Isoprenoids (terpenoids) represent one of the largest groups of natural products. They are found in all living organisms and comprise both primary and secondary metabolites (Rodríguez-Concepción & Boronat, 2002; Kirby & Keasling, 2009). Sterols, phytohormones as gibberellins or abscisic acid, phytol (e.g. as a side chain in chlorophyll a), prenylated quinones and many carotenoids are examples of primary metabolites that are isoprenoids or contain isoprenoid moieties. In higher plants, the majority of isoprenoids have secondary functions in the protection against pathogens or herbivores, or in the competition with other plants (Rodríguez-Concepción & Boronat, 2002). Isoprenoid biosynthesis occurs in three stages: (1) the biosynthesis of C5 precursors, (2) the formation of polyprenyl pyrophosphates and (3) their further processing.
In macroalgae, isoprenoids constitute the majority of natural products (Maschek & Baker, 2007). Here, the most important subclasses are sesquiterpenes (C15) and diterpenes (C20). In addition, triterpenes (C30) are found in the form of polyethers such as thyrsiferol, enshuol or teurilene (Fernández et al., 2000). In multicellular red algae, such as the prolific genus Laurencia, many isoprenoid natural products are halogenated. Algal polyether toxins can be formed by two different biosynthetic routes, originating either from triterpenes or from polyketides (Vilotijevic & Jamison, 2009; see also section on complex polyketides). Isoprenoid secondary metabolites are less common in microalgae than macroalgae (Maschek & Baker, 2007).
Biosynthesis of activated isoprenoid precursors
In this first stage of isoprenoid biosynthesis, the activated C5 precursor isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP) can be formed via the classical mevalonate pathway or the alternative methylerythrol phosphate (MEP) pathway (Bouvier et al., 2005). In higher plants, cytosol and plastid play complementary roles in isoprenoid biosynthesis: For example, the mevalonate pathway in the cytosol is involved in the formation of sterols and sesquiterpenes, while the MEP pathway in the plastid leads to phytol and carotenoids (Fig. 2a; Lichtenthaler, 1999). Coordinate regulation of the two pathways and exchange of intermediates between the compartments is poorly understood and seems to depend on the conditions and tissue (Bouvier et al., 2005). Experiments with inhibitors specific for mevalonate or MEP enzymes and the characterization of pathway mutants indicate that limited amounts of isoprenoid precursors can be exchanged between plastid and cytosol (Rodríguez-Concepción, 2010). There is biochemical evidence for transport of IPP, geranyl pyrophosphate and farnesyl pyrophosphate across plastid envelope membranes (Bick & Lange, 2003; Flügge & Gao, 2005), but so far, no prenyl pyrophosphate transporter has been identified in any organism. Both mevalonate and MEP pathways are also present in the brown alga E. siliculosus (Cock et al., 2010). In microalgae, the situation is somewhat different, however, and Table 2 summarizes data on the distribution of the two pathways in different species. To evaluate available genome sequences, we used blastp (Altschul et al., 1997) to search for HMG-CoA reductase and DXP reductoisomerase, enzymes of the mevalonate pathway and the MEP pathway, respectively. For the most part, the data yielded a consistent picture: Algae generally have both pathways as do higher plants, with the exception of chlorophytes, which only have the MEP pathway (Table 2). The proposal that only a single MEP pathway operates in the chloroplast of chlorophytes (Schwender et al., 2001) is supported by the fact that all genome sequences listed in Table 1 only encode a single DXP reductoisomerase. Moreover, for C. reinhardtii, V. carteri, Ostreococcus lucimarinus and Ostreococcus tauri, the other enzymes of the MEP pathway have also been shown to be encoded by unique genes (Frommolt et al., 2008). In one plausible, yet speculative scenario, cytosolic biosynthesis of phytosterols and other isoprenoids in chlorophytes may depend on the export of prenyl pyrophosphates from the plastid. An isotope labelling experiment suggests that the mevalonate pathway is also absent from the chlorophyte Caulerpa taxifolia, a macroalga that consists of a single multinucleate cell: 13CO2 was found to be incorporated into the backbone of the sesquiterpene caulerpenyne, a major secondary metabolite of C. taxifolia, whereas no enrichment was observed when the alga was fed with 1-13C-acetate (Pohnert & Jung, 2003).
Figure 2. Biosynthesis and examples of isoprenoids in algae. (a) Simplified scheme of isoprenoid biosynthesis and its compartmentation in higher plants and algae. Note that chlorophytes likely possess only a plastidic MEP pathway for the formation of DMAPP and IPP, while most other algae and higher plants have both mevalonate and MEP pathways (see Table 2 and main text for details). (b) Examples of algal secondary isoprenoids. FPP, farnesyl pyrophosphate; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; G3P, glyceraldehyde 3-phosphate. (a) Adopted from Rodríguez-Concepción & Boronat (2002), and modified.
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Table 2. Isoprenoid precursor biosynthesis in microalgaea
|Mevalonate||MEP||Genome sequence||Isotope labelling||Inhibitor study|
|Klebsormidium flaccidum||+||+|| ||Schwender et al. (2001)|| |
|Mesostigma viride||+||+|| ||Schwender et al. (2001)|| |
|Spirogyra sp.||+||+|| ||Schwender et al. (2001)|| |
|Botryococcus braunii|| ||+|| ||Sato et al. (2003)|| |
|Chlamydomonas reinhardtii|| ||+||Merchant et al. (2007)||Lichtenthaler (1999), Schwender et al. (2001)|| |
|Chlorella sp.|| ||+||Blanc et al. (2010)||Lichtenthaler (1999), Schwender et al. (2001)b|| |
|Dunaliella salina|| ||+|| || ||Capa-Robles et al. (2009)|
|Gloeotilopsis planctonica|| ||+|| ||Schwender et al. (2001)|| |
|Micromonas sp.|| ||+||Worden et al. (2009)|| || |
|Ostreococcus sp.|| ||+||Derelle et al. (2006), Palenik et al. (2007)|| || |
|Scenedesmus obliquus|| ||+|| ||Lichtenthaler (1999), Schwender et al. (2001)|| |
|Tetraselmis striata|| ||+|| ||Schwender et al. (2001)|| |
|Trebouxia asymmetrica|| ||+|| ||Schwender et al. (2001)|| |
|Volvox carteri|| ||+||Prochnik et al. (2010)|| || |
|Cyanidioschyzon merolae|| ||+||Matsuzaki et al. (2004)|| || |
|Cyanidium caldarium||+||+|| ||Lichtenthaler (1999)|| |
|Galdieria sulphuraria||+||+||Barbier et al. (2005)|| || |
|Aureococcus anophagefferens||(+)c||+||Gobler et al. (2011)|| || |
|Fragilariopsis cylindrus||+||+||JGI|| || |
|Haslea ostrearia|| ||+|| || ||Massé et al. (2004)d|
|Nitzschia ovalis||+||+|| ||Cvejić & Rohmer (2000)|| |
|Ochromonas danica||+||+|| ||Lichtenthaler (1999)|| |
|Phaeodactylum tricornutum||+||+||Bowler et al. (2008)||Cvejić & Rohmer (2000)|| |
|Rhizosolenia setigera||+||+|| ||Massé et al. (2004)|| |
|Thalassiosira pseudonana||+||+||Armbrust et al. (2004)|| || |
|Emiliania huxleyi||+||+||JGI|| || |
|Euglena gracilis||+||+|| ||Kim et al. (2004)|| |
The mevalonate pathway may also be absent from Haslea ostrearia and C. merolae, which may therefore represent exceptions among the heterokonts and red algae, respectively (Table 2). From the genome sequence, it is predicted that C. merolae only contains HMG-CoA synthase (and IPP isomerase), but none of the other four enzymes of the mevalonate pathway (Matsuzaki et al., 2004). Similarly, it is currently unclear whether the mevalonate pathway is present at all in H. ostrearia. While isotope labelling experiments were unsuccessful, this diatom was also treated with fosmidomycin. This inhibitor of the MEP pathway inhibited the biosynthesis of several isoprenoids including a sterol, which usually emerges from the mevalonate pathway (Massé et al., 2004). Observations from chlorophytes, H. ostrearia and C. merolae thus suggest that loss of the mevalonate pathway is not uncommon in algae. A model for the evolution of isoprenoid precursor biosynthesis in land plants and algae was presented recently (Grauvogel & Petersen, 2007).
Biosynthesis of polyprenyl pyrophosphates and isoprenoid end products
In the second stage of isoprenoid biosynthesis, linear C10–C25 polyprenyl pyrophosphates are synthesized from IPP and DMAPP by a family of closely related short-chain prenyltransferases (Vandermoten et al., 2009). The 1′-4 condensation of DMAPP with a single IPP molecule by geranyl pyrophosphate synthase (GPPS) yields the monoterpene precursor geranyl pyrophosphate, while condensation of two molecules of IPP with DMAPP by farnesyl pyrophosphate synthase (FPPS) results in the sesquiterpene precursor farnesyl pyrophosphate. Geranylgeranyl pyrophosphate synthase (GGPPS) from fungi and animals needs farnesyl pyrophosphate as the acceptor substrate for IPP, whereas the plastid-localized GGPPS in plants catalyses the successive addition of three IPP molecules to DMAPP (Vandermoten et al., 2009). The occurrence and distribution of the different short-chain prenyltransferases in algal genomes has not yet been examined. The fundamental role of farnesyl pyrophosphate and geranylgeranyl pyrophosphate as precursors of primary metabolites (sterols and carotenoids), however, suggests that all algae contain orthologues of FPPS and GGPPS. In agreement with this assumption, we detected candidates for FPPS, GGPPS and also for GPPS in the currently available algal genomes (Table 1) by blastp searches with the corresponding protein sequences from Arabidopsis.
In the third stage, the various polyprenyls can be further metabolized to a vast array of isoprenoids. For example, farnesyl pyrophosphate and geranylgeranyl pyrophosphate can be dimerized to yield squalene (the precursor of sterols and triterpenes) and phytoene (the precursor of carotenoids), respectively (Bouvier et al., 2005). While the genetic basis of the biosynthetic pathways of sterols and carotenoids – the two major classes of primary isoprenoids in plants and algae – has been examined in detail by comparative genomics (Frommolt et al., 2008; Desmond & Gribaldo, 2009), much less is known about the occurrence and formation of secondary isoprenoids in microalgae. Emission of volatile monoterpenes (C5) from higher plants has been known for several decades and some of its ecological consequences are well characterized, whereas emission of monoterpenes from axenic cultures of unicellular marine algae has only recently been demonstrated (Yassaa et al., 2008). Highest monoterpene emissions were found in the chlorophyte Dunaliella tertiolecta and in the diatoms P. tricornutum and Fragilariopsis kerguelensis, whereas emissions from three other diatoms, E. huxleyi and two cyanobacteria were low or insignificant. Shipboard measurements confirmed the marine production of monoterpenes and isoprene, even though emissions were over an order of magnitude lower than in terrestrial forests (Yassaa et al., 2008). These findings and their physiological and ecological significance call for more detailed investigation.
Monoterpenes and sesquiterpenes have also been isolated from strains of the chlorophyte genus Chlorella for the purpose of biochemical taxonomy (Liersch, 1976 and references therein; some of the strains are now classified as Scenedesmus species (Huss et al., 1999)). The diatom Pseudonitzschia multiseries, which is closely related to Nitzschia species (cf. Table 2), is assumed to be the producer of the neurotoxin domoic acid (see Fig. 6), although a bacterial origin can at present not be ruled out (Bates et al., 2004). Labelling studies with 13C-acetate suggest that domoic acid is formed by condensation of geranyl pyrophosphate with an activated glutamate derivative (Douglas et al., 1992). As it is known from higher plants that labelled acetate is readily incorporated into mevalonate-derived cytosolic isoprenoids, but poorly incorporated into MEP-derived plastidic isoprenoids (Lichtenthaler, 1999), the low incorporation of the 13C-label from acetate into the isoprenoid moiety of domoic acid is in agreement with biosynthesis of geranyl pyrophosphate via the MEP pathway in P. multiseries.
A restricted number of diatoms synthesize highly branched isoprenoids (HBIs) that belong to the sesterterpene (C25) and triterpene subclasses (Fig. 2b; Damsté et al., 2004). HBI alkenes are considered specific diatom markers in marine sediments, where they are widely found. Interestingly, the diatom Rhizosolenia setigera makes C25 haslenes and C30 rhizenes via the mevalonate route, whereas H. ostrearia appears to use the MEP route (Massé et al., 2004). Apart from information on precursor biosynthesis, little is known about pathway intermediates or enzymes involved in HBI biosynthesis, and enzymes responsible for the formation of the additional T branch in these molecules have not been identified yet.
The molecular details of the biosynthesis of secondary isoprenoids in algae are essentially unknown. In land plants, their formation is initiated by members of a large family of terpene synthases (Bohlmann et al., 1998; Trapp & Croteau, 2001). Using protein sequences of three different terpene synthases from Arabidopsis for blastp searches, we were not able to find candidates for algal terpene synthases in any of the algal genomes listed in Table 1. As the diatom P. tricornutum was one of the microalgae reported to emit various monoterpenes, albeit at low rates (Yassaa et al., 2008), P. tricornutum and other algae may have evolved terpene synthases unrelated to the enzymes from land plants. Alternatively, the low rates of monoterpene production in P. tricornutum may result from nonenzymatic reactions (Wise et al., 2002). Notably, an aqueous protein extract from the red macroalga Ochtodes secundiramea was shown to catalyse the formation of the monoterpene myrcene from geranyl pyrophosphate, and the enzyme could be partially purified (Wise et al., 2002). However, no algal monoterpene synthase has yet been identified (Wise, 2003).
Carotenoids are tetraterpenes (C40) best known as primary metabolites that are produced by all photosynthetic organisms, and that are involved in light harvesting and photoprotection (Grossman et al., 2004). In addition, carotenoids are abundant within lipid globules in the eyespot apparatus, a primitive visual system that exists in most flagellate chlorophytes (Kreimer, 2009). Secondary carotenoids not required for photosynthesis and localized either in plastoglobules or in cytosolic lipid droplets are produced by many green microalgae under stress conditions and can be accumulated to high levels (Lemoine & Schoefs, 2010). Some species are even used to produce carotenoids industrially. For example, the unicellular chlorophytes Haematococcus pluvialis and Dunaliella salina are used for the production of astaxanthin (Fig. 2b) and β-carotene, respectively (Del Campo et al., 2007). These high-value products are used as additives in food and animal feed. While β-carotene in D. salina accumulates inside the plastid in interthylakoid lipid globules (Del Campo et al., 2007), astaxanthin is usually found in cytosolic lipid droplets and is esterified with fatty acids (Lemoine & Schoefs, 2010). A variety of functions have been suggested for astaxanthin, ranging from storage of carbon and energy to protection against reactive oxygen species or UV irradiation (Lemoine & Schoefs, 2010).
A genome-based examination of carotenoid biosynthetic genes indicated that C. reinhardtii may also be able to synthesize ketocarotenoids (Lohr et al., 2005). Experiments subsequently revealed that the formation of ketocarotenoids in C. reinhardtii is confined to maturing zygospores (Lohr, 2008). Under nutrient deprivation, the zygotes develop into thick-walled resting spores that accumulate ketocarotenoids and massive amounts of neutral lipid. Unlike H. pluvialis, zygospores of C. reinhardtii contain 4-ketolutein (Fig. 2b) as the major ketocarotenoid, but astaxanthin was also detected (Lohr, 2008; S. Werner, M. Bauch, A. Hallmann, V. Schmitt and M. Lohr, unpublished results). The availability of the complete genome sequence and an elaborate molecular toolbox for C. reinhardtii will facilitate proteomic, transcriptomic and reverse genetic approaches to gain a deeper understanding of the molecular mechanisms of the concomitant accumulation of ketocarotenoids and lipids in green algae.
Algae are currently being explored as a renewable source of biofuel (Radakovits et al., 2010; Lü et al., 2011). While triacylglycerides attract considerable interest in this regard, the isoprenoid pathway also delivers compounds that could be used as biodiesel (Fortman et al., 2008). For example, the unicellular chlorophyte Botryococcus braunii accumulates large amounts of fatty acids and isoprenoids, with hydrocarbon contents of up to 60% dry weight (Metzger & Largeau, 2005). B. braunii strains are grouped into three chemical races. In race B, triterpenes such as the botryococcenes (Fig. 2b) are the predominant hydrocarbons, whereas race L produces lycopadiene, a tetraterpene hydrocarbon. In addition to these hydrocarbons, B. braunii makes related ether lipids. For example, twelve lycopanerols derived from lycopadiene have been described in race L (Metzger & Largeau, 2005). In B. braunii, the majority of hydrocarbons is located in the cell wall and extracellular globules derived therefrom (Largeau et al., 1980; Weiss et al., 2010).
Understanding the regulation of isoprenoid metabolism is the key to its potential manipulation
Sufficient knowledge on how the routing of polyprenyl precursors at critical branch points of the biosynthetic network of isoprenoid formation is regulated will be a prerequisite for increasing the yield of desired isoprenoids in biotechnological applications. For cytosolic isoprenoids, the triterpene secondary metabolism competes with sterol metabolism for the common precursor farnesyl pyrophosphate. Similarly, the formation of mono- and diterpenes in the plastid competes with carotenoid biosynthesis. One mechanism of controlling the allocation of precursors to different pathways may be the formation of multi-enzyme complexes that channel the substrate across the branch point towards a specific class of isoprenoids. There is experimental evidence that phytoene synthase forms a complex with GGPPS (Cunningham & Gantt, 1998 and references therein; Welsch et al., 2010), thereby shunting isoprenoid precursors to the carotenoid pathway. Another way to achieve a higher flux through certain pathways could be an increased expression of key pathway genes. In Arabidopsis, five of the 40 putative terpene synthase genes are arranged in tandem with genes encoding bona fide GGPPS (Aubourg et al., 2002). As GGPPS provides the substrate of the terpene synthases, the parallel duplication of genes for terpene synthase and GGPPS in Arabidopsis supports a central role of GGPPS and potentially other polyprenyl synthases in controlling the flux towards different branches of the isoprenoid pathway. Coordinate regulation between mevalonate and MEP pathways in species with both pathways is another aspect that needs to be examined more thoroughly. Details of the regulation of the two pathways in microalgae may differ from that in land plants, because isoprenoids like gibberellins and strigolactones, which serve as phytohormones in land plants, appear to be absent from unicellular algae (Blanc et al., 2010) or are likely to have different functions in these species.
In summary, many interesting questions about isoprenoid biosynthesis in algae and other plants remain unresolved. As described, pathway regulation and exchange of compounds between compartments are incompletely understood. Furthermore, enzymes responsible for the formation of secondary isoprenoids in algae are still largely unidentified. Research on isoprenoid biosynthesis may help to exploit algae as source of both natural products and biofuel.