erg1L37P, and erg1Q443UAG (derived from the strain KLN1 with disrupted chromosomal copy of ERG1 gene) were grown aerobically for 24 h in YPD media. Cells were stained with Nile Red and their images were taken by confocal microscopy and evaluated as described in Materials and methods. Bar: 10 μm. 9 View largeDownload slide Lipid droplet content and morphology in erg1 mutants with defective squalene epoxidase. Strains ERG1,erg1L37P, and erg1Q443UAG (derived from the strain KLN1 with disrupted chromosomal copy of ERG1 gene) were grown aerobically for 24 h in YPD media. Cells were stained with Nile Red and their images were taken by confocal microscopy and evaluated as described in Materials and methods. Bar: 10 μm. In conclusion, we demonstrated that squalene epoxidase encoded by the ERG1 gene is an excellent target for increasing squalene production in yeast S. cerevisiae. This was particularly evident in two erg1 mutants that showed high accumulation of squalene without compromised vitality. Inhibition of squalene epoxidase activity in anaerobically grown S. cerevisiae yielded only small accumulation of squalene that could be increased by ergosterol depletion of anaerobic cells. This increase in squalene levels in ergosterol-depleted anaerobic cells indicates the existence of feedback inhibition of presqualene pathway by ergosterol. Partial inhibition of squalene epoxidase by treatment with specific inhibitor terbinafine yielded high accumulation of squalene with saturation limit of about 700 μg per 109 cells. However, this high accumulation of squalene was associated with defective growth in terbinafine-treated cells. Similarly, high levels of squalene were observed in two mutants in the ERG1 gene encoding squalene epoxidase, erg1L37P and erg1Q443UAG. Both mutants were hypersensitive to terbinafine and showed reduced squalene epoxidase activity. These erg1 mutants appeared to be well adapted to low squalene epoxidase activity and high squalene levels as accumulation of squalene was not accompanied with disturbed growth. Squalene levels in erg1 mutants were further significantly increased by modification of cultivation temperature. Accumulation of squalene in cells with reduced squalene epoxidase activity affected also the development of lipid droplets as storage compartment for neutral lipids. These cells showed generally increased content of lipid droplets that was associated with lipid droplet aggregation under some conditions. Our results proved that genetic modification squalene epoxidase activity is an excellent tool that can be used for increasing squalene production in the yeast S. cerevisiae to levels interesting for biotechnological applications. Acknowledgement We thank F. Turnowski (Karl Franzens University Graz, Austria) for providing us yeast strains, K. Višacká (Comenius University Bratislava, Slovakia) for help with confocal microscopy, and B. Bilčík (Institute of Animal Biochemistry and Genetics SAS, Slovakia) for the statistical analysis. 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TI - Squalene epoxidase as a target for manipulation of squalene levels in the yeast Saccharomyces cerevisiae
JF - FEMS Yeast Research
DO - 10.1111/1567-1364.12107
DA - 2014-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/squalene-epoxidase-as-a-target-for-manipulation-of-squalene-levels-in-z4HIyQu60O
SP - 310
EP - 323
VL - 14
IS - 2
DP - DeepDyve
ER -