Lipids and lipid metabolism in eukaryotic algae

Progress in Lipid Research Progress in Lipid Research 45 (2006) 160–186 www.elsevier.com/locate/plipres

Review

Lipids and lipid metabolism in eukaryotic algae Irina A. Guschina, John L. Harwood

*

School of Biosciences, Cardiff University, P.O. Box 911, Cardiff CF10 3US, UK Received 2 December 2005; accepted 4 January 2006

Abstract Eukaryotic algae are a very diverse group of organisms which inhabit a huge range of ecosystems from the Antarctic to deserts. They account for over half the primary productivity at the base of the food chain. In recent years studies on the lipid biochemistry of algae has shifted from experiments with a few model organisms to encompass a much larger number of, often unusual, algae. This has led to the discovery of new compounds, including major membrane components, as well as the elucidation of lipid signalling pathways. A major drive in recent research have been attempts to discover genes that code for expression of the various proteins involved in the production of very long-chain polyunsaturated fatty acids such as arachidonic, eicosapentaenoic and docosahexaenoic acids. Such work is described here together with information about how environmental factors, such as light, temperature or minerals, can change algal lipid metabolism and how adaptation may take place. Ó 2006 Elsevier Ltd. All rights reserved. Keywords Algae; Oxylipins; Chlamydomonas reinhardtii; Arachidonic acid; Eicosapentaenoic acid; Docosahexaenoic acid; Fatty acid synthesis; Environmental effects

Abbreviations: AA, arachidonic acid (C20:4n-6); APCI-MS, atmospheric pressure chemical ionization mass spectrometry; ASQD, 2 0 -Oacyl-sulfoquinovosyldiacylglycerol; BTA1Cr, betaine lipid synthase (from Chlamydomonas reinhardtii); DAG, diacylglycerol; DGCC, diacylglyceryl carboxyhydroxymethylcholine; DGGA, diacylglyceryl glucuronide; DGDG, digalactosyldiacylglycerol; DGTA, diacylglyceryl hydroxymethyl-N,N,N-trimethyl-b-alanine; DGTS, diacylglyceryltrimethylhomoserine; DHA, docosahexaenoic acid (C22:6n-3); DI, inhibiting dose; DPA, docosapentaenoic acid (C22:5n-6); EDA, eicosadienoic acid (C20:2n-6); EPA, eicosapentaenoic acid (C20:5n-3); ESI-ITMS, electrospray ionization ion trap mass spectrometry; ETrA, eicosatrienoic acid (C20:3n-3); FAD, fatty acid desaturase; HETE, hydroxyeicosatetraenoic acid; HEPE, hydroxyeicosapentaenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; GC–EIMS, gas chromatography–electron impact mass spectrometry; GL, galactosylglycerides (mainly MGDG, DGDG); Kcs, b-ketoacyl-coenzyme A synthase (of fatty acid elongation); LOX, lipoxygenase; LPA, lyso-phosphatidic acid; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic acid; PAR, photosynthetically active radiation; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PHEG, phosphatidyl-O-[N-(2-hydroxyethyl) glycine]; SAM, S-adenosyl-L-methionine; SQDG, sulphoquinovosyldiacylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIP, phosphatidylinositolphosphate; PIP2, phosphatidylinositolbisphosphate; PLD, phospholipase D; TAG, triacylglycerol. * Corresponding author. Tel.: +(0)44 2920874108; fax: +(0)44 2920874116. E-mail address: Harwood@cardiff.ac.uk (J.L. Harwood). 0163-7827/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.plipres.2006.01.001

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recent advances in algal lipid biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. New compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Oxylipins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Red algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Diatoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Brown algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4. Euglenophyta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The green algae Chlamydomonas reinhardtii as a model system for studying lipid metabolism and functions in photosynthetic organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Algae as a source of polyunsaturated fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Synthesis of eicosapentaenoic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Synthesis of docosahexaenoic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Arachidonic acid occurrence and formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Species with high levels of several very long-chain PUFAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Identification of genes involved in the biosynthesis of PUFAs in algae . . . . . . . . . . . . . . . . . . . . . . . Lipid metabolism in algae with no very long-chain PUFAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of the environment on lipid metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Nutrients and nutritional regimes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Other growth conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Anthropogenic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contents 1. 2.

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1. Introduction Algae are important constituents of many ecosystems ranging from marine and freshwater environments to desert sands and from hot springs to snow and ice. They account for more than half total primary production at the base of the food chain worldwide [1]. Algae are a very varied and highly specialized group of organisms. Moreover, the systematics of algae is based on the kinds and combinations of photosynthetic pigments present in different algal species. The chemical nature of the storage products and algal cell walls also play an important role in the definition of the various algal groups. According to systematic classification, the thousands of eukaryotic algal species are grouped into nine divisions with the largest classes being Chlorophyceae (green algae), Phaeophyceae (brown algae), Pyrrophyceae (dinoflagellates), Rhodophyceae (red algae) and Chrysophyceae (yellow-green algae) [1]. It is generally accepted that the ability of algae to adapt to environmental conditions is reflected in an exceptional variety of lipid patterns as well as with their ability to synthesize a number of unusual compounds [2]. Moreover, further development of modern analytical methods combining various chromatographic techniques with sensitive detection systems and mass spectrometry, as well as new derivatization procedures, has led to significant progress in the identification of new and unusual classes of lipids and fatty acids in algae in recent years. For previous information on algal lipid biochemistry refer to [3–6]. In this review we will concentrate on information reported in the last decade or so. 2. Recent advances in algal lipid biochemistry 2.1. New compounds Due to advances in lipid analytical methods in the past two decades, the structural determination and identification of a number of new algal compounds have been reported. Long-chain (C35–C40) alkenones and

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their derivatives have been identified in haptophyte Chrysotila lamellose using GC–EIMS (gas chromatography–electron impact mass spectrometry) [7]. These compounds were relatively stable during aging of C. lamellose in contrast to sterols which undergo intensive abiotic degradation as indicated by accumulation of significant amounts of 24-methylcholesta-5,22E-dien-3b-ol-7-one and 24-ethylcholesta-5,22E-dien-3b-ol-7one. On the basis of these detailed analysis, some further evaluation of long-chain alkenones as indicators of paleoceanographic conditions was suggested [7]. The structural elucidation of some galactolipids from the freshwater dinoflagellate Glenodinium sanguineum and from a marine diatom of the genus Chaetoceros has been reported using HPLC/ESI-ITMS (high performance liquid chromatography–electrospray ionization ion trap mass spectrometry) [8]. This method was successfully used to establish the regiochemical distribution of the acyl groups on the glycerol backbone of algal glyceroglycolipids and was suggested as a convenient tool to estimate the contribution of the plastidial and cytosolic pathways to the biosynthesis of galactolipids in algae [8]. In addition, an unusual polar lipid has been isolated from the red alga Gracilaria verrucosa and its inositol phosphoceramide structure proposed, as based on GC–MS (gas chromatography–mass spectrometry) analysis [9]. Acetonitrile chemical ionization tandem MS has been demonstrated to be a valuable methodology in order to determine the location of double bonds in minor fatty acids which may not exceed 1% of total fatty acids in a golden alga, Schizochytrium spp. [10]. In addition, two marine green algae, Codium dwarkense and Codium taylorii, were analysed for fatty acids by GC–MS using serially coupled capillary columns with different stationary phase polarities. This method showed the presence of more than 40 volatiles, including low molecular and dioic compounds [11]. Some papers have been published recently describing studies of unusual hydrocarbons and ether lipids from a green colonial microalga Botryococcus braunii [12,13]. These hydrocarbons are classified as: (1) n-alkadienes and trienes, (2) triterpenoid botryococcenes and methylated squalenes, (3) a tetraterpenoid, lycopadiene [14]. In addition to above mentioned hydrocarbons and classic lipids such as fatty acids, glycerolipids and sterols, these algae synthesize a number of ether lipids closely related to hydrocarbons [14]. The review of Metzger and Largeau [14] summarizes the available information on algal biodiversity, the chemical structure and biosynthesis of hydrocarbons and ether lipids and biotechnological studies related to hydrocarbon production [14]. The distribution and structural characterization of halogenated (chlorinated, fluorinated, brominated and iodinated) fatty acids and their derivatives in different organisms including algae have been reviewed in detail by Dembitsky and Srebnik [15]. For example, three chlorinated epoxy monocyclopentyl fatty acids, named egregiachlorine A–C, have been isolated from marine brown alga, Egregia menziesii. Chlorosulfolipids have been reported notably for the red alga Ochromonas danica where they accounted for 15% of the total lipids. Later, these lipids were isolated from more than 30 species of both freshwater and marine red, green and brown algae, as well as macrophytic and microalgal species. Z- and E-forms of 3-bromo-2-heptanoic acid and Z- and E-forms of 3-bromo-2-nonanoic acids have been found in the red algae Bonnemaisonia nootkana, Bonnemaisonia hamifera and Trailliella intricata (see [15]). Simple iodinated acetic and acrylic acids and their ethyl esters have been isolated from the marine red algae Asparagopsis taxiformis and Asparagopsis armata. Their biological potential as related to anticancer, antifungal and antibacterial properties are discussed in this review [15]. Several species of green microalgae have been examined recently with an emphasis on the occurrence and structural analysis of cell wall-insoluble non-hydrolysable biopolymers called algaenans [16–18]. The presence of these highly resistant biopolymers in the trilaminar outer wall is associated with some protective mechanisms against microbial attack in algae [16]. The long-chain x-hydroxy fatty acids have been shown to be the main structural blocks of algaenan in the freshwater green microalgae [16,17]. Analysis of ester-bound lipids from the cell wall of the green algae, Tetraedron minimum, Scenedesmus communis and Pediastrum boryanum, revealed C30–C34 mono- and diunsaturated x-hydroxy fatty acids [16]. Allard and Templier [18] compared neutral lipid profiles of algaenan-producing and algaenan-devoid species in an attempt to use the non-polar lipid composition (and particularly the unusual long-chain lipids) as specific indicators of the presence of algaenan. Detailed analysis of hydrocarbon, free fatty acid and alcohol distributions, as well as fatty acid methyl and/or ethyl esters, methyl ketones and monoesters, has been undertaken but no relationship between non-polar lipid patterns of trilaminar outer wall-containing microalgae and the presence or absence of algaenan was been found [18].

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A novel lipid constituent was isolated from the brown alga Fucus serratus and identified as phosphatidyl-O[N-(2-hydroxyethyl) glycine] (PHEG) [19]. The fatty acid composition showed 82 mol% arachidonic, 9% eicosapentaenoic (EPA) and 6% arachidic acids. These were located at both the sn-1 and sn-2 positions of the glycerol moiety. PHEG was present in 30 different species, representing the 16 orders of brown algae, in amounts ranging from 8 to 25 mol% of the total phospholipids and was suggested to be a characteristic lipid constituent of brown algae [19]. Two unusual compounds, diacylglyceryl carboxyhydroxymethylcholine (DGCC) and diacylglyceryl glucuronide (DGGA), were identified in the lipid fraction of Pavlova lutheri [20]. DGCC was enriched in palmitate and EPA, and DGGA contained predominantly oleate, docosapentaenoic and docosahexaenoic (DHA) acids. Analysis of subcellular membrane fractions demonstrated an accumulation of these lipids together with a betaine lipid, diacylglyceryl hydroxymethyl-N,N,N-trimethyl-b-alanine (DGTA), in non-plastid membranes [20]. 2.2. Oxylipins A recent hot topic for lipid research has been the chemistry, biochemistry, molecular biology and physiological role of hydroxy fatty acids and the oxygenated derivatives of fatty acids, known as oxylipins. In higher plants, two 18C fatty acids (linoleic and a-linolenic acids) serve as the most important precursors of oxylipins [21], whereas algae may transform 18C and/or 20C fatty acids [22,23]. In general, green algae metabolize 18C acids at C-9 and C-13 positions while brown algae metabolize both 18C and 20C acids, principally using lipoxygenases with an n-6 specificity (for review see [22]). 2.2.1. Red algae Some red algae (an example is Gracilariopsis lemaneiformis) metabolize 20C acids via 8-, 9- or 12-lipoxygenase (LOX)-initiated pathways, whereas other species contain oxylipins of the eicosanoid family produced by 5R-, 8R-, 9S-, 12S- and 15S-lipoxygenase action, as well as octadecanoids formed through 9S-, 11R- and 13Sactivity [22]. The last few years have seen a large increase in the number of detailed biosynthetic investigations of oxylipin formation in algae. An application of HPLC interfaced to electrospray ionization mass spectrometry for the analysis of eicosanoids in the red alga Glacilaria asiatica, allowed the identification of several new compounds which had not been reported previously for this species, namely LTB4(leukotriene B4), 8-HETE (hydroxyeicosatetraenoic acid) and 15-keto-prostaglandin E2 [24]. In the red alga, Rhodymenia pertusa , the presence and function of 5R-lipoxygenase acting on both arachidonic and eicosapentaenoic acids has been strongly supported by the identification of a few new eicosanoid metabolites, 5R,6S-diHETE (dihydroxyeicosatetraenoic acid), 5R,6S-diHEPE (dihydroxyeicosapentaenoic acid), 5-HETE and 5-HEPE (hydroxyeicosapentaenoic acid) [25]. Structures of these eicosanoids were deduced using NMR and GC–electron impact mass spectrometry of their TMS (trimethylsilyl) esters [25]. Based on the well-established functional roles of oxylipins in animals and higher plants, Bouarab et al. [26] hypothesized that these compounds are also involved with defense mechanisms in the red alga Chondrus crispis. These workers showed that the resistant haploid phase of C. crispis produced both 20C and 18C oxylipins when challenged by pathogen extracts. Several enzyme activities related to oxidative lipid metabolism, including LOX, were upregulated in C. crispis gametophytes, 24 h following challenge with pathogen extracts. So it was concluded, that those oxylipids identified in the study appeared as essential intermediates for the innate immunity of C. crispis [26]. At the molecular level, a cDNA encoding a putative 12-lipoxygenase was identified in the gametophyte of Porphyra purpurea [27]. In addition, polyenoic fatty acid isomerase, which converts arachidonic acid into a conjugated triene, has been purified and cloned from Ptilota filicina [28]. 2.2.2. Diatoms Progress in understanding the biological importance of oxylipins for various defense reactions in diatoms has been made in the last 10 years. Diatoms are abundant in most aquatic habitats and are considered to be the most important primary producers in sustaining marine food chains [29]. Moreover, seasonal blooms of phytoplankton are often dominated by these algae. Even though diatoms are thought

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of as a high-quality food source, a large body of evidence on the toxic effects of these algae has also been accumulated [29–40]. Three antiproliferative aldehydes inducing low hatching rates in copepods have been isolated from the diatoms Thalassiosira rotula, Skeletonema costatum and Pseudo-nitzchia delicatissima [30]. The compounds were identified by spectroscopic techniques as 2-trans-4-cis-decatrienal, 2-trans-4-trans-7-cis-decatrienal and 2trans-4-trans-decadienal and they have been suggested to be the probable agents of reproductive failure in copepods when diatoms were the major food source [30]. D’Ippolito et al. [33] applied a new derivatization procedure based on conversion of aldehydes into ethyl esters by the Wittig reaction with carbetoxyethylidene-triphenylphosphorane. This method was designed to allow the GC–MS detection of compounds with alkyl chains ranging from 8 to 16 carbon atoms and the procedure was also suitable for NMR and HPLC application [33]. Using this method, two new compounds, 2,4trans,trans-octadienal and trans,trans,cis-2,4,7-octatrienal were detected in the alga T. rotula [33]. In the marine diatom S. costatum, short-chain aldehydes (other than the ones already described) were responsible for the dramatic effects of this alga on the reproductive success of the copepod Temora stylifera [34]. Further investigations on the mechanisms of diatom-derived aldehyde actions, on the apoptogenic inductive reactions of aldehydes in copepods and sea urchin embryos and on their action on the fertilization in ascidian oocytes (including inducing teratogenic embryo modifications) have been carried out [35,36]. Moreover, recently, the cytotoxicity of diatom-originated oxylipins has been tested against different organisms, including bacteria, algae, fungi, echinoderms, molluscs and crustaceans [37]. It was concluded that the wide spectrum of physiological pathologies revealed may reflect the potent toxicity of diatom-derived oxylipins as related to their non-specific chemical reactivity towards nucleophilic biomolecules [37]. However, experiments with yeast suggested some efficient protection mechanisms have evolved in unicellular organisms which were related to the inability of the oxylipins to penetrate the cell wall/cell membrane and enter such cells [37]. With regard to defense reactions in diatoms, it has been shown that aldehydes are not synthesized in intact diatom cells but were produced only after mechanical cell disruption/wounding [31]. This fact was thought to reflect a mechanism to avoid self-toxicity [31]. Later studies revealed that the activation of oxylipin-based chemical defense in the diatom T. rotula was initiated by phospholipase A2 with the release of polyunsaturated eicosanoic fatty acids from phospholipids [32]. In this alga, only non-esterified 20C fatty acids were shown to serve as substrates for the synthesis of 2,4-decadienal and 2,4,7-decatrienal by the action of lipoxygenase coupled to hydroperoxide lyase [32]. D’Ippolito et al. [29] described the biochemical pathway for the synthesis of bioactive aldehydes from complex lipids in the marine diatom S. costatum [29]. This pathway is similar to that in higher plants and operates through hydrolysis of glycolipids and release of eicosapentaenoic acid and 16C polyunsaturated fatty acids with the subsequent lipoxygenase/hydroperoxide lyase action to produce metabolites [29]. Lipoxygenase-mediated formation of unsaturated acyclic and alicyclic hydrocarbons and polyunsaturated aldehydes has been demonstrated in several species of freshwater diatoms after activation of lipoxygenase reactions by osmotic stress [40]. In the fresh water diatom Gomphonema parvulum, the lipoxygenase-based production of volatile hydrocarbons, such as the 11C hydrocarbon hormosirene and 5Z,7E-9-oxo-nona-5,7-dienoic acid, was also dependent on the use of eicosapentaenoic acid as a substrate, as found for marine species [41]. Recently, a possible nontoxic role for diatom aldehydes as chemical signals during stress or unfavorable growth conditions within the phytoplankton community has also been suggested after a demonstration that Thalassiosira weissflogii produced aldehyde, 2-trans,4-trans-decadienal (which is usually involved in the wound-activated response of diatoms to copepod grazing), could induce a degenerative process in the diatom itself [38]. This process included some modifications of cell membrane characteristics and led to cell death by a mechanism resembling apoptosis [38]. An osmoregulatory role for these metabolites has been also proposed [23]. 2.2.3. Brown algae In Laminaria angustata, 9C aldehydes were formed exclusively from the 20C fatty acid, arachidonic acid, while 6C aldehydes could be derived either from 18C or from 20C fatty acids [42]. These volatile aldehydes are known to be responsible for fresh green and cucumber-like flavours and aromas in foodstuffs. The

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intermediates in their biosynthesis were identified using chiral HPLC as 12S-HPETE (hydroperoxyeicosatetraenoic acid) and 15S-HPETE. The fatty acid hydroperoxide lyase that catalyses the formation of 9C aldehydes from 12S-HPETE has been suggested to have a high specificity [42]. It also has been shown that nhexanal can be formed from linoleic acid by a pathway similar to that of higher plants, namely via a sequential action of lipoxygenase and fatty acid hydroperoxide lyase [43]. Comparative studies of volatile compounds, including ketones and aldehydes, have been undertaken in seven species of brown algae from the Black Sea [44]. 2.2.4. Euglenophyta Using the microalga Euglena gracilis as a model, LC/APCI-MS (atmospheric pressure chemical ionization mass spectrometry) has been applied to analyze and identify algal hydroxy fatty acids [45]. 15-, 12-, 8- and 5HETEs (hydroxyeicosatetraenoates) were identified as major compounds and the formation of these metabolites was followed during incubation of cell-free extracts with arachidonic acid [45]. 3. The green algae Chlamydomonas reinhardtii as a model system for studying lipid metabolism and functions in photosynthetic organisms For several decades, algae have been intensively used as model photosynthetic organisms for studies of a number of physiological and metabolic processes, e.g., photosynthesis, respiration, nitrogen assimilation, heavy metal homeostasis and tolerance. The haploid green alga C. reinhardtii should be mentioned as an important example of such an organism [46,47]. Over the past 15 years, the number of molecular biology methods that can be applied to Chlamydomonas have increased and several different techniques are now available for genetic transformation [48]. Thus, a genetic approach, using insertional mutagenesis by plasmid transformation, has been used to study the biosynthesis of the sulfolipid, 2 0 -O-acyl-sulfoquinovosyldiacylglycerol (ASQD), in C. reinhardtii and to identify genes essential for its synthesis [49]. n-3 Octadecatetraenoic acid was the main acid attached to the head group of ASQD. Furthermore, analysis of sulphoquinovosyldiacylglycerol (SQDG) molecular species indicated that there were two pools of the latter lipid (16:0/16:0 and 18:1/16:0) of which only the second appeared to be the substrate for an acyltransferase forming ASQD. When studying the biosynthesis of this lipid in C. reinhardtii, it was suggested that the acyltransferase responsible for acylation of SQGD could be localized in the outer envelope of the plastid, where it would have access to SQDG made in the plastid and also to fatty acids derived from the ER [49]. Algae mutants defective in a certain lipid or a fatty acid have been shown to be powerful tools to investigate the functions and physiological roles of lipids. Thus, within the past decade, experiments with C. reinhardtii mutants deficient in SQDG or in D3-trans-hexadecenoic acid-containing phosphatidylglycerol have led to further progress in understanding the specific role of these lipids in thylakoid membranes, namely in the functioning of Photosystem II [50–52]. Moreover, a mutant of C. reinhardtii which was impaired in fatty acid desaturation of chloroplast lipids (designated as hf-9) has been used to study the role of lowered unsaturation levels in such lipids with regard to high temperature tolerance of photosynthesis in this alga [53]. C. reinhardtii has a unique lipid composition in comparison to higher plants [54]. In particular, phosphatidylcholine and phosphatidylserine are not present in its membranes and a major membrane lipid in this alga is a betaine lipid, diacylglyceryltrimethylhomoserine (DGTS) [54]. This makes the organism a very useful system for studying the biosynthesis of phosphatidylethanolamine which is independent of both phosphatidylcholine and phosphatidylserine metabolism, as well as the biosynthesis of DGTS itself [55,56]. A cDNA which encodes the ethanolaminephosphotransferase protein has been sequenced from C. reinhardtii. Although the enzyme coded by the cDNA is clearly an ethanolaminephosphotransferase (as noted above C. reinhardtii contains phosphatidylethanolamine but not phosphatidylcholine), the enzyme had more activity with CDPcholine than CDP-ethanolamine as a substrate [55]. Biosynthesis of DGTS has been studied in C. reinhardtii using [14C-carboxyl]-S-adenosyl-L-methionine (SAM) [56]. It was shown that the DGTS head group was synthesized utilizing SAM for both the homoserine and the methyl moieties and that enzyme activity was associated with the microsomal fraction [56]. Recently, the discovery of the betaine lipid synthase (BTA1Cr) gene, as well as analysis of the bifunctional protein, that it encoded, has been reported for C. reinhardtii [57]. Heterologous expression of BTA1Cr led to

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DGTS accumulation in Escherichia coli (which normally lacks this lipid) and allowed in vitro analysis of the enzymatic properties of BTA1Cr [57]. As a continuation of investigations of membrane lipid biosynthesis in C. reinhardtii, the synthesis of phosphatidylinositol (PI) in this alga was examined [58]. The characterization of inositol incorporation into PI provided evidence for the presence of both pathways which had been established in plant and animal tissues previously [58]. One reaction utilized CDP-diacylglycerol and was catalyzed by PI synthase (CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase). The second reaction was the phosphatidylinositol:myo-inositol exchange reaction, in which a free inositol was exchanged for an existing inositol headgroup. Partial characterization of these enzymes has been made and the microsomal fraction was shown to be the major site of PI biosynthesis in C. reinhardtii [58]. The advantages of Chlamydomonas spp. as a model system for the study of lipid-based signal transduction have been demonstrated by a number of studies over the last few years. Arisz et al. [59] analysed the polar glycerolipids of Chlamydomonas moewusii with a particular emphasis on phosphatidic acid (PA), phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) because of their known roles in intracellular signal transduction [60,61]. The breakdown of PIP2 by phospholipase C and the formation of PA by the action of phospholipase D, are known as stress-stimulated reactions in plants [62] and these activities were demonstrated in C. moewusii [63]. In this species, PA was found to account for 0.67 mol% of the phospholipids, and a PI:PIP:PIP2 ratio of 100:1.7:1.3 was found [59]. The pool of phosphoinositides was investigated, and, in addition to two isoforms (PI-3-P and PI-4-P) which had been previously recognized in plants, a third, PI-5-P, was identified in C. moewusii [64]. Its level rapidly increased when the algae were subjected to hyperosmotic stress, suggesting a role for PI-5-P in osmotic-stress signaling [64]. Study of the substrate preference of stress-activated phospholipase D (PLD) and its contribution to PA formation revealed that PE was the main substrate of PLD activity in C. moewusii, in response to various stimuli [65]. Osmotic stress in C. moewusii also resulted in the accumulation of lyso-phosphatidic acid (LPA) in a dose- and time-dependent manner [66]. Based on kinetic and inhibitor studies, it was concluded that synthesis of LPA resulted from the activation of a phospholipase A2 that specifically hydrolysed PA. These authors also proposed a potential role of LPA as a lipid signal molecule in algae [66]. 4. Algae as a source of polyunsaturated fatty acids Currently, the production of polyunsaturated fatty acids (PUFA) by marine and freshwater microalgae is the subject of intensive research and increasing commercial attention [67,68]. Fish oil is a major source for the commercial production of these fatty acids but, since there is an increasing demand for purified PUFAs, some alternative sources are being sought. Moreover, the quality of fish oil depends on fish species, season/climate, geographical location of catching sites and the quality of food consumed. In addition, in some cases there is a danger of contamination by lipid-soluble environmental pollutants. Furthermore, in order to purify PUFAs from complex low-grade crude fish oils, expensive and relative difficult techniques (such as adsorption chromatography, fractional or molecular distillation, enzymatic splitting, low temperature crystallization, supercritical fluid extraction and urea complexation) may have to be applied [67]. Some species of freshwater and marine algae contain large amounts of high-quality PUFAs and are widely used at the moment to produced PUFAs for aquaculture operations. They can grow heterotrophically on cheap organic substrates, without light, under well-controlled cultivation conditions. The following strategies are considered important in order to increase the use of algae for commercial production of PUFAs in the near future: the further selection and screening of oleaginous species, improvement of strains by genetic manipulation, optimization of culture conditions and development of efficient cultivation systems [67]. Moreover, it is often important to know whether PUFAs are present within membrane lipids, e.g., phospho- or glycolipids, or if they are present in the cytosol as part of triacylglycerols [68]. 4.1. Synthesis of eicosapentaenoic acid The heterotrophic production of eicosapentaenoic acid (C20:5n-3) by microalgae has been recently reviewed in some detail including its distribution in different algae species, systems for mass cultivation of

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algae, factors influencing production of EPA, as well as improvement of microalgal strains for EPA production [67]. As to the biosynthesis of this acid in microalgae, the elucidation of biosynthetic routes for EPA in the red alga Porphyridium cruentum has been made using externally supplied fatty acids and radiolabelled precursors [69,70]. Exogenously supplied fatty acids were incorporated into algal lipids and were further metabolized along x-3 and x-6 pathways [69]. In the x-6 pathway, c-linolenate was produced following desaturation of linoleic acid, then elongated to dihomo-c-linolenate and, subsequently, desaturated to arachidonate and further to EPA. In the x-3 pathway, linoleate was first desaturated to a-linolenate which was then converted to 18:4n-3, 20:4n-3 and EPA [69] (Fig. 1). In a study with radiolabelled precursors, the cells of P. cruentum were pulse-labelled with various radiolabelled fatty acid precursors. EPA-containing galactolipids of both ‘‘prokaryotic’’ and ‘‘eukaryotic types’’, were shown to be the major end products of biosynthesis [70]. In the prokaryotic molecular species, EPA/arachidonic acid and 16C fatty acids were esterified to the sn-1 and sn-2 positions of galactolipids, respectively, whereas in eukaryotic species both positions were occupied by EPA or arachidonic acid [70]. However, it was suggested, that both eukaryotic and prokaryotic lipid species were formed in the two pathways (x-3 and x-6: Fig. 1) which involved cytoplasmic and chloroplastic lipids. Cytoplasmic linoleoyl-PC might be converted into either arachidonyl-PC in the x-6 pathway or into EPA-PC, which would then have their diacylglycerol (DAG) moieties transferred to chloroplasts. In the chloroplasts, the DAGs could be galactosylated to the respective monogalactosyldiacylglycerol (MGDG) molecular species and further desaturation of C20:4(n-6)-MGDG by a chloroplastic D17 (x-3) desaturase would then result in the formation of EPA-containing galactolipids [70]. At lower biomass concentrations, D17 (x-3) desaturation of AA to EPA was enhanced in both prokaryotic and eukaryotic species of MGDG and digalactosyldiacylglycerol (DGDG) under low light conditions [71]. A reduction in growth temperature led to an increase in the proportion of eukaryotic molecular species of MGDG, especially 20:5/20:5 MGDG, suggesting a possible adaptive role of eukaryotic molecular species to low growth temperature [71]. The involvement of PC as a substrate for the first dedicated step of the proposed x-3 pathway inP. cruentum has been validated by using salicylhydroxamic acid which inhibited D6 desaturation [72]. This inhibitor, as well as the herbicide SAN 9785, (which inhibits chloroplastic desaturation of linoleate to a-linolenate) has been also used to treat cultures of the eustigmatophyte Monodus subterraneus in order to elucidate the biosynthesis of EPA using radiolabelled acetate or linoleic acid [73]. The results indicated that PC was mostly involved in the desaturation of 18C fatty acids, whereas PE and DGTS were substrates for the further desaturation of 20C PUFAs which resulted in EPA production in this alga species. [73]. Furthermore, it was suggested that PE and DGTS were the donors of C20:5/C20:5-DAG (diacylglycerol) and C20:4/C20:5-DAG, respectively, which might be imported to the chloroplast after which their fatty acids were incorporated into eukaryotic-type molecular species of MGDG [73].

Fig. 1. Two pathways for the biosynthesis of eicosapentaenoic acid in eustigmatophytes (modified from [73]). The most active route is shown with bold arrows. D, desaturase; E, elongase.

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A similar role of PC and PE in the desaturation of 18C and 20C fatty acids, respectively, along the EPA biosynthetic pathway has been also suggested for another eustigmatophyte, Nannochloropsis spp. [74]. However, the role of DGTS (which contains 50–58% of EPA in a wild-type Nannochloropsis spp.) has been proposed to differ from that in M. subterraneus where only 24% of EPA was found [73–75]. A mutant of Nannochloropsis spp. (JS1) which was completely devoid of EPA was used for comparison in experiments which studied the biosynthetic pathway for EPA in Nannochloropsis spp. [75]. The alteration in the fatty acid composition of the mutant membrane lipids as well as their [14C]acetate labelling kinetics were interpreted as indicating that EPA was synthesized only in an extrachloroplastic location [75]. A very high content of EPA (over 90% of total fatty acids) has been reported for DGTS from the marine green alga, Chlorella minutissima [76]. Because of this high content it will be obvious that EPA is found at both the sn-1 and sn-2 positions of DGTS, in contrast to its asymmetric distribution in other algae. Furthermore, the large amount of DGTS (from 10% to about 44% of total lipids, depending on a monthly cycle) was accompanied by the presence of PC as the major phospholipid in contrast to a previously observed reciprocal relationship between DGTS and PC in other organisms [76,77]. In C. minutissima, the DGTS level showed a marked rhythmic fluctuation with time which was inversely correlated with the level of MGDG, the other major lipid in this alga. Moreover, the fatty acid composition of the latter was very similar to that of DGTS and, taken together, these findings also support the proposed role of DGTS as a donor of EPA for chloroplast lipids in eustigmatophytes (see above) [76]. A mutant of P. cruentum, which has been selected on the basis of impaired growth at suboptimal temperatures, was used to study lipid metabolism in this alga [78]. Lipid analysis of the mutant revealed diminished proportions of EPA and of the eukaryotic molecular species of MGDG and elevated proportions of TAG as compared with the wild-type. Pulse-labelling of the wild-type algae with radioactive fatty acid precursors showed an initial incorporation of the fatty acids into PC and TAG. In the chase period, the label of these lipids decreased with time while that of chloroplastic lipids, mainly MGDG, continued to increase. In the mutant, however, the labelling of TAG after the pulse was higher than that of the wild-type and decreased only slightly in the subsequent chase. It was concluded that in wild-type P. cruentum, TAG can supply acyl groups for the biosynthesis of eukaryotic species of MGDG [78]. Very long-chain (>20C) polyunsaturated fatty acid production and partitioning of such acids into TAGs have also been studied in the marine microalgae Nannochloropsis oculata (Eustigmatophyceae), Phaeodactylum tricornutum and Thalassiosira pseudonana (Bacillariophyceae), and the Haptophyte P. lutheri [79]. Differences in the time-course of production and incorporation of DHA and EPA into TAGs were found in these species. Such differences were not only observed between species but also during the different phases of growth within a species. In N. oculata, 90% of the total cellular fatty acids were found to be present in TAGs at the end of stationary phase. Levels of EPA in this alga remained fairly constant during the exponential phase but showed a significant increase when the cells entered stationary phase. 68% of the total EPA was accumulated in TAGs at the end of stationary phase in comparison to only 8% during the exponential phase of growth [79]. The authors suggested that the pathways responsible for the synthesis of EPA and partitioning of the latter into TAGs were both induced upon the transition of N. oculata from the exponential to the stationary phase of growth [79]. In P. tricornutum, on the other hand, the amount of EPA per cell did not show any significant increase upon transition to the stationary phase, although there was a significant increase in the amount of EPA in TAGs (from 3% to 40% of the total cellular EPA) [79]. In another diatom, T. pseudonana, total FA content increased significantly in stationary phase. In this growth stage, 74% of the total cellular FAs were present in the TAG fraction. The amount of EPA partitioning into TAGs also showed a significant increase, from 16% to 67% after 150 h in stationary phase [79]. In contrast to these four species, the total FA content per cell decreased upon transfer to stationary phase in P. lutheri. By the end of this stage, 52% of the total FAs were present in TAGs. Although there was an overall decrease in FA levels, EPA content (as micrograms of fatty acids per cell) increased during the incubation period [79]. These data indicate the wide variety in the details of lipid accumulation and EPA levels in different algal species and the difficulty for general extrapolation of results from one organism to another. Detailed analysis of the lipid classes of P. lutheri, cultivated in a semicontinuous mode, revealed that nonpolar lipids and glycosylglycerides were the major constituents, representing about 57% and 24% of the total lipids on a fatty acid basis, respectively [80]. Phospholipids accounted for 10% of the total fatty acids. Acylated

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steryl glycosides and diphosphatidylglycerol were identified in this species for the first time. The distribution of PUFAs within individual lipid classes revealed that EPA was mainly accumulated in MGDG (45% of the total fatty acids) and TAG (33% of the total fatty acids) [80]. A significant amount of very long-chain PUFAs has been found in lipids of chlorarachniophytes [81]. These algae are unicellular organisms isolated from warm marine environments and characterized by an amoeboid morphology that may be the result of secondary endosymbiosis of a green alga by a non-photosynthetic amoeba or amoeboflagellate. The fatty acids from acylglycerolipids including chloroplast-associated glycolipids, storage triacylglycerols and cytoplasmic membrane-associated polar lipids, were characterized from four species representing three chlororachniophyte genera: Bigelowiella natans, Gymnochlora stellata, Lotharella amoeboformis and a Lotharella sp., to determine whether the fatty acid profiles may be indicative of a chlorophyte origin [81]. The polar lipid fraction did not contain any phospholipids commonly found in eukaryotic algae and consisted mainly of phosphorus-free lipids, including the betaine lipids (as concluded from a positive Dragendorf reagent test). The major fatty acids of this fraction were palmitate and C22:5n-3. EPA together with palmitic acid were the major acids in the glycolipid (MGDG, DGDG) and triacylglycerol fractions. The authors concluded that the results of their study supported a chlorophyte endosymbiont origin but Chlorella could not be considered as the endosymbiont source since the fatty acid composition of glycolipids from those chlororachniophytes which were analysed did not correspond to any species of Chlorella examined to date [81]. However, it should be mentioned that C. minutissima has been reported to contain about 90% of EPA in its DGTS [76] and could, in theory, be a possible endosymbiont for chlororachniophytes. Although no data are available on the fatty acid distributions in various non-polar and galactolipids in this species, the high levels of EPA in DGTS would be consistent with some of the suggestions made for the endosymbiont origins in chlororachniophytes [81]. Due to its ability to accumulate up to 30% of the total acids as EPA, a marine diatom P. tricornutum has been widely used as a food organism in aquaculture [82]. In addition, it has been shown that lowering temperature during its growth was a significant factor affecting the level of EPA accumulated in this organism. When temperature was shifted from 25 to 10 °C for 12 h, EPA concentration increased by 120% compared with the control [82]. A detailed study of fatty acid distribution among the different acyl lipid classes and the influence of culture age and nitrogen concentration on this distribution was made in steady-state continuous cultures of P. tricornutum [82]. Hexadecenoic acid and EPA were the two major fatty acids in this microalga, and, together, they accounted for about 50% of total fatty acids. Only slight changes in the fatty acid composition were observed when the culture age changed. The only trends were moderate increases in palmitate, hexadecatrienoate and EPA and a decrease in hexadecenoate from older to younger cells [83]. However, average cell age had a strong impact on lipid classes, producing changes in the amounts of TAG (from 43% to 69% of total lipids) and in galactolipids (from 20% to 40%) at different cultivation stages. In general, the content of polar lipids tended to decrease with culture age [83]. The effect of nitrogen concentration was assayed by using different dilutions of the standard (high enough to support a nitrogen-saturated, photo-limited growth) medium used. When the nitrogen concentration was decreased, saturated and monounsaturated fatty acids accumulated [83]. The fraction of galactosylglycerides also reduced from 21% to 12% with corresponding increases in the content of both non-polar and phospholipids from about 73% to 79% and from 6% to 8%, respectively. In earlier studies, it was established that EPA can be synthesis in P. tricornutum by the classical x-6-pathway (Fig. 1), the classical x-3-pathway (Fig. 1), a pathway relying on intermediates from both of these pathways and by an alternative x-3-pathway involving D9-elongation and D8-desaturation [84]. Using [14C]linoleic acid in pulse-chase experiments, it has been suggested that the third route was the most active one, i.e. D12D

x3D

D6D

D6E

D5D

C18 : 1n-9 ! C18 : 2n-6 ! C18 : 3n-3 ! C18 : 4n-3 ! C20 : 4n-3 ! C20 : 5n-3 EPA productivity in the microalgae Navicula saprophila, Rhodomonas salina and Nitzschia sp. has been studied under photoautotrophic, heterotrophic and mixotrophic conditions [85]. Under photoautotrophic conditions, the proportions of EPA as a percentage of total fatty acids were 20.1% for N. saprophila, 15.4% for R. salina and 24.7% for Nitzschia sp. Mixotrophic conditions in the presence of acetic acid was found to promote a high growth rate and high EPA content of the biomass in the Nitzschia sp. [85].

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4.2. Synthesis of docosahexaenoic acid The chloroplast-less heterotrophic marine microalga Crypthecodinium cohnii has been proposed (and commercially used) as a source of another very long-chain PUFA of the x-3 family, docosahexaenoic acid, C22:6n-3 (DHA). This alga accumulates relatively large amounts of lipids (20% of biomass.) and has very unique fatty acid composition (see [86]). The level of DHA in this alga is about 30–50% of the total fatty acids but no other PUFAs, which are common intermediate products in the cascade of elongation and desaturation reactions from 18C precursors, were present above 1% [86]. This remarkable metabolic characteristic makes C. cohnii a useful organism for studying the biosynthesis of DHA in algae. De Swaaf et al. [86], when studying the optimization of DHA production inC. cohnii, showed that sea salt concentrations above half the average seawater salinity were required for good growth and lipid accumulation. The range of glucose concentrations, supporting good growth, was between 25 and 84.3 g/l. They also showed that, although higher temperatures (30 °C) favored growth, lipid accumulation was higher at a slightly lower (27 °C) incubation temperature [86]. The potential use of ethanol as a carbon source for large-scale production of DHA has been studied in batch-fed cultures of C. cohnii, with the highest value (53 mg/l/h) for DHA accumulation being reported [87]. The biosynthesis of DHA in this species has been studied by heavy isotope (13C) labelling and desaturase inhibitor experiments [88]. In contrast to previous work where a strong positional preference of C22:6 in TAG for the sn-1 and sn-3 positions was reported [89], the labelling study revealed a uniform intramolecular distribution of DHA on the glycerol backbone of lipids in C. cohnii. [13C]Label from both of the precursors used, [1-13C]acetate and [1-13C]butyrate, were incorporated regularly into DHA and all the odd carbon atoms but not the even ones were enriched [88]. When [1-13C]oleic acid was added to the growth medium, it was incorporated into the lipids but was not converted into DHA. The authors suggested that the system in C. cohnii responsible for DHA production may synthesize it only de novo with a two-carbon unit as the basic building block [88]. Specific desaturase inhibitors (norflurazon and propyl gallate) inhibited lipid accumulation but the fatty acid patterns were not altered in contrast to the effect of these inhibitors on fatty acid synthesis in the arachidonic-producing fungus, Mortierella alpina [88]. The authors explained their results by the presence of three tightly regulated separate systems for fatty acid production by C. cohnii, namely (1) for the biosynthesis of saturated fatty acids; (2) the conversion of saturated fatty acids into monounsaturated fatty acids and (3) the de novo synthesis of DHA [88]. In connection with the latter system, the very low level of any C18 PUFA in C. cohnii (see above and 86) was suggested to indicate that the pathway to DHA is tightly regulated so that very few intermediates escape. (However, see the discussion of the polyketide synthase pathway later.) Another alga, P. lutheri, has been found to have DHA distributed in significant quantities throughout its lipids. Within these, DHA was highest in TAG (27%), diphosphatidylglycerol (22%) and betaine lipids (21%) [80]. Certain species of thraustochytrids are being currently explored as potential sources of very long-chain PUFAs, especially for the nutritional enrichment of food products and use as feed additives in aquaculture [90–93]. Thraustochytrids are a common type of marine microheterotroph isolated from sub-tropical mangroves, cold and cool water, and littorals. They produce substantial amounts of DHA and docosapentaenoic acid (DPA), C22:5n-6 [94,95]. After growth optimization of Schizochytrium spp., both in flask and fermenter cultures, the highest DHA and DPA levels accumulated at the rate of 2.0 and 0.44 g/l per day, respectively, in the medium used. The lipid extracted from the cells was about 50% of the dry biomass and contained 93% TAG. DHA content of the total lipid fraction was 34% of the fatty acids [94]. Following growth of Schizochytrium sp. strain SR21 in a medium containing 12% glucose, the lipid content was 77.5% of dry biomass and DHA accounted for 35.6% of total fatty acids [95]. Non-polar lipids accounted for 95% of the total lipids in this strain. PC, PE and PI were the major phospholipids identified in relative amounts of 74%, 11% and 5%, respectively. 75% of PC molecular species were 1-palmitoyl-2-DHA-PC and 1,2-diDHA-PC [95]. The fatty acid profiles of three strains of S. mangrovei, which were recently isolated from decaying leaves in the intertidal zone of Hong Kong mangroves, showed that the percentage of DHA varied from 32% to 39% of total fatty acids, depending on strain. Myristate, palmitate and DPA, together with DHA, were the main fatty acids. Only slight changes in cell fatty acid composition were found due to growth stage [92]. In another thraustochytrid, Thraustochytrium aureum, TAG was again the dominant lipid and it contained about 40% DHA [96].

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The biosynthesis of DHA is thought to involve D6 desaturation of a-linoleate to C18:4n-3, followed by elongation to C20:4n-3 and D5 desaturation to EPA. The further reactions were not clearly established until recently [97]. The conventional assumption was that 20:5n-3 was elongated to 22:5n-3 and then converted to 22:6n-3 by the final D4 desaturation [98]. (Note that this differs from the Sprecher pathway in animals [99], see below.) Since thraustochytrids accumulate large amounts of DHA and its precursor DPA, they served as model organisms for studying the mechanism of DHA synthesis. The identification of a D4 desaturase from Thraustochytrium spp. by Qiu et al. [98] finally provided unambiguous evidence for the conversion of 22:5n-6 to DHA in this organism (Fig. 2). By contrast, the pathway for DHA biosynthesis in mammals, the Sprecher pathway, which is D4 desaturase independent, involves two consecutive elongations, D6 desaturation in the endoplasmic reticulum and a two-carbon shortening via limited b-oxidation in peroxisomes (Fig. 2) (see [98]). Recently an alternative pathway for DHA biosynthesis catalyzed by polyketide synthase was reported to occur in Schizochytrium spp. [100]. When this organism was labelled with [1-14C]acetate, DHA contained 31% of the label recovered in fatty acids, and this percentage remained constant during the 10–15 min of [1-14C]acetate incorporation and the subsequent 24 h of culture growth [100]. Similarly, DPA represented 10% of the label throughout the experiment. The incorporation kinetics of 14C-labelled palmitoleic, oleic

Mammals

Algae

Fig. 2. Comparison of the aerobic pathway for DHA biosynthesis in mammals and algae.

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and a-linolenic acids showed that over 90% of the label was accumulated into TAG and phospholipids. 24 h after the fatty acid supplementation, about 98% of the radioactivity was recovered as the original fatty acid supplied and no label was detected in DHA or DPA indicating that there was no precursor-product relationship between the 16- or 18-carbon fatty acids used and the 22-carbon PUFAs [100]. [1-14C]Malonyl-CoA was incorporated into DHA, DPA and saturated fatty acids of a cell-free homogenate of Schizochytrium. The same biosynthetic activities were retained in a 100,000 g supernatant fraction but no activities were found in the membrane pellet. Thus, the authors concluded that no membrane-bound desaturases or fatty acid elongases were involved in the biosynthesis of DHA and DPA in this organism [100]. Addition support for the polyketide synthase pathway was provided by the sequencing of 8500 randomly selected clones from a Schizochytrium cDNA library which failed to reveal the expected number and types of various desaturases in the aerobic pathway whereas polyketide synthase-homologous sequences were well represented [100]. The polyketide pathway has been clearly demonstrated in the marine bacterium Shewanella, as well as in Schizochytrium [100], but it remains to be seen how many other algae use this mechanism for EPA or DHA synthesis. 4.3. Arachidonic acid occurrence and formation Whereas very long-chain x-3 fatty acids are quite abundant in microalgae, those of the x-6 family like C20:3n-6 and C20:4n-6 (or arachidonic acid, AA), are almost excluded from the lipids of fresh water algae and account for only a few percent of total fatty acids in the marine species [101]. The green alga Parietochloris incisa (Trebouxiophyceae) isolated from the snowy slopes of Mt. Tateyama (Japan) has been shown, unusually, to contain 33.6% AA of the total fatty acids in the logarithmic phase and 42.5% in the stationary phase [101]. Other major fatty acids identified were palmitate, oleate and linoleate. The polar lipid profile was similar to that of other Chloroccoccales and included the galactolipids, MGDG and DGDG, the sulfolipid sulfoquinovosyldiacylglycerol (SQDG), the phospholipids PC, PE, PG, PI and phosphatidic acid (PA). A betaine lipid, DGTS, has also been identified in P. incisa. TAG was a major lipid class even in logarithmic phase, where it accounted for 43% total fatty acids [101]. In the logarithmic phase, AA was a major component of both the galactosylglycerides and its proportion decreased significantly during the stationary phase. A similar decrease in the proportion of AA during the stationary phase was noted for all the phospholipids [101]. In contrast, TAG accumulated larger amounts of AA in the stationary phase (from 43% to 47%) at the expense of palmitate. Based on the well-established accumulation of PUFAs during responses to different ecological factors (e.g., high light intensity and UV radiation, low temperature), the authors suggested that the capacity of some algae to store very long-chain PUFAs in TAG could be a reserve which would allow the organisms to adapt to any further rapid changes in its environment [101]. P. incisa, which tends to inhabit harsh habitats, was taken as a good example to support such a hypothesis, as well as a later study when induction of AA accumulation was caused by nitrogen starvation [102]. Further investigations were carried out to provide evidence in support of the hypothesis that P. incisa accumulates AA and TAG simultaneously in order to allow this alga to utilize AA from TAG for membrane reconstruction during adaptation to changed environmental conditions [101]. In experiments with [1-14C]arachidonic acid, it was shown that a reduction in temperature led to a rapid transfer of label from TAG to polar lipids whereas, at 25 °C, the label stayed relatively unchanged in both lipid classes [103]. Treatment of the cultures of P. incisa with SAN 9785, which inhibits the chloroplastic x-3-desaturation, also caused a transfer of AA from TAG to galactolipids [103]. The authors thought that this result was consistent with their idea that stress could elicit the use of AA from TAG to permit membrane biogenesis and renewal [103]. P. incisa has also been used to examine metabolic pathways for the biosynthesis of arachidonic acid [104]. Thus, pulse-chase labelling with [1-14C]acetate showed its incorporation via the de novo synthesis of fatty acids as well as for elongation of 16C and 18C PUFAs. When the cultures were labelled with [1-14C]oleic acid, most of the label was located in non-chloroplast (microsomal) lipids suggesting that the first step of any lipidlinked fatty acid desaturation would take place on these lipids [104]. Furthermore, PC and DGTS appeared to be the major substrates for the sequential D12 and D6 desaturations of oleic acid to c-linolenate. The latter fatty acid has been suggested to be released from its lipid carrier after which it could be elongated to C20:3n-6 (presumably as an acyl-CoA substrate) which could then be reincorporated into PE and PC and

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finally desaturated by a D5 desaturase to AA (Fig. 3) [104]. In contrast to the pathway for conversion of linoleate to AA, MGDG probably acted as a substrate for the chloroplastic D12 and x-3 desaturations in P. incise which were common to higher plants and many green algae [104]. 4.4. Species with high levels of several very long-chain PUFAs The lipid composition of the diatom, S. costatum, has been recently re-examined by Berge et al. [105]. The membrane lipids contained high levels of MGDG, SQDG and PC. In the non-polar lipid fraction, TAGs, sterols and a C18:1 fatty alcohol were identified. During the exponential growth phase, PUFAs were the dominant fatty acids and the authors reported that C20:5n-3, C16:3n-4 and C16:4n-1 accounted for about 61% of the total fatty acids. (However, it should be noted that the authors did not properly identify the fatty acids. It seems unlikely that C16:4n-1 exists (it has never been reported in any organism) and is, more likely, C16:4n-3). DHA accumulated at the relative proportion of 5.6% [105]. EPA was located mainly in polar lipids varying from 30.2% in MGDG to 56.8% in DGDG while only 11.8% was found in TAG. DHA was identified in the large proportion in phospholipids, especially in PE (34.7%) but was practically absent in galactolipids. In TAG, its proportion accounted for 2.3% [105]. Lipid and fatty acid composition has been analyzed in the stationary-phase cultures of the microalgal flagellate Isochrysis galbana and the red alga P. cruentum, which accumulate large amounts of EPA+DHA and AA+EPA, respectively [106]. Non-polar lipids (TAG) and galactolipids (MGDG, DGDG) were the major lipid classes, representing 40–45% of the total, whereas phospholipids represented only 10–20%. In P. cruentum, TAG and MGDG contained the larger amounts of AA (24.2% and 21.0%, respectively), while DGDG and SQDG contained the larger amounts of EPA (20.3% and 19.2%, respectively). In I. galbana, 38.6% and 27.7% of EPA accumulated in PC and MGDG, respectively. In this species, the percentage of DHA reached 64% in PE and 39% in PC [106]. Variations in lipid classes and fatty acid composition of the diatom Chaetoceros muelleri and Isochrysis sp. grown in a semicontinuous system have been studied during the entire process of hatchery cultivation in relation to an annual cycle [107]. At the beginning of the cultivation process for C. muelleri, in a small volume of culture, TAG, sterol, saturated, monounsaturated and PUFAs, particularly EPA, were high, sharply dropping when the growth medium was increased. An inverse relationship was noted for the galacto- and phospholipids, resulting in no significant effect of the culture volume on the total lipid content in this alga. In Isochrysis spp., the total lipid content increased with the volume of culture mainly due to a rise in polar lipid classes. At the same time, saturated and PUFAs accumulated [107]. The fatty acid profiles of six Codium species, collected from south east Australia, have been analyzed [108]. The major fatty acids were C16:0, C16:3n-3, C18:1n-9, C18:3n-3, C18:2n-6 and C20:4n-6. The total content of PUFAs ranged from 17.2% to 54.5%. Significant variations in individual fatty acid contents in relation to species, season and location were revealed [108].

Fig. 3. Alternative pathways for the biosynthesis of arachidonic acid in P. incisa (based on [103]). E, elongation; D, desaturase.

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The lipid composition of ice algae, which were dominated by assemblages of Melosira arctica and Nitzschia frigida, as well as phytoplankton from the pelagic zone (which were rich in flagellates) have been examined [109]. Total lipids from the Melosira assemblages had higher percentages of C16:4 and EPA than those from the Nitzschia assemblages. The phytoplankton from the pelagic zone contained less 16C PUFAs and EPA but more 18C PUFAs and DHA than the ice algae. Spring samples of ice algae showed that the major fatty acids of the non-polar lipid fraction were C16:0, C16:1n-7 and EPA whilst the glycolipid fraction was characterized by higher levels of EPA and 16C PUFAs [110]. Phospholipids contained higher levels of DHA than either glyco- or non-polar lipids although EPA was still the major PUFA. For samples collected in the autumn, EPA was the major PUFA in all lipid fractions. In general, the fatty acid compositions of the lipid fractions from spring and autumn algal samples were broadly similar and were consistent with diatoms being the predominant group in the ice algae studied. The high level of non-polar lipids observed in both spring and autumn samples was suggested to be a characteristic of ice algae regardless of season [110]. 4.5. Identification of genes involved in the biosynthesis of PUFAs in algae A cDNA for chloroplast x-6 (D12-) desaturase, which catalyzes the desaturation of monoenoic to dienoic acids in chloroplasts, has been isolated from C. reinhardtii [111]. The amino acid sequence showed 46–51% homology to those of the higher plant plastid x-6 and the cyanobacterial D12 desaturases. Introduction of the cloned genomic counterpart of this cDNA, designated as des6, into a Chlamydomonas mutant with defects in chloroplast x-6 desaturation (and also in activities of Photosystems I and II) complemented the desaturation mutation, indicating that the des6 gene codes for the chloroplast x-6 desaturase [111]. Two cDNA clones corresponding to D12 and x-3 fatty acid desaturase (FAD) genes (designated as CvFad2 and CvFad3, respectively) have been isolated from Chlorella vulgaris C-27 based on the sequence information from genes encoding for plant D12 and x-3 FADs which desaturate oleate to linoleate and linoleate to a-linolenate, respectively [112]. The deduced amino acid sequence ofCvFad2 showed about 66% similarity to those of higher plant microsomal D12 FADs and about 35% to plastidial D12 FADs. Accumulation of linoleic acid occurred whenCvFad2 was expressed in S. cerevisiae [112]. The predicted protein of CvFad3 had about 60% similarity to the microsomal and plastidial x-3 FADs and lower similarity to D12 FAD. Although CvFad3 seemed to encode a microsomal x-3 FAD (based on the features of the amino acid sequences of the Cand N-terminal regions and also fatty acid analysis of polar lipids in transgenic tobacco plants expressing this gene) rather than a plastidial one, the authors could not conclude the exact localization of the protein in Chlorella from their results [112]. It has also been shown that both genes (for the D12 and x-3 FADs) were upregulated by low temperature but in different ways. The level of the transcript of CvFad2 increased gradually during cold exposure and, after 24 h, it had reached 3.2 times the initial level. The level of the transcript CvFad3 increased up to 5.4 times the initial level after only 3 h of cold exposure but then gradually decreased [112]. This study showed clearly that the two desaturase genes were induced by low temperature and suggested the possibility that they might be involved in the development of low temperature freezing tolerance in Chlorella [112]. The marine diatom P. tricornutum has been used for cloning genes encoding fatty acid desaturases involved in EPA biosynthesis [113]. The coding sequences of two desaturases have been identified using a combination of PCR, mass sequencing and library screening. Both protein sequences contained the typical features of membrane-bound front-end desaturases (three conserved histidine clusters, an H to Q substitution in the third histidine-box and a cytochrome b5 domain fused at the N-terminus) [113]. The full length clones were expressed in S. cerevisiae and characterized as D5- and D6-fatty acid desaturases. In addition, it has been shown that these desaturases were not specific for the desaturation of a single fatty acid and had no preference for either the x-3- or the x-6-pathway viz. they accepted and modified every potential substrate encountered. Based on this and previous data, the authors suggested that such an indiscriminate use of x-3- and x-6-fatty acids may be a general feature of front-end desaturases [113]. A cDNA encoding a fatty acid elongating component (IgASE1), which was specific to linoleic and a-linolenic acids, has been isolated from the prymnesiophyte micro-alga I. galbana, and a D8 desaturation pathway has been suggested as a major route for EPA and DHA biosynthesis in this alga [114]. Transgenic yeastexpression of IgASE1 converted C18:2n-6 and C18:3n-3 acids to eicosadienoic acid (EDA; C20:2n-6) and

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eicosatrienoic acid (ETrA; C20:3n-3), respectively [114]. IgASE1 contained a variant histidine box (his-box) with glutamine replacing the first histidine of the conserved histidine-rich motif (HXXHH) as found in other equivalent proteins [115]. Site-directed mutagenesis was used to determine the importance of glutamine and other variant amino acid residues in the his-box. The results showed that all the residues were required for optimum enzyme activity but their substitution did not affect the substrate specificity [115]. Expression of IgASE1 in Arabidopsis also resulted into accumulation of EDA and ETrA in all transgenic plant tissues/ organs examined, with the novel 20C fatty acid content reaching 15 mol% of total leaf fatty acid [116]. Positional analysis of the various lipid classes revealed that 20C acids were excluded from the sn-2 position of chloroplast galactolipids, MGDG and DGDG, the chloroplast phospholipid PG and seed triacylglycerol, whereas they were enriched in the same position in PC. In general, the 20C PUFA content largely mirrored the ‘‘eukaryotic’’ component of the various lipid classes. Also, the positional distribution in PA was more similar to that observed in the chloroplast lipids than PC. This would be consistent with the author’s previous suggestion that the DAG moiety returned to the chloroplast may have originated from PA [116]. The results of the fatty acid distribution in MGDG and DGDG were consistent with discrete ‘‘prokaryotic’’ and ‘‘eukaryotic’’ pools of MGDG and DGDG synthesized at the inner and outer chloroplast membranes of Arabidopsis, respectively [116]. Two cDNA sequences that encoded D4 and D5 polyunsaturated fatty acid desaturases have been identified in Thraustochytrium sp. [98]. The cloning and functional characterization of a cDNA encoding a very longchain PUFA D4-desaturase has also been reported for P. lutheri [117]. Heterologous expression in yeast demonstrated that this enzyme desaturated 22:5n-3 and 22:4n-6 in to 22:6n-3 and 22:5n-6, respectively, and was equally active with both substrates. Expression of D4-desaturase varied during growth and was 4-fold higher during the mid-exponential and stationary phases [117]. The identification of genes involved in the two-step conversion of EPA into DHA has been reported by Pereira et al. [118]. From Pavlova spp., a gene (pavELO) encoding a novel elongase which catalysed the conversion of EPA into x-3-docosapentaenoic acid (DPA) in yeast was identified. In addition, a novel D4-desaturase gene (IgD4) was isolated from I. galbana which was capable of converting x-3-DPA into DHA, as well as adrenic acid (22:4n-6) into x-3-DPA. These two genes were functioning together to carry out the two-step conversion of EPA into DHA as yeast co-expression studies showed [118]. In a study by Meyer et al. [119], in vivo radiolabelling studies in combination with a-oxidation were used to produce biochemical evidence for the involvement of a D4-desaturase in the synthesis of DHA from DPA in E. gracilis and Thraustochytrium spp. These organisms efficiently converted exogenously supplied [2-14C]docosapentaenoic acid to DHA. Hydrogenation and a-oxidation of the labelled DHA isolated from these species showed that it was the result of direct D4-desaturation and not substrate breakdown and resynthesis [119]. Mass sequencing of a cDNA library from E. gracilis resulted in the isolation of a D4-desaturase cDNA clone. When expressed in yeast, the desaturase converted DPA to its D4-desaturated product DHA. The enzyme activity was not limited to 22C fatty acids, since it also desaturated 16C fatty acids. It is interesting to note that the enzyme seems to require the presence of a D7-double bond in the substrate [119]. In the marine microalga T. pseudonana, D11-desaturase activity was detected [120]. This desaturase was characterized as a cytochrome b5 front-end desaturase active exclusively on palmitic acid [120]. A further study on desaturases from T. pseudonana revealed the presence of 11 open reading frames which showed similarity to functionally characterized fatty acid front-end desaturases [121]. Phylogenetic analysis showed that two of the T. pseudonana desaturase sequences grouped with proteobacterial desaturases that lack a fused cytochrome b5 domain. From the remaining gene sequences, three have been reported to encode D6-, D5- and D4-desaturases involved in the production of DHA, and one encoded a D8-sphingolipid desaturase with strong preference for dihydroxylated substrates [121]. Two types of elongases were isolated from T. pseudonana and Osteococcus tauri (marine phytoplanktonic alga): one specific for the elongation of (D6-)C18-PUFAs and one specific for (D5-)C20-PUFAs , showing highest activity with EPA [122]. By co-expression of the D6- and D5-elongases from these algae respectively with D5- and D4-desaturases from two other algae (P. tricornutum and E. gracilis) the synthesis of DHA was successfully implemented in stearidonic acid-fed yeast [122]. Recently, the first acyl-CoA D6-desaturase cloned from a photosynthetically active organism, has been reported for O. tauri [123]. Genomic DNA of this fully sequenced alga was used to amplify a gene coding

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for a typical front-end desaturase involved in PUFAs biosynthesis. Very high desaturation activity with D6regioselectivity was revealed when expressed in S. cerevisiae [123]. Kinetics of desaturation with parallel analysis of acyl-CoAs and total acyl groups showed that the desaturase product was detected in the acyl-CoA pool five minutes after addition of the exogenous substrate to the yeast medium and long before its appearance in the total fatty acids. Coexpression of this desaturase with an acyl-CoA elongase and the lipid-linked D5-desaturase also confirmed this enzyme as an acyl-CoA D6-desaturase. Its protein sequence contained all the signatures of a microsomal membrane-bound front-end desaturase, but the O. tauri enzyme was not related to any previously described sequences for functionally characterized D6-desaturases [123]. Finally, it is noteworthy, that identification and functional characterization of enzymes involved in biosynthesis of long-chain PUFAs in algae has an important biotechnological significance in that they may represent new and promising tools for the biotechnological production of very long-chain PUFAs in transgenic oilseed crops [122–125]. The successful reconstitution of the D8-desaturation pathways for both x-6 and x-3 very long-chain PUFA biosynthesis in Arabidopsis thaliana has been reported. In this case, A. thaliana was transformed with a D9-elongase from I. galbana, a D8-desaturase from E. gracilis and a D5-desaturase from Mortierella alpine. The transgenic plants accumulated appreciable amounts of AA and EPA [126]. The transgenic production of significant amounts of very long-chain PUFAs in Brassica juncea seeds was achieved using a series of transformations including constructs with D5-desaturase and D6-elongases from Thraustochytrium sp. [127]. 5. Lipid metabolism in algae with no very long-chain PUFAs Some of the earliest work on fatty acid biosynthesis was carried out with the green alga, C. vulgaris [e.g., [6,128–130]] and such organisms continue to offer great possibilities for elucidating the metabolism and functions of acyl lipids. Biosynthesis of glycerolipids has been recently investigated in Chlorella kessleri with a special emphasis on the fatty acid distribution at the sn-1 and sn-2 positions in membrane lipids [131]. In galactolipids, 18C acids almost exclusively occupied the sn-1 position of MGDG, whereas both 16C and 18C acids were esterified to the sn-2 position. So-called ‘‘prokaryotic’’ and ‘‘eukaryotic’’ lipid species accounted for 65% and 35% of MGDG, respectively. For DGDG, 68% of the molecular species contained ‘‘eukaryotic’’ combinations of fatty acids as revealed by analysis of fatty acid composition at sn-2 position. Metabolism was studied a pulse-chase radiolabelling experiment with [2-14C]acetate as a precursor [131]. It was found that MGDG and DGDG were labelled well as the sn-1-18C–sn-2-16C (or C18/C16) species at the start of the chase, suggesting the synthesis of these galactolipids within chloroplasts via a prokaryotic pathway. The sn-1-18C–sn-2-18C (C18/C18, i.e., eukaryotic) species of these lipids gradually accumulated radioactivity at later times with a concomitant decrease of radioactivity in the C18/C18 species of PC. The authors explained their results by the supply of DAG from PC for galactolipid synthesis as proposed in the eukaryotic pathway [131]. So, C. kessleri was concluded to be similar to a group of higher plants (e.g., spinach) which contain hexadecatrienoate and which synthesize chloroplast lipids by the cooperation of prokaryotic and eukaryotic pathways and was distinct from most other green algae which used a mainly prokaryotic pathway for the synthesis of chloroplast lipids. The authors proposed that the physiological function of the eukaryotic pathway in C. kessleri was to supply chloroplast membranes with C18:3/C18:3-MGDG for optimal functioning, and that the acquisition of this pathway by green algae was favorable for the eventual evolution of land plants [131]. The composition and positional distribution of fatty acids in the polar lipids from four strains of Chlorella ellipsoidea, which differed in chilling susceptibility and frost hardiness, have been analyzed [132]. Analysis of the polar lipid fraction from chilling-sensitive, chilling-resistant and chilling-sensitive revertant strains of C. ellipsoidea revealed that the sum of palmitic and trans-3-hexadecenoic acids in PG was about 60% for the sensitive strains and 53% for the resistant strain. The amount in the resistant mutant strains is much higher than for the PG composition in cold-resistant plants where 20% would be a more typical value [133]. A difference in the total amount of fatty acid unsaturation which was apparently associated with chilling sensitivity of C. ellipsoidea was also shown for PC and PE. During hardening, the content of C16:3 at the sn-2 position of both DGDG and MGDG or of C18:3 at the sn-1 position in SQDG, DGDG and MGDG increased, and a similar tendency was noted for PC and PE. It is interesting that the chilling-resistant and unhardened strains

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of C. ellipsoidea contained a ‘‘eukaryotic’’ combination of fatty acids in PG, which was proposed to indicate a special role in chilling resistance in this algal species [132]. We studied the lipid metabolism in four green algae which were lichen photobionts. There were two species each of Trebouxia spp. and of Coccomyxa spp. and we examined metabolism by following incorporation of radiolabel from [1-14C]acetate [134]. All the algal species studied were able to accumulate the typical storage lipid, TAG, but their lipid metabolism was much more concentrated towards the maintenance of membrane lipids. Phospholipid synthesis was important in all algae, with PG and PC labelled the most. Total phosphoglyceride labelling was 30–35% of total polar lipids in Coccomyxa and 60–78% in Trebouxia spp. The typical chloroplast glycosylglycerides MGDG, DGDG and SQDG were appreciable labelled in all species. A betaine lipid DGTS was found in all the algae, except Trebouxia aggregata, but was very minor in T. erici and C. mucigena. Both Coccomyxa spp. contained a rhamnose-lipid together with another sugar-containing, but unidentified, lipid. All the algal photobionts had palmitic as their main saturated fatty acid, oleate as their main monoenoic acid and linoleic and a-linolenic as the main polyunsaturated fatty acids. All these species also contained significant amounts of various 16C unsaturated fatty acids, including hexadecatetraenoate [134]. 6. Effects of the environment on lipid metabolism 6.1. Nutrients and nutritional regimes The effect of nutrient-limitation on the lipid and fatty acid composition of C. moewusii was found to result mainly in alterations in the fatty acid composition of the chloroplast lipids, PG and MGDG [59]. The PUFAs C16:3, C16:4 and C18:3, characteristic of the plastidic galactolipids, and C16:1(D3-trans), specific for plastidic PG, decreased in nutrient-limited medium. The concomitant rise in C16:0 and C18:1 which was found was suggested to reflect synthesis of non-polar storage lipids since these fatty acids predominated in such lipids in C. moewusii [59]. Growth characteristics have been shown to have a significant impact on the fatty acid profiles of Chlamydomonas spp. when cultivated at 20 °C [135]. In this alga, the concentration of PUFAs decreased progressively when the growth conditions changed from photoautotrophic via mixotrophic to heterotrophic [135]. In addition, the lipid class and fatty acid compositions of E. gracilis have been analyzed after cultivation under various conditions of autotrophy and photoheterotrophy, in order to estimate the contribution of lactate (a carbon source) and ammonium phosphate (a nitrogen source) to lipid metabolism [136]. Effects of increasing ammonium phosphate concentration on lipid composition were observed only when lactate was depleted. These effects resulted in an increased content of galactolipids rich in polyunsaturated 16C and 18C fatty acids, and in an enhanced MGDG to DGDG ratio. Nitrogen had no influence on the content of medium chain (12– 14C) acids but induced a reduction of 22C acids [136]. In the absence of ammonium phosphate in the cultural medium, increasing lactate concentrations were accompanied by a decrease in all plastid lipids, whereas the content of storage lipids (enriched in myristate and palmitate) increased. Formation of 18C PUFAs appeared to be reduced, as indicated by accumulation of oleate [136]. Nutrient-limitation in the freshwater diatom Stephanodiscus minutulus was found to have pronounced effects on its lipid composition [137]. This alga was grown under silicon, nitrogen, or phosphorus limitation. All of the nutrient-limited cells showed an increase in triacylglycerols and a decrease of polar lipids as expressed as percentages of total lipids [137]. Nitrogen starvation and, surprisingly, also very high levels of nitrogen (15 mM) increased crude lipid content (as a percentage of dry weight) in Ulva pertusa [138]. Increasing the nitrogen concentration decreased the proportion of the major PUFAs C16:4n-3 and C18:4n-3 and increased the proportion of palmitate, C18:1n-7 and linoleate. Conversely, phosphorus starvation decreased the proportion of palmitate and increased that of C16:4n-3 with no effect on the total lipid content of the seaweed [138]. The effect of phosphorus-limitation has been studied in seven species of marine algae which belong to five different classes of microalgae [139]. Phosphorus-limitation led to increased lipid content in P. tricornutum, Chaetoceros sp. and in P. lutheri, but a decreased lipid content in the green flagellates, Nannochloris atomus and Tetraselmis sp. More severe nutrient-limitation resulted in a higher relative content of palmitate and oleate and a lower relative content of C18:4n-3, EPA and DHA [139]. In contrast, an enhanced level of unsaturated fatty acids in all those individual

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lipids analyzed (PC, PG, DGDG, MGDG, SQDG) has been reported in phosphorus-starved cells of the green alga C. kessleri [140]. The effect of sulfur- and phosphorus-limitation on the acidic lipids in thylakoid membranes has been studied in C. reinhardtii [141]. Those cells grown with a limited sulfur source, as compared with those grown under normal conditions, showed a loss of most of their SQDG. In these cells, PG content increased by 2-fold, thus, compensating for the reduced level of another anionic lipid, SQDG. C. reinhardtii cells exposed to limited phosphorus showed a 40% decrease in PG and a concomitant increase in the SQDG content as compared with the control samples. These results have been suggested to imply the existence of a mechanism which keeps the total content of SQDG and PG constant under both phosphorus- and sulfur-limiting conditions. Moreover, it also has been proposed that SQDG can substitute for PG to some extent in order to sustain the functional activity of chloroplast membranes [141]. It is generally accepted now, that phosphate-limitation usually causes the replacement of membrane phospholipids by non-phosphorus glycolipids and betaine lipids (the latter shown in the photosynthetic bacteria, Rhodobacter sphaeroides [142]) and, thus, represents an effective phosphate-conserving mechanism [143]. However, when the lipid metabolism of four green algal lichen photobionts with different phosphorus status was examined by labelling from [1-14C]acetate, only minor alterations were noted [134]. Two levels of phosphate (0.017, 1.7 mM) were used and, although growth and total lipid labelling were impaired in low phosphate media, there were only minor changes in the relative rates of phosphoglyceride labelling and hardly any decrease in the relative labelling of PG. In this case, it was concluded that the algae maintained their phosphoglyceride synthesis because there were significant endogenous phosphorus stores in the algae as revealed by X-ray probe electron microscopy [134]. The effect of CO2 concentration on glycerolipid synthesis has been studied in C. kessleri [144]. In this study, cells were grown under ordinary air (low-CO2 cells) or under CO2-enriched air (high-CO2 cells) and labelled from [14C]acetate. The fatty acid distribution at the sn-1 and sn-2 positions of each of the major lipids (MGDG, DGDG, PC, PE), as well as the patterns of labelled fatty acids and lipids, were compared. In comparison to high-CO2 cells, low-CO2 cells showed elevated contents of a-linolenate, especially at both the sn-1 and sn-2 positions of MGDG and DGDG, and also at the sn-2 positions of PC and PE. When the cells were labelled with [14C]acetate, slower rates of linolenate labelling of the major lipids, together with the lower incorporation into total membrane lipids was observed in the low-CO2 cells compared to the high-CO2 cells. It was suggested that the higher unsaturation levels in low-CO2 cells were at least partially due to repressed fatty acid synthesis, which allowed desaturation of pre-existing fatty acids. In both galactolipids, the contents of ‘‘eukaryotic’’ molecular species in low-CO2 cells were higher than those in high-CO2 cells, indicating relatively greater metabolic flow through this pathway compared to the ‘‘prokaryotic’’ pathway for galactolipid biosynthesis. The authors proposed that, together with the repression of fatty acid synthesis, the increased synthesis of C18/C18 species of galactolipids, which could then be used as substrates for chloroplast-located desaturation, contributed to the higher contents of linolenate in low-CO2 cells compared to the high-CO2 cells [144]. The effect of CO2 concentration on the fatty acid composition of lipids has also been studied in wild-type C. reinhardtii and its cia-3 mutant strain which is devoid of the chloroplast carbonic anhydrase (chlCA) and deficient in a CO2-concentrating mechanism [145]. In the mutant, a high content of PUFAs was found when it was grown at a high CO2 concentration. In both strains, there was some increase in the total PUFA content as a result of the decrease in CO2 concentration from 2% to 0.03%. However, in the mutant, the increase in PUFAs was less pronounced and certain components (e.g., C16:4n-3) did not change. Thus, in this study, a correlation between the induction of the CO2-concentrating mechanism and an acceleration of fatty acid desaturation was suggested and, of course, this was less obvious in the mutant devoid of chlCA which is one of the components of the CO2-concentrating system [145]. The effect of CO2 on the content and composition of lipid fatty acids and chloroplast lipids, in particular, in the unicellular halophilic green alga Dunaliella salina (known to be susceptible to CO2 stress) have been investigated [146]. Even a one-day-long increase in the CO2 concentration from 2% to 10% was shown to provoke an increase in the total amount of fatty acids on a dry weight basis by 30%. Differences in the fatty acid content and composition indicated increased fatty acid synthesis de novo but an inhibition of their elongation and desaturation. This led to an increase in the relative content of saturated fatty acids at 10% CO2. After one-daylong CO2 stress, the MGDG/DGDG ratio increased 4-fold while the ratio of x-3/x-6 fatty acids, as well as the

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proportion of trans-16:1D3 in PG increased sharply. The authors suggested that these changes might represent an adaptation of the photosynthetic membranes to ensure effective photosynthesis in D. salina under these conditions [146]. 6.2. Other growth conditions The effect of ambient temperature on the composition of intracellular fatty acids and the release of free fatty acids into a medium by two microalgae, C. vulgaris and B. braunii has been studied under batch culture conditions [147]. Both species responded to the temperature regimes by similar changes in their intracellular fatty acid composition, showing a decrease in the relative content of more unsaturated fatty acids, especially trienoic ones, when temperature increased but there were no significant changes in the composition of extracellular unsaturated free fatty acids detected for these algae following temperature elevation [147]. There was no effect of temperature on the content of the acidic lipids SQDG and PG when studied in C. reinhardtii [141]. A shift in temperature from 25 to 10 °C led to an increase in the relative proportion of oleate but a decrease in linoleate and parinate (C18:4), together with a significant increase in the relative proportion of ergosterol in the green alga Selenastrum capricornutum [148]. These rather complex changes emphasize that a simple correlation of increased unsaturation with lower growth temperatures is often not seen. More subtle alterations are usually found. Lipids and fatty acids in cultures of the haptophyte I. galbana grown at 15 and 30 °C were analyzed and compared [149]. Total lipids accumulated at a higher rate at 30 °C with a slight decrease in the proportion of non-polar lipids, an increase in the proportion of glycosylglycerides but no change in the proportion of phospholipids. Cells grown at 15 °C contained higher levels of a-linolenate and DHA with a corresponding decrease in linoleate, monounsaturated and saturated fatty acids [149]. The fatty acid content of four tropical Australian microalgal species, diatom Chaetoceros sp., two cryptomonads, Rhodomonas sp. and Cryptomonas sp. and an unidentified prymnesiophyte, cultured at five different temperatures have been reported [150]. EPA was identified in all species with the highest amount in the prymnesiophyte where its percentage decreased at higher temperatures. All species had lower percentages of DHA at higher temperatures. The Chaetoceros sp. and the prymnesiophyte contained moderate amounts of AA which accumulated to its higher levels at growth temperatures within the range 27–30 °C [150]. The effect of different levels of light and salinity on fatty acid composition was studied in three seaweeds U. pertusa (Chlorophyta), Grateloupia sparsa (Rhodophyta) and Sargassum piluliferum (Phaeophyta) [151]. In U. pertusa, exposure to a combination of high light intensity and low salinity resulted in a large decrease in the total (mg/g dry weight) fatty acid content. Growth at high light intensity increased the content of most saturated fatty acids (myristate, pentadecanoate, palmitate and iso-heptadecanoate). In G. sparsa, low light and high salinity exposure caused significant increases in the total fatty acid content, total PUFAs, total saturated and total monoenoic acids compared to normal salinity. High light intensity increased the levels of myristate, oleate, vaccenate, EPA and total n-3 fatty acids. In S. piluliferum, high light intensity resulted in a decrease in the levels of almost all fatty acids whereas higher salinity enhanced the levels of C18:4n-3, AA and EPA as well as total n-3 and n-6 acids [151]. In Nannochloropsis sp., the degree of unsaturation of fatty acids decreased with increasing irradiance, with a 3-fold decrease of the percentage of total n-3 fatty acids (from 29% to 8% of total fatty acids), caused mainly by a decrease of EPA [152]. The effect of light (high levels of 300 lmol photons/m/s or low levels of 6 lmol photons/m/s) was studied in the filamentous green alga, Cladophora spp. [153]. The total phospholipid content of Cladophora decreased under high light exposure with a concomitant increase of the concentration of non-polar lipids, mainly TAG. The concentrations of two acetone-soluble polar lipids (probably, MGDG and DGDG) were significantly enhanced in low light conditions. Analysis of total fatty acids showed that low light caused a decrease in the relative percentage of palmitate and an increase in those of palmitoleate and a-linolenate [153]. Further studies of light exposure were made with the diatom T. pseudonana and, again, significant changes in fatty acid and lipid composition were observed. The light regimes used were 100 lmol photon/m/s on a 12:12 h light:dark (L:D) cycle; 50 lmol photon/m/s on a 24:0 h L:D cycle and 100 lmol photon/m/s on a 24:0 h L:D cycle [154]. Cells grown under 100 lmol photon/m/s continuous light showed high accumulation of TAG and a reduced percentage of the total polar lipids. The fatty acid composition of logarithmic phase

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cells grown under the two continuous light regimes were similar, but the 100 lmol photon/m/s 12:12 L:D cells contained a higher proportion of PUFAs and a lower proportion of saturated and monounsaturated fatty acids mainly due to different levels of palmitate, palmitoleate, C16:4, C18:4n-3 and EPA. With the onset of stationary phase, cells grown under 100 lmol photon/m/s continuous light contained increased proportions of saturated and monounsaturated fatty acids and decreased amounts of PUFAs. Concentrations (as % dry weight) of myristate, palmitate, palmitoleate, EPA and DHA increased in all cultures during stationary phase [154]. Not only reduced light but also darkness has been studied to see if lipid content is altered. In the green algae S. capricornutum, dark treatment resulted in a decrease in the relative proportion of oleate and an increase in linoleate, together with an increase in the relative proportions of D7-chondrillastenol and a decrease in chondrillasterol [148]. For the dinoflagellate Prorocentrum minimum, dark exposure gradually reduced the content of TAG and galactosylglycerides (GL), whereas the total content of phospholipids changed little (PC, PE and PG decreased; PS, PA and PI increased) [155]. An increase in the activity of b-oxidation and isocitrate lyase paralleled the decrease of TAG and GL. These observations were suggested to indicate that TAG and GL were utilized as alternative carbon sources by the cells under non-photosynthetic growth conditions [155]. Variations in the lipid composition of the marine red alga Tichocarpus crinitus exposed to different levels of photon irradiance have been noted [156]. The contents of both storage and structural lipids were affected significantly by light intensity. Exposure of the alga to low light conditions [8–10% of the incident photosynthetically active radiation (PAR)] induced an increase in the abundance of some of the cell membrane lipids, especially SQDG, PG and PC, while growth of algae at higher light intensities (70–80% of PAR) resulted in a 1.5-fold increase in the level of storage TAGs. There were no significant differences in the fatty acid composition of the total lipid pool in T. crinitus grown under different light conditions. However, the content of the most unsaturated acid, EPA, was slightly higher in the algae under low light compared to that at high light. Exposure of algae to low light caused an increase in the content of EPA in MGDG and a decrease of that in PG. The concentration of trans-C16:1 acid in PG increased in T. crinitus grown under high light intensity [156]. In the green alga Ulva fenestrate, grown at 24% PAR, the amounts of MGDG, SQDG and PG increased 2– 3.5 times compared to algae cultivated at 80% PAR [157]. On the other hand, the content of DGDG and betaine lipid showed little dependence on light intensity as did the relative proportions of fatty acids in TAG, MGDG and SQDG. Variations in the fatty acid compositions of DGDG and PG and changes in the amounts of different lipid classes were responsible for the differences in the total fatty acid composition with various light intensities. The biggest changes were seen with palmitate and C16:4n-3 [157]. Some algae are capable of growing under quite extreme conditions of pH and it is, therefore, not surprising that there have been studies to see whether moderate pH changes can affect lipid metabolism in non-extremophiles. For Chlorella spp., alkaline pH stress resulted in TAG accumulation and a decrease in membrane lipid classes (and, presumably, membranes) [158]. The lipid and fatty acid composition of a Chlamydomonas sp. isolated from a volcanic acidic lake and C. reinhardtii obtained from an algal collection (Institute of Applied Microbiology, Tokyo) were compared, and the effects of the pH of the medium on lipid and fatty acid composition of these Chlamydomonas spp. were studied [159]. The fatty acids in polar lipids in the unidentified Chlamydomonas sp. were more saturated that those of C. reinhardtii. The relative percentage of TAG in the total lipid content in Chlamydomonas spp. grown in medium at pH 1 was higher than that in cells grown at higher pHs. It was suggested, that the increase in saturation of fatty acids in membrane lipids of Chlamydomonas may represent a possible adaptation mechanism for low pH in order to decrease membrane lipid fluidity [159]. Not only are some algae well adapted to extreme pHs, they can also tolerate high salt. D. salina is an example of the latter. Azachi et al. [160] showed that expression of b-ketoacyl-coenzyme A (CoA) synthase (Kcs) (which catalyzes the first and rate-limiting step in fatty acid elongation) increased in D. salina cells transferred from 0.5 to 3.5 M NaCl. They suggested that salt adaptation in Dunaliella entailed modifications of the fatty acid composition of algal membranes, and lipid analysis indicated that microsomes, but not plasma membranes or thylakoids, from cells grown in 3.5 M NaCl contained a considerably higher ratio of 18C (mostly unsaturated) to 16C (mostly saturated) fatty acids compared with cells grown in 0.5 M NaCl. The salt-induced Kcs, jointly with fatty acid desaturases, was thought to play a role in adapting intracellular membrane com-

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partments to function in the high internal glycerol concentrations used to balance the external osmotic pressure created by high salt [160]. 6.3. Anthropogenic factors Environmental pollutants are know to affect algal lipid metabolism (see [5,6]) and heavy metals have been studied in this regard (see [5,6] for references). A recent study has evaluated the potential toxic effects of cadmium (2 lg/ml) on membrane lipids of E. gracilis. This was studied using autotrophic, heterotrophic (in the dark) and mixotrophic (in the light) cultures [161]. Cadmium caused an increase in the total lipid content per cell in all three culture systems. Among the membrane lipids, sterol content was lower in cadmium-treated cells cultivated under illumination. There were no changes in the total phospholipid content, although the cardiolipin levels were altered in all three cell types and, in mixotrophic cells, there was an increase in PG [161]. E. gracilis has also been shown to display somewhat different sensitivities to copper and zinc. Thus, the apparent LC50 for copper was 0.22 mM while that for zinc was 0.88 mM [162]. A higher accumulation of lipids per cell was observed at the DI50 (inhibiting dose) concentration for metal-treated cells [162]. Heavy metal exposure (Cu2+, Zn2+ and Cd2+) led to an increase in oleate (with all three metals) and altered the relative proportions of linoleate and C18:4 (with the changes being metal-specific) in S. capricornutum. Metal treatment also significantly increased D22 desaturation of 24-ethyl sterol-components [144]. The ability of heavy metals (copper or lead at 10 lM) to alter lipid metabolism was evaluated in four algal lichen photobionts following short term exposure [163]. The algae were grown under normal or deficient phosphate conditions to assess any interactions with the heavy metal stress. Given the frequent sensitivity of algae to copper and lead, there were surprisingly small changes on lipid metabolism, as assessed by radiolabelling from [1-14C]acetate. The main effects which were seen in a number of cases, were an overall inhibition of total lipid labelling and a relative increase in the labelling of triacylglycerols in the non-polar fraction. Both of these changes can be viewed as reflecting general toxicity of heavy metals, and the Coccomyxa species were more sensitive than Trebouxia species [163]. 7. Conclusions It will have been noted that research on algal metabolism continues to be very active but that many of the studies have involved lesser-known and novel species. In part, this has been driven by biotechnological considerations where special synthetic capabilities are desired. Moreover, there has been increasing emphasis on the use of molecular biology, not only to understand the regulation of lipid formation and the functions of individual components, but also to provide cDNAs for special enzymes which can then be used for the future production of useful genetically-modified organisms. It is likely that these trends will continue in the future and, because of that, we will gain new insights into the diverse and fascinating world of algal lipid metabolism. References [1] [2] [3] [4] [5] [6] [7] [8]

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