Molecular mechanisms of the coordination between astaxanthin and fatty acid biosynthesis in Haematococcus pluvialis (Chlorophyceae)

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1 The Plant Journal (2015) 81, doi: /tpj Molecular mechanisms of the coordination between astaxanthin and fatty acid biosynthesis in Haematococcus pluvialis (Chlorophyceae) Guanqun Chen 1,,, Baobei Wang 1,2,, Danxiang Han 1,, Milton Sommerfeld 1, Yinghua Lu 2, Feng Chen 3 and Qiang Hu 1, * 1 Laboratory for Algae Research and Biotechnology, College of Technology and Innovation, Arizona State University, 7001 E. Williams Field Road, Mesa, AZ 85212, USA, 2 Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, No. 422, Siming South Road, Xiamen, Fujian, , China and 3 Institute for Food & Bioresource Engineering, College of Engineering, Peking University, 298 Chengfu Road, Haidian, Beijing , China Received 13 June 2014; revised 21 September 2014; accepted 21 October 2014; published online 29 October *For correspondence ( huqiang@asu.edu). These authors contributed equally to this work. Present address: Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada. SUMMARY Astaxanthin, a red ketocarotenoid with strong antioxidant activity and high commercial value, possesses important physiological functions in astaxanthin-producing microalgae. The green microalga Haematococcus pluvialis accumulates up to 4% fatty acid-esterified astaxanthin (by dry weight), and is used as a model species for exploring astaxanthin biosynthesis in unicellular photosynthetic organisms. Although coordination of astaxanthin and fatty acid biosynthesis in a stoichiometric fashion was observed in H. pluvialis, the interaction mechanism is unclear. Here we dissected the molecular mechanism underlying coordination between the two pathways in H. pluvialis. Our results eliminated possible coordination of this inter-dependence at the transcriptional level, and showed that this interaction was feedback-coordinated at the metabolite level. In vivo and in vitro experiments indicated that astaxanthin esterification drove the formation and accumulation of astaxanthin. We further showed that both free astaxanthin biosynthesis and esterification occurred in the endoplasmic reticulum, and that certain diacylglycerol acyltransferases may be the candidate enzymes catalyzing astaxanthin esterification. A model of astaxanthin biosynthesis in H. pluvialis was subsequently proposed. These findings provide further insights into astaxanthin biosynthesis in H. pluvialis. Keywords: Haematococcus pluvialis, fatty acid biosynthesis, carotenoid, microalgae, gene transcription, metabolic coordination, acyl CoA diacylglycerol acyltransferase. INTRODUCTION The red ketocarotenoid astaxanthin (3,3 0 dihydroxy-b, b carotene-4,4 0 dione) has 13 conjugated double bonds alternating with single bonds, and thus possesses strong antioxidant ability, neutralizing free radicals and scavenging reactive oxygen species (Fassett and Coombes, 2012; Han et al., 2013). This secondary carotenoid has important applications in the nutraceutical, cosmetic, food and feed industries, with a total market value of over 240 million US dollars (Guerin et al., 2003; Li et al., 2011). Astaxanthin is synthesized by a limited number of plants and microorganisms, of which microalgae are the major resource. Some microalgal species have been reported to accumulate The Plant Journal 2014 John Wiley & Sons Ltd astaxanthin under environmental stress or adverse culture conditions such as high light (HL), nutrient deprivation, and high salinity (Han et al., 2013). Among these species, the unicellular green microalga Haematococcus pluvialis accumulates the largest amounts of astaxanthin (up to 4% of dry weight). The physiological functions of astaxanthin and the carotenogenesis pathway in this species have been investigated, and H. pluvialis is used as a commercial astaxanthin producer and a model species for exploring astaxanthin biosynthesis in photosynthetic organisms (Boussiba, 2000; Lemoine and Schoefs, 2010; Han et al., 2013). 95

2 96 Guanqun Chen et al. The biosynthesis of free astaxanthin in microalgae usually includes five steps: (i) formation of isopentenyl diphosphate, the so-called active isoprene, (ii) stepwise condensation of isoprene units to form the first carotenoid, phytoene, (iii) subsequential desaturation resulting in lycopene formation, (iv) cyclization reactions that generate b carotene, and (v) synthesis of astaxanthin by the introduction of oxygen. A number of genes involved in carotenogenesis have been cloned and partially characterized from H. pluvialis, including genes encoding phytoene synthase (PSY), phytoene desaturase, lycopene cyclase, b carotene ketolase and b carotene hydroxylase (CrtR b) (Han et al., 2013). Transcription of these genes increased under stress, resulting in accumulation of astaxanthin, which in turn plays an important role in protecting the alga from stress (Steinbrenner and Linden, 2003; Li et al., 2008, 2010). Like other photosynthetic organisms, b carotene is synthesized in the chloroplast of H. pluvialis. In contrast, the synthesis of astaxanthin from b carotene is associated with extraplastidic compartments at as yet unidentified subcellular locations (Lang, 1968; Grunewald and Hagen, 2001; Grunewald et al., 2001; Collins et al., 2011; Han et al., 2013). In H. pluvialis, approximately 95% of astaxanthin molecules are esterified with fatty acids and stored in triacylglycerol (TAG)-rich cytosolic lipid bodies (LBs) (Yuan and Chen, 2000; Holtin et al., 2009). Under stress conditions, H. pluvialis accumulates large amounts of fatty acids and glycerolipids (Saha et al., 2013). De novo fatty acid synthesis in green microalgae and higher plants occurs in the chloroplast, starting from conversion of acetyl CoA to malonyl CoA. In most plants and microalgae, this step is catalyzed by a multi-subunit acetyl CoA carboxylase that comprises four transcriptional autocoordinated subunits: biotin carboxylase (BC), biotin carboxyl carrier protein, a carboxyltransferase and b carboxyltransferase (Bao et al., 1997; Ke et al., 2000; James and Cronan, 2004). Malonyl CoA is subsequently used to drive condensation reactions to elongate the acyl groups to saturated 16:0- and 18:0-acyl carrier proteins. To produce an unsaturated fatty acid such as oleic acid, a double bond is introduced by a soluble enzyme stearoyl acyl carrier protein desaturase (SAD). Fatty acids are subsequently exported into the cytosol, where they are converted to acyl CoAs, and then transferred to the endoplasmic reticulum (ER) for glycerolipid assembly and other utilizations (Chapman and Ohlrogge, 2012; Li et al., 2013). Although recent biochemical evidence indicated that the synthesis of astaxanthin and fatty acids are stoichiometrically coordinated in H. pluvialis (Schoefs et al., 2001; Zhekisheva et al., 2002, 2005), the mechanism of the coordination between these two pathways is unclear. The main objective of this study was to dissect the molecular mechanism of the coordination between astaxanthin and fatty acid biosynthesis in H. pluvialis. Using specific inhibitors, the inter-dependence of these two pathways was investigated at both gene transcript and metabolite levels. Subsequently, the contribution of esterification to astaxanthin synthesis was determined through in vivo and in vitro experiments, the subcellular location of astaxanthin biosynthesis and esterification was identified, and candidate enzymes catalyzing the astaxanthin esterification reactions were proposed. These findings provide further insights into the biosynthesis and function of astaxanthin in H. pluvialis. RESULTS Confirmation of the coordination of fatty acid and astaxanthin biosynthesis in H. pluvialis In order to dissect the molecular mechanisms that underlie the functional coupling of astaxanthin and fatty acid biosynthesis in H. pluvialis, norflurazon and cerulenin, two specific inhibitors of b carotene and fatty acid synthesis, respectively (Schoefs et al., 2001; Zhekisheva et al., 2002), were applied to the wild-type (WT) and an astaxanthinover-producting mutant (MT) strain under HL to induce astaxanthin producing. A previous study indicated that the MT strain was identical to the WT with respect to cell morphology, pigment composition and growth kinetics during the early vegetative stage of the life cycle, but had a higher astaxanthin content and a lower rate of cell mortality under HL (Hu et al., 2008). The optimal concentrations of cerulenin and norflurazon were 7.2 and 0.1 lm, respectively. These concentrations had little negative effect on growth of the WT and MT strains under low light (LL), but noticeably reduced growth under HL (Table 1). Astaxanthin synthesis occurred under HL, and 3.92 and 7.65 mg g 1 of astaxanthin accumulated in the WT and MT, respectively, at 72 h (Figure 1a,b). When WT and MT cells were treated with norflurazon, the astaxanthin content was reduced by 42% and 36%, respectively. Likewise, addition of cerulenin to the WT and MT cultures resulted in 33% and 42% reductions in the astaxanthin content, respectively. A basal level of total fatty acids (TFA) of % by cell dry weight was measured in the WT and MT strains under LL, and remained more or less constant during 3 days of cultivation. Under HL, the total amount of fatty acids increased to % over the same period of time. Addition of norflurazon or cerulenin to the HLexposed cultures substantially reduced the fatty acid content in both strains (Figure 1c,d). The fatty acid compositions of the WT and MT strains under LL and HL and in the presence of norflurazon or cerulenin under HL are shown in Table 2. It was apparent that saturation of fatty acids increased when the H. pluvialis cultures were shifted from LL to HL. As such, C16:0, C18:1 and C18:2 increased, whereas C16:1, C16:2, C16:3, C16:4, C18:3 and C18:4 decreased considerably in the two strains under HL

3 Coordination of astaxanthin and fatty acid synthesis 97 Table 1 Cell number (x 10 4 ) of wild-type (WT) and mutant (MT) Haematococcus pluvialis grown under various conditions Time (days) Strain Conditions WT LL HL HN HC MT LL HL HN HC Algae were cultured under low light (LL) for 4 days, and then transferred to high light (HL) with or without inhibitors, or kept under LL as a control. HN, HL + norflurazon; HC, HL + cerulenin. Values are means standard deviation of triplicates. Figure 1. Accumulation of astaxanthin and total fatty acids (TFA) in Haematococcus pluvialis. (a) Astaxanthin content of the wild-type strain (WT); (b) astaxanthin content of the mutant (MT); (c) TFA content of the wild-type strain; (d) TFA content of the mutant. LL, low light; HN, HL + norflurazon; HC, HL + cerulenin. Values are means standard deviation of triplicates. (a) (b) (c) (d) compared to LL. However, norflurazon and cerulenin had little effect on the fatty acid composition of the cells under HL. In order to further assess the correlation of astaxanthin and fatty acid biosynthesis in H. pluvialis, a linear regression was established between stress-induced fatty acid content (TFA accumulated under HL minus that accumulated under LL) and stress-induced astaxanthin content (astaxanthin accumulated under HL minus that accumulated under LL). As shown in Figure S1, linear correlations (R 2 > 0.94) were obtained in WT and MT under HL, HL + norflurazon (HN) or HL + cerulenin (HC) culture conditions, which provided evidence of the coupling of astaxanthin and fatty acid biosynthesis in H. pluvialis under HL. The coupling of fatty acid synthesis and carotenogenesis was not coordinated at the transcript level Previous studies have indicated that astaxanthin biosynthesis in H. pluvialis is related to increased gene transcription (Steinbrenner and Linden, 2001, 2003; Li et al., 2008, 2010; Vidhyavathi et al., 2008). Here we hypothesized that astaxanthin and fatty acid synthesis pathways may be coordinated at the gene transcription level. To test this hypothesis, the transcripts of PSY and CrtR b, which

4 98 Guanqun Chen et al. Table 2 Fatty acid composition of Haematococcus pluvialis grown under low light (LL), after 24 h of induction under high light (HL), or under HL in the presence of norflurazon (HN) or cerulenin (HC) WT MT Fatty acids HL HN HC LL HL HN HC LL C14: C15: C16: C16: C16: C16: C16: C18: C18: C18: C18:3 (n 6) C18:3 (n 3) C18: C20: C20: C20:4 (n 6) C20:5 (n 3) TFA (%) All values are means standard derivation of triplicates. WT, wild-type; MT, mutant; HL, high light intensity; HN, HL + norflurazon; HC, HL + cerulenin; LL, low light. TFA (%) = total fatty acids (mg) per mg cell dry weight 9 100%. encode two key enzymes in astaxanthin biosynthesis (Steinbrenner and Linden, 2001; Li et al., 2010), were quantitatively measured by real-time RT PCR. In addition, BC and SAD, two key genes in de novo fatty acid synthesis (Chapman and Ohlrogge, 2012), were cloned from H. pluvialis. The methods are described in Appendix S1, and the results are shown in Figures S2 and S3. These two genes were used as gene markers to monitor the transcript levels of genes in the fatty acid synthesis pathway by real-time RT PCR. The transcripts of PSY and CrtR b increased in both strains under HL, with maximum expression achieved after 24 h (Figure 2). Norflurazon significantly stimulated their expression: at 24 h, the transcript levels of PSY and CrtR b, respectively, were 2.9 and 6.2 times higher in WT (Figure 2a, c) and 2.0 and 1.8 times higher in MT (Figure 2b,d) compared with those under HL. In contrast, cerulenin only had slight effects on the expression of both genes in WT and MT. The expression of PSY was slightly lower under HC (Figure 2a,c) than under HL, whereas that of CrtR b was slightly higher at 24 h (Figure 2b,d). Similarly, the expression of BC and SAD was enhanced under HL in both strains, with the highest expression levels 24 h after induction by cerulenin (Figure 2e h). In summary, the carotenoid biosynthesis inhibitor norflurazon stimulated the transcription of carotenogenesis genes (PSY and CrtR b) but not that of BC and SAD, which indicated that inhibition of fatty acid biosynthesis by norflurazon is not due to decreased transcripts of the genes encoding fatty acid biosynthetic enzymes. Likewise, addition of cerulenin caused severe inhibition (60 70%) of astaxanthin synthesis in H. pluvialis cells, but did not remarkably alter the expression of PSY and CrtR b. Taken together, these results indicate that stress-stimulated astaxanthin and fatty acid biosynthesis were not coordinated at the gene transcription level. The coordination of fatty acid and astaxanthin synthesis occurs at the metabolite level As the interaction of fatty acid and astaxanthin synthesis did not occur at the transcript level (Figure 2), this functional coupling may be a feedback interaction at the metabolite level, i.e. depletion of fatty acids in cerelenintreated cells may result in an excess of free astaxanthin, which in turn may inhibit the carotenogenesis enzymes and eventually result in abolishment of de novo astaxanthin synthesis. As 95% of the astaxanthin accumulated under HL stress was esterified with fatty acids (Holtin et al., 2009), if the pathway coordination is truly coordinated at the metabolite level, esterification of free astaxanthin may relieve the feedback inhibition of carotenogenesis by the free astaxanthin. To test this hypothesis, exogenous fatty acids were individually added to the H. pluvialis cultured under HC. As MT always showed similar trends to the WT with regard to coupling of fatty acid and astaxanthin biosynthesis, only the WT was used in the following experiments. As shown in Figure 3, astaxanthin biosynthesis was recovered in a concentration-dependent manner by

5 Coordination of astaxanthin and fatty acid synthesis 99 Figure 2. Transcripts of four genes in the astaxanthin and fatty acid biosynthesis pathways in Haematococcus pluvialis. (a) PSY content in the wild-type strain (WT); (b) PSY content in the mutant strain (MT); (c) CrtR b content in the wild-type strain; (d) CrtR b content in the mutant strain; (e) BC content in the wild-type strain; (f) BC content in the mutant strain; (g) SAD content in the wild-type strain; (h) SAD content in the mutant strain. Relative amounts were calculated and normalized with respect to the transcript levels of the 18S gene. LL, low light; HL, high light; HN, high light lm norflurazon; HC, high light lm cerulenin. Values are means standard deviation of triplicates. (a) (c) (b) (d) (e) (f) (g) (h) providing palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2) or a linolenic acid (C18:3 (n 3)), with recovery rates of 88.2%, 70.5%, 52.9% and 54.4%, respectively. Interestingly, these exogenous fatty acids are the major fatty acid species in H. pluvialis cells (Table 2), and the most common acyl molecules in astaxanthin esters (Holtin et al., 2009). In contrast, providing palmitoleic acid (C16:1) and stearic acid (C18:0), minor fatty acid species of H. pluvialis cells (Table 2) and astaxanthin esters (Holtin et al., 2009), did not restore the abolished astaxanthin biosynthesis (Figure 3). Taken together, stimulating esterification by providing the major fatty acid species of H. pluvialis restores astaxanthin synthesis in cells in which de novo fatty acid synthesis was inhibited, confirming that the coupling of fatty acid and astaxanthin biosynthesis pathways is feedback-coordinated at the metabolite level. To further test whether the pathway coordination occurred at the metabolite level, the in vivo effect of cerulenin on production of free astaxanthin was investigated. As shown in Figure 4, H. pluvialis grown under HC accumulated a greater amount of free astaxanthin compared to that under HL. Taken together, in vivo inhibition of fatty acid accumulation by cerulenin resulted in an increase of free astaxanthin; stimulating esterification by providing the major fatty acid species of H. pluvialis restores astaxanthin synthesis in the cells in which de novo fatty acid synthesis is inhibited. Therefore, the coupling of fatty acid and astaxanthin biosynthesis pathways is feedback-coordinated at the metabolite level. Biosynthesis of free astaxanthin and astaxanthin esters both occurred at the ER The feeding experiments indicated that synthesis of astaxanthin and fatty acids were coordinated at the metabolite level. Although it is known that fatty acid biosynthesis occurs in chloroplasts, the subcellular localization of

6 100 Guanqun Chen et al. (a) (b) Figure 3. Effect of exogenous fatty acid on the synthesis of astaxanthin inhibited by cerulenin in Haematococcus pluvialis. Short lines above the x axis indicate the positions of bars when the values were zero (C18:0; c18:2 and C18:3 (n 3)). Values are means standard deviation of triplicates. (c) Figure 4. Effect of cerulenin on the content of free astaxanthin in wild-type Haematococcus pluvialis. HL, high light; HC, high light lm cerulenin. Values are means standard deviation of triplicates. astaxanthin synthesis and esterification remained unknown. We separated H. pluvialis subcellular fractions by continuous sucrose flotation gradient ultra-centrifugation. As indicated by immunoblot assay with organellespecific protein markers, the ER membranes were enriched in fractions A E, mitochondria were enriched in fraction H only, and thylakoid membranes were present in all fractions (Figure 5a). These fractions were subsequently used in in vitro enzymatic assays to identify the subcellular location of astaxanthin synthesis and esterification. When exogenous b carotene was used as the substrate, the enzymatic activities for astaxanthin formation were found only in Figure 5. Subcellular locations of astaxanthin synthesis and esterification. (a) Immunoblot analysis of subcellular fractions. BIP, AOX1 and PsbA were used as markers for the ER, mitochondria and chloroplast, respectively. (b) Enzymatic activity resulting in formation of total astaxanthin. (c) Net changes of free astaxanthin and astaxanthin esters. Subcellular components were separated by sucrose gradient centrifugation (sucrose concentration 0 60%) and labeled as (A) 10% sucrose, (B) 15%, (C) 20%, (D) 25%, (E) 30%, (F) 35%, (G) 45%, and (H) > 60% sucrose (pellets). Total membrane protein (T) was used as a control. Values are means standard deviation of triplicates. fractions A D in which the ER membranes were enriched (Figure 5b). Although fractions A D were contaminated by thylakoid membranes (Figure 5a), an association of astaxanthin synthetic enzymes with the thylakoid membranes may be excluded, because fractions containing thylakoid membranes without ER membranes did not show any

7 Coordination of astaxanthin and fatty acid synthesis 101 detectable astaxanthin synthesis and esterification. Therefore, these results indicate that both astaxanthin synthesis and esterification occur at the ER. Furthermore, astaxanthin esters were always the predominant product, whereas only a small amount of free astaxanthin was detected in the reactions (Figure 5c), consistent with the fact that astaxanthin esters were the predominant species of total astaxanthin in H. pluvialis. (a) The esterification step is essential for astaxanthin formation in H. pluvialis To further test the hypothesis that the esterification process plays a key role in the coordination of astaxanthin and fatty acid synthesis at the metabolic level, the effect of exogenous fatty acids on astaxanthin synthesis was studied by an in vitro enzymatic assay to eliminate possible effects of other factors from living cells. As synthesis of free astaxanthin and astaxanthin esters both occurred at the ER (Figure 5), only ER membranes were used in the enzyme assay. Oleic acid (C18:1) and palmitic acid (C16:0) are two substrates that may be preferred for astaxanthin esterification (Figure 3), and thus were used as substrates in the in vitro assays. As shown in Figure 6(a), the enzymatic activity for astaxanthin synthesis increased with addition of oleic acid. The maximum enzymatic activity was detected in the presence of 2.08 C18:1, and was three times that of the control without any exogenous fatty acids. Similarly, the enzymatic activity increased with addition of palmitic acid, and the maximum enzymatic activity was 2.5 times higher than that of the control (Figure 6a). Interestingly, addition of exogenous fatty acids, regardless of concentration and type, had no significant influence on the amount of free astaxanthin, whereas astaxanthin esters were substantially increased (Figure 6b,c). As inhibition of in vivo astaxanthin synthesis in H. pluvialis under HC was reversed by exogenous fatty acids (Figure 3), the contribution of esterification to this recovery was analyzed in an in vitro assay. As shown in Table 3, without exogenous fatty acids, the enzymatic activity of the crude ER membranes isolated from cerulenin-treated cells (HC) was only 59% of that under HL, in line with the reduced cellular astaxanthin content (Figure 1a). With supplied oleic acid (2.08 mm) or palmitic acid (2.08 mm), the enzymatic activity of the crude ER membranes isolated from the cerulenin-treated cells increased by and 2.93-fold, respectively, comparable to that for the control (H. pluvialis cultured under HL with the exogenous fatty acid added at the same concentration). These in vitro experiments suggested that the abolished astaxanthin synthesis under HC conditions was caused by inhibition of astaxanthin esterification, due to depletion of fatty acids. Taken together, the esterification process was shown to be an essential step to facilitate astaxanthin formation in H. pluvialis. (b) (c) Figure 6. Effect of exogenous fatty acids on astaxanthin formation in the in vitro assay. (a) Enzymatic activity leading to the formation of total astaxanthin; (b) net change in free astaxanthin; (c) net change in astaxanthin esters. Crude ER membranes were extracted from H. pluvialis grown under high light (HL). Values are means standard deviation of triplicates. Diacylglycerol acyltransferases (DGATs) may catalyze the esterification of astaxanthin The molecular identity of the enzymes responsible for esterification of carotenoids (including astaxanthin) in microalgae remains unknown. In mammals, retinol, a degradation product of carotenoid, may be esterified to

8 102 Guanqun Chen et al. Table 3 Effect of exogenous fatty acids on the in vitro activities (lg mg protein 1 h 1 ) of astaxanthin-producing enzymes (a) No fatty acid C18:1 C16:0 HL HC HL, high light; HC, HL + cerulenin. Values are means standard deviation of triplicates. form retinyl ester (Shih et al., 2009). This step is catalyzed by a membrane-binding diacylglycerol acyltransferase 1 (DGAT1), which has acyl CoA:retinol acyltransferase activity (Yen et al., 2005; Burri and Neidlinger, 2007; Shih et al., 2009). Murine DGAT1 also had other acyltransferase activities, including acyl CoA monoacylglycerol acyltransferase and wax synthase activities for synthesis of diacylglycerol and wax esters, respectively (Yen et al., 2005). Similarly, DGAT2, another membrane-binding DGAT in mammals, plants and microorganisms, also utilizes a broad range of acyl acceptors (Liu et al., 2012). For instance, Thraustochytrium aureum DGAT2 uses fatty alcohols as acceptors of acyl CoA substrates in a wax synthase-type reaction (Zhang et al., 2013). Recently, two phytyl ester synthases were identified in Arabidopsis; they have both DGAT1 and DGAT2 activities and are involved in fatty acid phytyl ester synthesis (Lippold et al., 2012). In tomato (Solanum lycopersicum), PALE YELLOW PETAL 1 (PYP1), a homolog of Arabidopsis phytyl ester synthase, encodes a carotenoidmodifying protein that plays a vital role in production of xanthophyll esters in tomato anthers and petals (Ariizumi et al., 2014). To determine whether DGATs mediate the esterification of astaxanthin in H. pluvialis, xanthohumol, an inhibitor of both DGAT1 and DGAT2 (Inokoshi et al., 2009), and A922500, a DGAT1-specific inhibitor (Zhao et al., 2008; King et al., 2009), were introduced into the in vitro enzymatic assays, respectively. When xanthohumol was added at concentrations ranging from 7.69 to mm), the enzymatic activity for formation of total astaxanthin (both free astaxanthin and its esters) decreased concomitantly with an increase in free astaxanthin and a decrease in astaxanthin esters (Figure 7). When the concentration of xanthohumol was increased to 76.9 mm, the synthesis of astaxanthin esters was completely inhibited (Figure 7c). When lm A was added to the reaction system, the enzymatic activity decreased by 55% (Figure 7a), accompanied by accumulation of free astaxanthin and a decrease in astaxanthin esters (Figure 7b,c). However, higher concentrations of A did not result in more severe inhibition of enzymatic activity (Figure 7a). These results indicate that both DGAT1 and DGAT2 are probably responsible for esterification of astaxanthin at the ER in H. pluvialis. DISCUSSION (b) (c) Figure 7. Effect of DGAT inhibitors on astaxanthin synthesis in the in vitro assay. (a) Enzymatic activity leading to the formation of total astaxanthin; (b) net change in free astaxanthin; (c) net change in astaxanthin esters. One fold represents the lowest concentration of inhibitors: 7.69 mm (5 lmol incubation 1 ) for the DGAT1 and DGAT2 inhibitor xanthohumol and lm (10 nmol incubation 1 ) for the DGAT1-specific inhibitor A922500, respectively. Incubation without any DGAT inhibitor was used as a control. Values are means standard deviation of triplicates. Although the dependence of astaxanthin accumulation on de novo fatty acid biosynthesis has been observed over the past 15 years (Mendoza et al., 1999; Schoefs et al., 2001; Zhekisheva et al., 2002, 2005), the molecular mechanism underpinning coupling of these two pathways is

9 Coordination of astaxanthin and fatty acid synthesis 103 unknown. In this study, we confirmed coordination of the HL-induced astaxanthin and fatty acid biosynthesis using suitable inhibitors, eliminated the possibility of interdependence at the transcriptional level, and showed that this interaction comprised feedback coordination at the metabolite level. We further showed that astaxanthin esterification plays an essential role in this interaction, that both free astaxanthin biosynthesis and esterification were associated with the ER, and that DGATs may be the enzymes catalyzing the process of astaxanthin esterification. Many metabolic pathways, such as de novo biosynthesis of the primary metabolite fatty acids in plants and production of some secondary metabolite antibiotics in Streptomyces species, are coordinated by feedback inhibition of the end-products (Shintani and Ohlrogge, 1995; Liu et al., 2013). End-product inhibition of carotenogenesis was also reported in other microorganisms, including Phycomyces species and the yeast Phaffia rhodozyma (Bejarano et al., 1988; Schroeder and Johnson, 1995). In H. pluvialis, astaxanthin is the predominant carotenoid, and most of the astaxanthin combines with fatty acids to generate the end-product astaxanthin ester (Holtin et al., 2009). Feedback coordination explains the severe decrease in fatty acid biosynthesis during inhibition of carotenogenesis, because when astaxanthin biosynthesis is blocked by an inhibitor under HL stress, a considerable proportion of the stress-induced fatty acids accumulated to excess in the cell. Likewise, when fatty acid biosynthesis was inhibited by cerulenin under HL, the final product of carotenogenesis in H. pluvialis is free astaxanthin, which, in excess, inhibits carotenogenesis by feedback coordination (Figures 1 and 2, and Figure S1). Our in vivo and in vitro experiments further indicated that astaxanthin esterification may stimulate enzymatic reactions toward formation of astaxanthin from b carotene by relieving the end-product feedback inhibition of carotenogenesis. The final concentration of free astaxanthin in the in vitro assays was substantially lower than that of astaxanthin esters, suggesting that free astaxanthin is an intermediate product (Figure 6). Upon formation of astaxanthin esters at the ER membranes, they were sequestrated into cytoplasmic LBs (Santos and Mesquita, 1984), where the products are spatially segregated from the enzymes involved in astaxanthin biosynthesis in the chloroplast (from acetyl CoA to b carotene) and the ER (from b carotene to astaxanthin esters). This step may further accelerate free astaxanthin synthesis and astaxanthin esterification. Taken together, astaxanthin esterification coordinates stress-induced carotenogenesis and fatty acid biosynthesis at the metabolic level, and is an essential process for augmenting astaxanthin accumulation in H. pluvialis under stress. It should be noted that astaxanthin accumulation may also be regulated by TAG accumulation. LBs are the location for astaxanthin accumulation, in which TAG serves as the solvent for astaxanthin (Zhekisheva et al., 2005; Collins et al., 2011). Therefore, TAG is required for accumulation of astaxanthin ester in LBs (Zhekisheva et al., 2005). In the present study, astaxanthin biosynthesis under HC was reduced by 65% at 12 h compared to HL, whereas the TFA content was only reduced by 4.2% (Figure 1a,c). To minimize the influence of TAG on astaxanthin accumulation, this time point was selected for the fatty acid feeding experiment. The TFA content of WT H. pluvialis under HC was reduced by 35.5% and 33.7% at 48 and 72 h, respectively, compared to that under HL only (Figure 1c). Therefore, TAG accumulation and LB generation are inhibited by cerulenin under high light, which at least partially contributes to the decrease in astaxanthin production in H. pluvialis in this study. Although astaxanthin has important physiological functions in H. pluvialis, the subcellular localization of astaxanthin biosynthesis remains controversial. In this study, we demonstrated that synthesis of free astaxanthin and astaxanthin esters from b carotene is associated with the ER (Figure 5). Previous studies have suggested that b carotene is exclusively synthesized in the chloroplast and then exported for synthesis of astaxanthin (Cunningham and Gantt, 1998; Grunewald and Hagen, 2001; Grunewald et al., 2001). A recent Raman microscopic analysis also indicated that b carotene is located in both the chloroplast and cytosolic LBs in H. pluvialis cells, whereas astaxanthin is mostly concentrated in cytosolic LBs (Collins et al., 2011). Thus, in living H. pluvialis cells, ER-bound enzymes may use the b carotene exported from the chloroplast as the substrate for astaxanthin biosynthesis and esterification as shown in our immunoblot results and in vitro assay (Figure 5). As ER is the major subcellular location of TAG biosynthesis in higher plants and probably microalgae, astaxanthin esters and the newly synthesized TAG are presumed to be synthesized in the ER cisternae, as indicated by electron microscopic examinations (Lang, 1968; Santos and Mesquita, 1984). When the quantities of astaxanthin and TAG exceed a certain threshold, this part of the ER may give rise to formation of tiny small astaxanthin-containing LBs, which then coalesce to form large LBs (Yen et al., 2008). The biosynthesis of astaxanthin in H. pluvialis from phytoene to b carotene is initiated within the chloroplast, and the b carotene is subsequently transferred to the ER and converted to astaxanthin. Our results strongly support the hypothesis that astaxanthin biosynthesis is feedback-inhibited by its end-product free astaxanthin, but the exact mechanism of how ER-localized astaxanthin feedback inhibits carotenogenesis-related enzymes within the chloroplast is unknown. Similar feedback regulation of enzymes in different cell organelles has also been reported for fatty acid biosynthesis in higher plants, which is

10 104 Guanqun Chen et al. initiated within the plastid up to the level of oleic acid (18:1). Oleic acid is subsequently transferred to the ER for further elongation and desaturation. The first step of de novo fatty acid biosynthesis in the plastid is catalyzed by acetyl CoA carboxylase. It has recently been reported that the activity of acetyl CoA carboxylase, and thus fatty acid synthesis in the plastid, is feedback-inhibited by hydroxy fatty acids produced on the ER in transgenic Arabidopsis, but the exact mechanism was not identified (Bates et al., 2014). With regard to carotenogenesis in H. pluvialis, PSY catalyzes the synthesis of phytoene, the first and rate-limiting step of carotenoid biosynthesis. However, the level of psy transcripts under HC is the same as that under HL (Figure 2a,b). Therefore, it is unlikely that the feedback inhibition is due to transcriptional regulation of psy. Although the inhibition of astaxanthin accumulation by cerulenin correlated with the increase in free astaxanthin (Figure 4), the level of b carotene under HC varied between 0.23 to 0.36 mg g 1 from 12 to 72 h, which was not significantly different to that under HL at the same time points. However, in a previous study, we found that b carotene is relocated between the cytosol and the chloroplast via an unknown cellular machinery (Collins et al., 2011). Therefore, although the overall content of b carotene in the cell does not change in response to cerulenin treatment, the accumulation of free astaxanthin may signal to the cell to relocate b carotene from the cytosol to the chloroplast and subsequently inhibit the enzymes involved in carotenogenesis within this organelle. If this is the case, trafficking of b carotene across the chloroplast envelope membranes would contribute to this process. In addition, there are also other possible mechanisms. Further identification of the metabolic signals that control various carotenogenesisrelated enzymes within the chloroplast, and thus carotene synthesis, in response to free astaxanthin accumulation may extend our understanding of xanthophyll biosynthesis in microalgae and generate alternative bioengineering strategies for increasing total astaxanthin production. Notably, our results do not rule out the possibility that astaxanthin biosynthesis and esterification also occur in other cellular fractions in addition to the ER. For instance, a previous study reported that H. pluvialis b carotene oxygenase, which catalyzed a committed step for astaxanthin production, was also located in chloroplast membranes and cytoplasmic LBs as suggested by immunoblotting analysis (Grunewald et al., 2001). When chloroplast membranes and LBs were compared, b carotene oxygenase activity was only detected in LBs (Grunewald et al., 2001). LBs are considered to originate from the ER in eukaryotes, which is supported by several functional studies and evidence that numerous ER-resident proteins, including TAG synthesis enzymes and LB structural proteins, are present in LBs (Murphy, 2001; Walther and Farese, 2009; Wilfling et al., 2013). The ER derived model of LB biogenesis may explain the detection of the enzymatic activities for astaxanthin synthesis in LBs of H. pluvialis (Grunewald and Hagen, 2001). In large mature LBs, the astaxanthin biosynthetic enzymes may continue to convert b carotene to astaxanthin, which is subsequently esterified to astaxanthin esters. The biochemical and subcellular localization analyses suggest that the enzymes catalyzing esterification of astaxanthin may belong to the DGAT families. The two membrane-binding DGATs, DGAT1 and DGAT2, are enzymes with multiple functions that are involved in a variety of acyltransferase activities. For instance, DGAT1 may transfer the acyl group from acyl CoA to retinol, a degradation product of carotenoids, to produce retinyl esters (Yen et al., 2005), and DGAT2 may use a fatty alcohol as CoA acceptor to produce a wax ester (Biester et al., 2012). In addition, tomato PEP1, which may have both DGAT1 and DGAT2 activities, catalyzes the esterification of xanthophyll (Ariizumi et al., 2014). In our study, the DGAT1-specific inhibitor A inhibited the synthesis of astaxanthin by 55% compared to the control, whereas the DGAT1 and DGAT2 inhibitor xanthohumol inhibited the formation of astaxanthin esters by 100% (Figure 7). The inhibition of astaxanthin ester synthesis led to accumulation of free astaxanthin in the enzymatic reaction system, which indicated that other candidates may also catalyze the esterification step, although their contribution may be much smaller than the candidates with DGAT activities. In addition, among the subcellular fractions, the ER membranes exhibited the highest activities of astaxanthin esterification (Figure 5), which is consistent with the subcellular localization of DGAT1 and DGAT2 (Liu et al., 2012). To further confirm the contribution of DGATs to astaxanthin esterification, molecular cloning of DGAT genes (including the DGAT1, DGAT2, PEP1 and unknown acyltransferases with DGAT activities), and characterization of the corresponding proteins, is necessary. In general, microalgae have multiple DGAT homologs (Chen and Smith, 2012). For instance, Chlamydomonas has one DGAT1 and five DGAT2 enzymes, and Nannochloropsis has two DGAT1 and 11 DGAT2 enzymes (Chen and Smith, 2012; Li et al., 2014). Based on the findings in this study, the model of astaxanthin biosynthesis proposed by Han et al. (2013) has been updated (Figure 8). This biological process involves multiple pathways, including carotenogenesis (from isopentenyl pyrophosphate to free astaxanthin), de novo fatty acid biosynthesis and esterification of astaxanthin. b carotene, the precursor for synthesis of astaxanthin, is presumed to be exported from the chloroplast to the ER, where it is converted to astaxanthin and then astaxanthin esters. DGATs are proposed as the candidate enzymes transferring acyl groups from acyl CoA to free astaxanthin to produce astaxanthin esters, which plays a key role in

11 Coordination of astaxanthin and fatty acid synthesis 105 coordination of carotenogenesis and de novo fatty acid biosynthesis at the metabolic level. In addition, the steps from b carotene to astaxanthin esters may also occur in LBs, as numerous ER-resident proteins, including astaxanthin biosynthetic enzymes, may be recruited to LBs during the biogenesis of LBs, and continue to contribute to the production of astaxanthin. EXPERIMENTAL PROCEDURES Strains and culture conditions Haematococcus pluvialis NIES144 (wild-type, WT) was obtained from the National Institute for Environmental Studies (Tsukuba, Japan). The mutant strain 2877 (MT), with high astaxanthin content, was obtained by N methyl-n nitro-n nitrosoguanidine mutagenesis (Hu et al., 2008). Algal species were grown in 250 ml Erlenmeyer flasks containing 100 ml basal growth medium at 22 C and low light (LL, 20 lmol photons m 2 sec 1 ) (Li et al., 2010). To study astaxanthin and fatty acid biosynthesis, exponentially growing cells (cell density of approximately cells ml 1 ) were exposed to continuous illumination of 150 lmol photons m 2 sec 1 (HL), HL + norflurazon (HN), or HL + cerulenin (HC) (Schoefs et al., 2001; Zhekisheva et al., 2002). Cultures under LL served as a control. Live cells in the cultures were counted using a hemocytometer under an Olympus BH 2 light microscope (Tokyo, Japan). Quantification of astaxanthin and fatty acids Free astaxanthin and astaxanthin esters were quantified by HPLC (Li et al., 2010). Fatty acid contents were determined by gas chromatography (GC) (Chen et al., 2007). Appendix S1 provides a detailed description of these methods. RNA isolation, cdna synthesis, and real-time RT PCR RNA isolation, cdna synthesis, and real-time RT-PCR were performed as described previously (Li et al., 2010). Primers are listed in Table S1. Appendix S1 provides a detailed description of these methods. Restoration of astaxanthin synthesis by feeding exogenous fatty acids Fatty acids (Sigma-Aldrich, were individually dissolved in 100 mm Tris/HCl buffer (ph 8.0) containing 0.1 g ml 1 bovine serum albumin, and added to WT cells grown under low light (LL) for 4 days. Cells were then induced under HL for 12 h to accumulate astaxanthin. Cultures under HL with neither exogenous fatty acids nor cerulenin served as positive controls, and cultures without fatty acids but with cerulenin (HC) served as a negative control. The recovery rate was calculated using the following formula: Recovery rate ¼ Astaxanthin ðsampleþ Astaxanthin ðhcþ 100 Astaxanthin ðhlþ Astaxanthin ðhcþ Preparation of crude ER membranes and subcellular fractions Crude ER membranes were prepared by a modified Michael s method (Lord, 1987). Briefly, H. pluvialis cells were collected by centrifugation at 3000 g for 5 min. The pellets were resuspended in ice-cold breaking buffer containing 10 mm Tris/HCl (ph 7.5), Figure 8. Model of astaxanthin biosynthesis and esterification in Haematococcus pluvialis. ACCase, acetyl CoA carboxylase; BKT, b carotene ketolase; CrtR b, b carotene 3,3 0 hydroxylase; DGAT, diacylglycerol acyltransferase; FAS, fatty acid synthase; FA, fatty acid; GPP, geranylgeranyl pryophosphate; IPP, isopentenyl diphosphate; LCY b, lycopene cyclase; PDS phytoene desaturase; PSY, phytoene synthase; SAD, stearoyl acyl carrier protein desaturase; TAG, triacylglycerol; ZDS, f carotene desaturase. 5 mm EDTA and 0.1% bovine serum albumin. The suspension was passed through a French press at an internal pressure of kpa. The homogenates were centrifuged at 3000 g for 5 min at 4 C to remove large cell debris and unbroken cells. The supernatants were used for preparation of ER membranes and other subcellular fractions. To prepare crude ER membranes, the supernatant was transferred to a fresh tube, and centrifuged at g for 12 min at 4 C to remove mitochondria. The resulting supernatant was layered onto a discontinuous sucrose gradient (18% and 25% sucrose in breaking buffer), and fractionated by ultra-centrifugation ( g, 60 min). The interface between breaking buffer and 18% sucrose, and the 18% sucrose layer, were collected, diluted with six volumes of breaking buffer, and centrifuged again at g for 60 min. The pellets were collected and used as crude ER membranes for all in vitro enzymatic assays except the experiment for identifying the subcellular location of synthesis of astaxanthin and astaxanthin esters. To prepare subcellular fractions, the supernatant from the 3000 g centrifugation after French press treatment was loaded onto the top of a continuous sucrose gradient (10 60% sucrose in breaking buffer), and centrifuged at g for 3 h. The membranes were separated into eight sucrose gradients: (A) 10%, (B) 15%, (C) 20%, (D) 25%, (E) 30%, (F) 35%, (G) 45%, and (H) > 60% (pellets). The 2 ml aliquots were collected from each layer of the sucrose gradients, diluted with six

12 106 Guanqun Chen et al. volumes of breaking buffer, and then centrifuged at g for 1 h to collect pellets containing crude membranes. The pellets were resuspended in suspension buffer (50 mm Tris/HCl buffer, ph 7.5, 0.33 M sorbitol). Enzymatic assay The enzymatic activity for astaxanthin synthesis was performed using Fraser s method with optimization (Fraser et al., 1997, 1998). The optimization process is described in Appendix S1. Briefly, 300 ll of the reaction solution (1 mm dithiothreitol, 0.5 mm FeSO 4, 5 mm ascorbic acid, 0.5 mm 2 oxoglutarate, 0.1% w/v deoxycholate) were mixed with 300 ll of crude ER membranes or other subcellular fractions (containing 700 lg protein), 2 mm NADPH and 2 mm ATP, and then equilibrated at 30 C for 5 min. Subsequently, 50 ll b carotene liposome suspension containing 100 lg b carotene was added. Reactions were performed at 250 rpm and 30 C in 10 ml round-bottomed glass tubes with a supply of oxygen (Figure S4). Reactions were terminated by adding 1.8 ml methanol. Reactions that were immediately terminated at 0 min and reactions containing water instead of crude proteins were used as negative controls. To avoid oxidation, all samples containing carotenoids were handled in dim light, and covered with aluminum foil during the reaction. The terminated reactions were first extracted with three volumes of 10% v/v diethyl ether in petroleum ether, and then with two volumes of chloroform. The organic extracts were pooled, dried under nitrogen gas, and dissolved in dichloromethane and methanol (25:75 v/v) for HPLC analysis. The enzymatic activity for astaxanthin formation was defined as production of total astaxanthin (both free astaxanthin and its esters) per mg protein per hour. To inhibit DGAT activity, xanthohumol (Enzo Life Sciences, and A ((1R,2R)-2-[[40-[[phenylamino) carbonyl] amino] [1,10- biphenyl]-4-yl] carbonyl] cyclopentanecarb- oxylic acid) (EMD Millipore, were added to the reaction mixture, respectively. Immunoblot analysis The subcellular fraction suspensions (100 ll) were centrifuged at g for 10 min at 4 C to collect the membranes, which were resuspended in a mixture of 60 ll buffer A (0.1 M dithiothreitol and 0.1 M Na 2 CO 3 ) and 40 ll buffer B (30% sucrose and 5% SDS), and vortexed at 3000 rpm for 30 min. Insoluble proteins were removed by centrifugation at g for 10 min. The protein concentration was measured using a CB X protein assay kit (G Biosciences, Proteins were separated by SDS PAGE on a 4 20% pre-cast polyacrylamide gel (Bio Rad, and transferred to a nitrocellulose membrane. Luminal binding protein (BIP), the C terminus of the D1 protein of photosystem II (PsbA) and alternative oxidase 1 (AOX1) were used as probes for the ER, thylakoid membrane and mitochondria membrane, respectively. Total membrane protein was used as a control. Antigen antibody complexes were visualized using an enhanced chemiluminescence substrate detection kit (Thermo, ACKNOWLEDGEMENTS We sincerely thank King-Wai Fan and Jiangxin Wang for technical assistance. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. Correlation of stress-induced fatty acid and astaxanthin biosynthesis. Figure S2. Alignment of microalgal and plant biotin carboxylase. Figure S3. Alignment of microalgal and plant stearoyl acyl carrier protein desaturase. Figure S4. Optimization of the in vitro enzymatic assay. Table S1. Primers used in this study. Appendix S1. Additional experimental procedures. 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