bio synthesis of triacylglycerols

[page 40] [International Journal of Plant Biology 2011; 2:e10]

Biosynthesis of Triacylglycerols(TAGs) in plants and algaeAlexandro Cagliari,1 Rogerio Margis,2,3

Felipe dos Santos Maraschin,4Andreia Carina Turchetto-Zolet,1,3

Guilherme Loss,3Marcia Margis-Pinheiro1,3

1Departamento de Genética,2Departamento de Biofísica, 3Centro de Biotecnologia, 4Departamentode Botânica, Universidade Federal do RioGrande do Sul, Brazil

Abstract

Triacylglycerols (TAGs), which consist ofthree fatty acids bound to a glycerol backbone,are major storage lipids that accumulate indeveloping seeds, flower petals, pollen grains,and fruits of innumerous plant species. Thesestorage lipids are of great nutritional andnutraceutical value and, thus, are a commonsource of edible oils for human consumptionand industrial purposes. Two metabolic path-ways for the production of TAGs have beenclarified: an acyl CoA-dependent pathway andan acyl-CoA-independent pathway. Lipidmetabolism, specially the pathways to fattyacids and TAG biosynthesis, is relatively wellunderstood in plants, but poorly known inalgae. It is generally accepted that the basicpathways of fatty acid and TAG biosynthesis inalgae are analogous to those of higher plants.However, unlike higher plants where individ-ual classes of lipids may be synthesized andlocalized in a specific cell, tissue or organ, thecomplete pathway, from carbon dioxide fixa-tion to TAG synthesis and sequestration, takesplace within a single algal cell. Another distin-guishing feature of some algae is the largeamounts of very long-chain polyunsaturatedfatty acids (VLC-PUFAs) as major fatty acidcomponents. Nowadays, the focus of attentionin biotechnology is the isolation of novel fattyacid metabolizing genes, especially elongasesand desaturases that are responsible forPUFAs synthesis, from different species ofalgae, and its transfer to plants. The aim is toboost the seed oil content and to generatedesirable fatty acids in oilseed crops throughgenetic engineering approaches. This paperpresents the current knowledge of the neutralstorage lipids in plants and algae from fattyacid biosynthesis to TAG accumulation.

Introduction

The present review describes the currentunderstanding of the fatty acid and triacylglyc-erol (TAG) biosynthesis in vascular plants andalgae. The two initial sections compare fattyacid profiles and the pathways for TAG accu-mulation present in these organisms, empha-sizing the peculiarities of each group. Thethird section summarizes some economicalapplications of TAG molecules. In addition, itdiscusses some biotechnological efforts aimedat the genetic manipulation of fatty acids andTAG content for the production of nutritionallyand industrially desirable oils in crop plantsand algae. Other classes of lipids, such ascarotenoids that also belong to the lipid classof compounds, are not discussed here. Forother lipid classes, readers can report to recentreviews on algae1-3 and on vascular plants.4-8

Fatty acid biosynthesis in plantsTriacylglycerols (TAGs), as a highly reduced

form of carbon, are an important energyreserve in plant seeds, providing nutrients forsubsequent germination and seedling develop-ment. These storage lipids are of great nutri-tional and nutraceutical value, and a commonsource of edible oils for human consumptionand industrial purposes.9

TAGs of most seeds usually contain thesame acyl groups that are found in membranelipids: palmitic acid (16:0), stearic acid (18:0),oleic acid (18:1D9), linoleic acid (18:2D9,12),and α-linolenic (18:3D9,12,15).10 These fattyacids are often referred to as common fattyacids.11 The biosynthesis of these five majorfatty acids occurs primarily in two subcellularcompartments: the de novo synthesis (de novosynthesis is defined as the synthesis of com-plex molecules from simple molecules, asopposed to being recycled after partial degra-dation; in such cases, synthesis of fatty acidsfrom acetyl-CoA, a non-fatty acid precursor) ofpalmitic and stearic acids and the desaturationof stearic acid to oleic acid occur in plastids,while the conversion of oleic acid to linoleicand then α-linolenic occurs in endoplasmicreticulum (ER).12,13

In plastids, fatty acids are synthesized fromacetyl-Coenzyme A (acetyl-CoA) in a three-step process: i) irreversible carboxylation ofacetyl-CoA by the action of acetyl-CoA carboxy-lase to form malonyl-CoA. Subsequently, themalonyl group is transferred to acyl carrier pro-tein (ACP) giving rise to malonyl ACP, the pri-mary substrate of the fatty acid synthase com-plex (Figure 1A).14 The formation of malonylCoA, catalyzed by the highly regulated plastidicacetyl CoA carboxylase complex, is the commit-ted step in fatty acid synthesis.15; ii) repeatedcondensation of malonyl-CoA with a growing

ACP-bound acyl chain by action of the fattyacid synthase complex, with the consecutiveaddition of two carbon units for each elonga-tion cycle to form 16:0-ACP (Figure 1A).14 Foreach cycle, four separated reactions are neces-sary. The first step corresponds to the forma-tion of 3-ketobutyl-ACP through the condensa-tion of acetyl-CoA with malonyl-ACP by ketoa-cyl synthase III (KAS III), followed by reductionto 3-hydroxylacyl-ACP, dehydration to an enoyl-ACP and a second reduction to form the elon-gated 4:0-ACP. Subsequent rounds of conden-sation reactions of 4:0-ACP with malonyl-ACPgiving rise 14:0-ACP and 16:0-ACP are cat-alyzed by KAS I enzyme.16; the elongation of16:0-ACP to form 18:0-ACP, catalyzed by KAS IIand the first desaturation step (that occurs inthe plastid) where a D9-desaturase is theenzyme responsible for the conversion of 18:0-ACP to 18:1-ACP (Figure 1A). These three fattyacids (16:0-ACP, 18:0-ACP and 18:1-ACP) arethen exported to the cytosol into the acyl-CoAand acyl-lipid pools.9,17 In some organisms, the

International Journal of Plant Biology 2011; volume 2:e10

Correspondence: Márcia Pinheiro Margis,Departamento de Genética, Universidade Federaldo Rio Grande do Sul, Prédio 43312, 91501-970,Porto Alegre, Brasil.Tel. +55.51.3308.9814.E-mail: [email protected]

Key words: fatty acid biosynthesis, TAG accumu-lation, lipid metabolism.

Acknowledgments: this project was supported byCNPq (Conselho Nacional de DesenvolvimentoCientífico e Tecnológico), CAPES (Coordenaçãode Aperfeiçoamento de Pessoal de NívelSuperior), FAPERGS (Fundação de Amparo aPesquisa do Estado do Rio Grande do Sul), FINEP(Financiadora de Projetos) and MCT (Ministériode Ciência e Tecnologia).

Contributions: AC is principal author in the con-ception, design, analysis and interpretation ofdata, and drafted the article. RM participated inthe conception and design, and critically revisedthe article. FM, AT-Z and GL revised the articleand contributed important intellectual content.MM-P participated in conception and design andfinal approval of the version to be published.

Received for publication: 28 March 2011.Revision received: 14 July 2011.Accepted for publication: 11 October 2011

This work is licensed under a Creative CommonsAttribution NonCommercial 3.0 License (CC BY-NC 3.0).

©Copyright A. Cagliari et al., 2011Licensee PAGEPress, ItalyInternational Journal of Plant Biology 2011; 2:e10doi:10.4081/pb.2011.e10

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[International Journal of Plant Biology 2011; 2:e10] [page 41]

elongation process can be extended and fattyacid chains containing up to 18 carbon atomscan be synthesized.14

The termination of fatty acid elongation iscatalyzed by acyl ACP thioesterases (acyl-ACPhydrolases) enzymes. Thioesterases catalyzesthe hydrolysis of acyl-ACP to produce free fattyacids, which are able to cross the plastidialenvelope to be reactivated as acyl-CoAs on theoutside of the organelle (Figure 1A).18 Inplants, two main thioesterase types weredescribed: the FatA class, which preferentiallyremoves oleate from ACP, and FatBthioesterases that are active with saturatedand unsaturated acyl ACPs, and, in somespecies, with shorter-chain-length acylACPs.19-21 The interplay between the fatty acidsynthase complex, D9 desaturase, and the twothioesterases determines the ratio of acylchains produced in the plastid for TAG forma-tion.10

After it has been exported from the plastids,oleic acid enters the cytosolic pool (Figure 1A)and is imported into the ER in association withCoA. Oleic acid is then available for conversionto linoleic and α-linolenic acid by the sequen-tial action of substrate-specific desaturas-es.13,14,17,22,23 Alternatively, other fatty acidchain modifications can occur in ER. Forexample, in Castor bean (Ricinus communisL.), oleic acid undergoes a hydroxylationprocess, through the activity of the oleatehydroxylase (FAH12) enzyme, yielding rici-noleic acid (C18:OH), an unusual hydroxylated(OH) fatty acid.22 Finally, part of the fatty acidsproduced in plastids and modified in the ER isretained as structural components of cellularmembranes (phospholipids of the ER andgalactolipids of plastids, for instance) and therest is transferred to TAG and accumulate asan energy source.17

Unusual fatty acidsDifferently from the conservative fatty acid

composition observed in the plant membranelipids, many evolutionarily divergentangiosperm species accumulate substantialamounts of unusual acyl chains in their seed-storage lipids. About 300 naturally occurringdifferent fatty acids have been described inseed oils. However, it has been estimated thatthousands more could be present throughoutthe plant kingdom.10,11

Unusual fatty acids correspond to those pre-senting chemical structures that deviate fromthe common fatty acids found in the majorityof plant species. The structures of the unusualfatty acid can vary in chain length from 8 to 24carbons. Alternatively, they can present modifi-cations along acyl chain composition, like dou-ble bonds in unusual positions or novel func-tional groups (such as hydroxy, epoxy, cyclic,halogen or an acetylenic group).11

In several plant species, unusual fatty acidsare the predominant fatty acids represented inthe seed oil composition. The reason for suchdiversity observed in seed oil constituents isunknown, but plants seem to be able to toleratehigh levels of unusual fatty acids in storagelipids because they are rapidly sequesteredinto oil bodies and, therefore, have no structur-al roles. The physical and chemical propertiesof many unusual fatty acids might explain, atleast in part, why they are excluded from themembrane lipids of seeds, and are absent fromother parts of the plant. It is hypothesized thatthey would disturb the structural integrity ofthe membrane bilayer and have deleteriouseffects on the cell. Consequently, storage andmembrane lipids have different fatty acid com-positions.11

Nature contains a wide variety of unusualfatty acids, some of which are important forindustry and human health. Producing theseunusual fatty acids in agronomical suitableplants has been a long standing goal for com-

panies and researchers involved in the field ofoilseed engineering.24-27

Saturated acyl chain fatty acidsSpecies of Araceae, Lauraceae, Lythraceae

and Ulmaceae often contain saturated acylchain lengths ranging from C8 to C14. Coconutoil, for example, possesses more than 90% ofits acyl chains composed by saturated fattyacids, generally with fatty acids chain lengthsranging from C8 to C16, predominantly lauricacid (12:0). Another example includesBrassicaceae, which shows a preferential pro-duction of very long chain saturated fatty acidswith carbon lengths of C20-C24.10

Medium-chain fatty acidsSome species of oleaginous plants are able

to accumulate high amounts of medium-chainfatty acids, generally showing fatty acid chainswith less than 16 carbons (Figure 2A).17

For common fatty acid formation, the grow-

Review

Figure 1. Fatty acid biosynthesis and Kennedy pathway for triacylglycerol biosynthesisin plants. (A) Fatty acid synthesis occurs in plastids through repeated steps of condensa-tion by the action of several enzymes that compound the fatty acid synthase complex(FAS), which promotes the consecutive addition of two carbon units for each elongationcycle to form 16:0. Then, the elongation of 16:0 occurs to form 18:0 and the first desat-uration step where 18:0 originates 18:1. The termination of fatty acid elongation is cat-alyzed by thioesterases enzymes and these three fatty acids are then exported to thecytosol into the acyl-CoA pools and are able to be accumulated as TAG molecules. (B)Kennedy pathway starts with acylation of glycerol-3-phosphate (G3P) to form lysophos-phatidic acid (LPA) through the action of sn-glycerol-3-phosphate acyltransferase (1).The second acyl-CoA dependent acylation is catalyzed through the catalytic action oflysophosphatidic acid acyltransferase (2), leading to the formation of phosphatidic acid(PA). Phosphatidic acid phosphatase (3) catalyzes the release of phosphate from PA toproduce DAG. The final acylation is driven by diacylglycerol acyltransferase (4) that con-verts DAG into TAG.

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ing acyl chain is terminated when it reaches alength of 16 or 18 carbons, by the action ofchain-length-specific thioesterases,28-30 whichcleaves the acyl group from the ACP to producea free fatty acid, thus terminating their elonga-tion. Plants that synthesize medium-chainfatty acids have an additional acyl-ACPthioesterase enzyme, which is responsible forthe premature cleavage of the acyl-chain fromACP, redirecting fatty acid synthesis from long(C16–C18) to medium (C8–C14) chains.11

Examples of the most commercially impor-tant medium-chain fatty acid oils include thepalm kernel and coconut oils, which containpredominantly lauric acid (12:0) (Figure 2A)in their fatty acid compositions.17 In addition,several species of the genus Cuphea are alsoknown to accumulate high amounts of medi-um-chain fatty acids, ranging from C8 to C14,usually with only one chain length dominatingin each species.31

Very long chain poly unsaturated fattyacidsPlant membrane sphingolipids contain sig-

nificant amounts of very long chain FAs (VLC-FA) that possess fatty acid chain lengths rang-ing from 20 to 26 carbons (Figure 2B).Unsaturated VLC-FAs are also found in thestorage oils of some plants (Cruciferaespecies) and in epicuticular and storage waxesters. They are synthesized outside of plastidsby successive rounds of elongation of a C18fatty acyl precursor by two carbons originatingfrom malonyl-CoA11 by a membrane-boundelongase complex.32 In comparison, very longchain polyunsaturated fatty acids (VLC-PUFAs), defined as fatty acids containing 20 ormore carbon atoms and three or more doublebonds, are almost completely absent in higherplants.33,34 The biosynthesis of VLC-PUFAsconsists of cycles of alternated desaturationand chain elongation, in which the desatura-tion reactions occur on acyl-PC substrates andthe elongation reactions occur on acyl-CoA.17

The primary biosynthesizing sources ofVLC-PUFAs are marine microorganisms suchas algae, which represent the base of an aquat-ic food network that results in the accumula-tion of VLC-PUFAs in fish oils.35,36 However,some fungi and lower plants can synthesizeVLC-PUFAs, and animals can convert dietaryfatty acids such as linoleic and α-linolenicacids to these more complex forms.26,34,37,38

From a nutritional point of view, the mostimportant VLC-PUFAs are arachidonic (ARA;ω6-20:4D5,8,11,14), eicosapentaenoic (EPA; ω3-20:5D5,8,11,14,17), and docosahexaenoic acid(DHA; ω3 22:6D4,7,10,13,16,19). VLC-PUFAs arenot only required as components of membranephospholipids in specific tissues or as precur-sors for the synthesis of the different groups of

eicosanoid effectors, but also contribute via amultiplicity of beneficial roles to the mainte-nance of good health, particularly by reducingthe incidence of cardiovascular diseases.39,40

Novel monounsaturated fatty acidsThe synthesis of common monounsaturated

fatty acids is catalyzed by a soluble plastidialdesaturase enzyme, which normally introducesa double bond between carbons 9 and 10 of aC18 acyl-ACP.11 However, some plants that areable to synthesize unusual monounsaturatedfatty acids present an additional desaturaseenzyme, which is closely related to the D9-desaturase, but introduces a double bond inpositions other than the ninth carbon from thecarboxyl group.10

Umbelliferae species, such as carrot andcoriander, are known to contain oils rich inpetroselenic acid (D6 18:1) (Figure 2C). Thisunusual monounsaturated fatty acid is theresult of the activity of a plastidial D4 desat-urase that introduces a double bond betweencarbons 4 and 5 of a C16 acyl-ACP, convertingpalmitoyl-ACP to D4 hexadecanoyl-ACP whichis then elongated to petroselinoyl-ACP andcleaved from ACP to produce the free fattyacid.41

Another curious example of this unusualdesaturation process is observed inMeadowfoam (Limnanthes alba) oil. Thisspecie accumulates oil with approximately65% of 20:1 acid in its fatty acid composition.The 20:1 acid possesses a double bond at C5carbon.42

Hydroxy, epoxy and acetylenic fattyacidsFatty acids with additional functional groups

in the acyl chain represent excellent feed-stocks for industry and have been used to pro-

duce innumerous bio-based products due totheir physical and chemical properties.17

The synthesis of the fatty acids containingfunctional groups such as hydroxyl, epoxy andacetylenic functional groups is achieved by theaction of a family of related enzymes.Structurally, these enzymes present similaritiesto extraplastidial membrane-bound D12-desat-urases (FAD2), and only four amino acid substi-tutions are sufficient to convert an 18:1desat-urase enzyme into an 18:1 hydroxylase enzyme.43

The synthesis of functional groups possess-ing fatty acids is thought to take place in theER and uses as a substrate fatty acids esteri-fied to the major membrane lipid phosphatidyl-choline (PC).11

Castor oil, for example, is rich in hydroxylat-ed fatty acid (OH) (Figure 2D), whereas someplants have epoxidated (Figure 2E) or methy-lated acyl chains in their TAGs.10 Examples ofplants that accumulate these kinds of fattyacids include Vernonia galamensis, Euphorbialagascae and Stokesia laevis that accumulate60-80% of an epoxy fatty acid known as verno-lic acid (cis-12-epoxyoctadeca-cis-9-enoicacid).43,44

Triacylglycerols biosynthesis inplantsTAGs are present in all eukaryotes, includ-

ing animals, plants, fungi and protists, andalso in some prokaryotes such Actinomy -cetes,45-47 Streptomyces species48 and mycobac-teria species.49 Due to its importance as aninert storage component, the biosynthesis ofTAGs is a common metabolism pathway thatappears to be conserved from bacteria tohumans.50

TAGs are quantitatively the most importantseed storage reserve in many plant speciesincluding oil crops such as sunflower

Review

Figure 2. Examples of unusual fatty acids produced by plants. (A) Medium-chain-fattyacid. (B) Very-long-chain-fatty acid. (C) Unusual double-bound position fatty acid. (D)Hydroxy-fatty acid. (E) Epoxy-fatty acid.

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(Helianthus annuus), oilseed rape (Brassicanapus), soybean (Glycine max), and maize(Zea mays).43,51 Besides their importance asenergy source, TAGs also represent an impor-tant source for building blocks for membranelipid biosynthesis.50 TAGs from plants areimportant sources for human nutrition, alsoproviding precursors for chemical industryproducts.14,43 TAGs are becoming increasinglyimportant raw materials for the production ofpaints, detergents, lubricants, biofuels andnylon precursors, and can also serve as renew-able biofuels as an alternative to crude oil.9

TAGs accumulated during seed maturationare stored in the seed oil bodies until germina-tion51-53 and can either be used for energy pro-duction through β-oxidation or serve as sub-strates for acylation reactions, such as phos-pholipid biosynthesis, for example.50

Pathways for TAG biosynthesisIn seed plants, glycerolipids can be synthe-

sized via two similar pathways that are knownas prokaryotic and eukaryotic systems on thebasis of their evolutionary origins. These twopathways occur in distinct subcellular com-partments and are similar in the two-stepenzymatic conversion of sn-glycerol-3-phos-phate (G3P) to phosphatidic acid (PA), but dif-fer in the subsequent conversion of PA intostructural, storage and signaling lipids.54

Although the two enzymatic pathways are sim-ilar, the enzymes that catalyze the acylationsare unique to their respective system.55

Plastidic enzymes are responsible for theprokaryotic system, while cytosolic enzymesare responsible for the eukaryotic system. Inthe prokaryotic pathway, fatty acids are direct-ly transferred from ACP to G3P, while in theeukaryotic pathway fatty acids are cleavedfrom the ACP by an acyl-ACP-thioesterase toform free fatty acids that are exported to thecytoplasm, esterified to CoA and then join theacyl-CoA pool. The acyl groups are then used bythe acyltransferases of the eukaryotic G3Ppathway in the ER to produce membrane andstorage lipids (TAGs).11 Moreover, theseenzymes can present structural differencesdepending on their subcellular localizations,forming independent clusters in phylogeneticstudies.56

Production of storage lipids in plantsinvolves de novo fatty acid synthesis in thestroma of plastids and subsequent incorpora-tion of the fatty acid into glycerol backboneleading to TAG in the ER.9

Two metabolic pathways for the productionof TAGs have been clarified: an acyl CoA-dependent pathway (Figure 1B) and an acyl-CoA-independent pathway (Figure 3).

Acyl CoA-dependent pathwayIn the acyl-CoA dependent pathway, com-

monly known as the Kennedy pathway (Figure1B), acyl-CoA is the substrate for successiveacylation reactions of the glycerol backbone,with the terminal step being the acylation ofsn-1,2- diacylglycerol (DAG) by DAG acyltrans-ferases (DGATs).57 The glycerol backbone forTAG assembly is derived from G3P which isproduced via the catalytic action of sn-glycerol-3-phosphate dehydrogenase (G3PDH) fromdihydroxyacetone phosphate (DHAP), derivedfrom glycolysis.18

The Kennedy pathway starts with acylation ofG3P to form lysophosphatidic acid (LPA)through the action of sn-glycerol-3-phosphateacyltransferase (G3PAT). The second acyl-CoAdependent acylation is catalyzed through thecatalytic action of lysophosphatidic acid acyl-transferase (LPAAT), leading to the formation ofphosphatidic acid (PA). Phosphatidic acid phos-phatase (PAP) catalyzes the release of phos-phate from PA to produce DAG.9 The final acyla-tion is driven by DGAT, using acyl CoA as an acyldonor, converting DAG to TAG (Figure 1B).14

Glycerol-3-phosphate acyltransferaseMembrane-bound Glycerol-3-phosphate

acyltransferase (G3PAT) is a soluble enzymethat initiates the fatty acid incorporationprocess by transferring fatty acids from eitheracyl-ACPs or acyl-CoA molecules to the sn-1position of G3P, forming LPA.10

In several plants, two G3PAT isoforms havebeen found in the plastidial and cytoplasmiccellular compartments. G3PATs can either beselective, preferentially using oleic acid as theacyl donor, or non-selective, using either oleicor the saturated palmitic acid at comparablerates. This differential substrate specificity forsaturated versus unsaturated fatty acids hasbeen implicated in the sensitivity of plants tochilling temperatures.58

Lysophosphatidic acid acyltransferaseLysophosphatidic acid acyltransferase

(LPAAT) catalyzes the transfer of the acyl-chain from the CoA ester to sn-2 of LPA, lead-ing to the formation of PA. There are twosequence-diverged gene subfamilies encodingLPAAT.10

LPAAT activity is associated, in plants, withmultiple membrane systems, including chloro-plasts, ER and the outer membrane of mito-chondria, which suggests their presence onseveral different isoforms.59,60 In plants, thisenzyme shows a preference for unsaturatedacyl chains,10 but it is also able to discriminateacyl groups having longer or shorter fatty acidchain lengths. In developing seeds of certainplants, special LPAATs can incorporate unusu-al acyl groups, such as in Castor bean, whichaccumulates oils esterified with unusual fattyacids.61

Phosphatidic acid phosphataseThe cytoplasmic enzyme Phosphatidic acid

phosphatase (PAP) dephosphorylates PA toyield DAG.62 DAG produced from the hydrolysisof PA is not only a direct precursor of TAG, butalso a substrate for the synthesis of membranephospholipids.63 In plants, two distinct PAPtypes involved in glycerolipid synthesis appearto exist in soluble and in membrane-associat-ed fractions (microsomes). The distribution ofthese forms seems to be affected by the cellu-lar metabolic status.64

PAP is also involved in general phospholipiddegradation and turnover. It was demonstratedthat PA accumulates in plants in a transientmanner in response to various forms of stress.The dephosphorylation activity of PAP resultsin the attenuation of the signaling function ofPA through its conversion to DAG, which can

Review

Figure 3. Acyl-CoA independent pathways for triacylglycerol (TAG) biosynthesis.Diacylglycerol transacylase (A) catalyzes the transfer of an acyl moiety between two dia-cylglicerol (DAG) molecules to form TAG and monoacylglycerol (MAG) as a co-product.Phospholipid: diacylglycrol acyltransferase (B) catalyzes the transfer of fatty acids fromthe sn-2 position of phospholipids (PL) to DAG to form TAG and lyso-phospholipids(LPL) as a co-product. Choline phosphotransferase (C) can contribute to TAG biosynthe-sis through a reversible conversion of PL into DAG. Then, the resulting DAG can be con-verted into TAG by the DGAT or PDAT enzymes.

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be important in the stress responses, especial-ly in the remodeling of membrane lipid compo-sition.64,65

Diacylglycerol acyltransferaseKennedy pathway’s final enzyme, diacylglyc-

erol acyltransferase (DGAT), catalyzes thethird acyl-CoA-dependent acylation reactionthat leads to the production of TAG from DAG.DGAT is an integral ER protein and has alsobeen shown to be present in oil bodies andplastids.66 Although DGAT1 and DGAT2 are themain types of DGAT enzymes, other different,structurally unrelated enzymes with DGATactivity have been described in plants.67,68 Asoluble DGAT from peanuts,69 a wax ester syn-thase/acyl-coenzyme A: Diacylglycerol acyl-transferase (WSD1) from Arabidospis43 and adistinct DGAT (DAcT) from Euonymus alatus.70

The substrate selectivity of DGAT depends onseveral factors, such as the acyl composition ofthe DAG pool, acyl-CoA concentration and tem-perature.9

DGAT1 was initially cloned from mousebased on its homology with mammalian acyl-CoA: cholesterol acyltransferase genes.71

Several homologs of DGAT1 have been clonedand characterized in animals and plants, andtheir functions have been verified by bothoverexpression and deletion approaches.72-75

A second family of DGAT genes (DGAT2),which have no sequence similarity with DGAT1,were first identified in the oleaginous fungusMorteriella ramanniana.76,77 Several DGAT2genes have been cloned and characterized fromanimals, fungi and plants.77-79 In animals,DGAT2 has different physiological functions invivo and presents a different temporal-spatialexpression profile compared to DGAT1.80-82 Inyeast, Sacharomyces cerevisae, the DGAT2enzyme is dominant in the stationary growthphase when the yeast is storing significantamounts of TAG.83,84 In addition, some experi-ments suggest a more important role of DGAT2expression in the accumulation of conjugatedand hydroxy fatty acid in seed oils.43,85-87

The roles of DGAT1 and DGAT2 in the oilproduction are apparently species-dependent.In plants, DGAT1 appears to be a major enzymegene for seed oil accumulation, while DGAT2appears to play significant roles in the selec-tive accumulation of unusual fatty acids, suchas epoxy and hydroxy fatty acid, into seed stor-age oils.43,86,88

A third enzyme is a soluble DGAT (DGAT3),which was only recently identified in peanutand other plant species.67,86 However, there islittle information about this new DGAT iso-form. An acyl-CoA-dependent acyltransferase,namely wax ester synthase/diacylglycerol acyl-transferase (WS/DGAT), was identified andpurified from the bacterium Acinetobacter sp.

strain ADP1, which can utilize both fatty alco-hols and DAG as acyl acceptors to synthesizewax esters and TAGs, respectively.88-90 A largenumber of genes with homology to thisAcinetobacter gene79,91 were identified inArabidopsis.DGATs may be one of the rate-limiting steps

in plant storage lipid accumulation,9,88,92 andthus appear to be crucial for mediating quanti-tative and qualitative aspects of seed oil syn-thesis in transgenic plants.67 In this scenarioDGAT seems to be a potential target for thegenetic modification of plant lipid biosynthesisin oilseeds for economic benefit.88 Studies inthis field demonstrated that overexpression ofDGAT1 resulted in increase,92 whereas sup-pression of DGAT activity resulted in adecrease in oil content in Arabidopsisseeds.75,93 In addition, DGAT activity seems tobe also important for the correct channeling ofunusual fatty acids into seed storage oils.67

Acyl CoA-independent pathwayAs an alternative to the Kennedy pathway,

nascent fatty acids may be first incorporatedinto membrane lipids at the plastid envelopeand/or in the ER and afterwards accumulatedas TAG molecules.14

Newly synthesized fatty acids can be incor-porated directly into PC via an acyl editingmechanism, rather than through PA and DAGintermediates.94 Acyl chains from PC can beincorporated into TAG, either through conver-sion back to DAG or by the action of a phospho-lipid: diacylglycerol acyltransferase (Figure 3)(PDAT).14 This enzyme is a member of thelecithin: cholesterol acyltransferase gene fam-ily91 that catalyzes the formation of TAG by anacyl transfer from the sn-2 position of phospho-lipids to DAG (Figure 3), with phos-phatidylethanolamine (PE) as the preferredacyl donor in both yeast and plants.95-97

PDAT activity has been reported in yeastmicrosomes and certain oilseeds.91,96,98 It wasdemonstrated that Arabidopsis PDAT is able toutilize different phospholipids as acyl donorsand accept acyl groups of chain lengths rang-ing from C10 to C22.43,97 In yeast, PDAT1 is amajor contributor to TAG accumulation duringthe exponential growth phase.76,83

The activity of PDAT enzymes (specially inoilseed plants) may play a critical role in theremoval of unusual fatty acids from membranephospholipids and transfer it into TAG mole-cules.9 It was suggested that the balance of theexpression of PDAT genes may represent animportant mechanism either for the mainte-nance of membrane lipid homeostasis, con-tributing to membrane lipid turnover or for theremoval of DAG, an effector molecule of thephosphatidylinositol signaling pathway thatpossesses a critical role for plant stress

responses.91

In another case of acyl-CoA-independenttransacylation, it has been postulated that aDAG transacylase (DGTA) catalyzes the trans-fer of an acyl moiety between two DAG mole-cules to form TAG, and monoacylglycerol(MAG) as a co-product (Figure 3),85,99 and thatthe reverse reaction participates in the remod-eling of TAGs.88,100 However, no gene encodingsuch as transacylase has been identified sofar.85

There is growing evidence for the presenceof an alternative acyl-independent pathway forTAG formation in plants, involving an enzymethat is normally related with membranebiosynthesis, named choline phosphotrans-ferase (CPT) or amino-alcohol phosphotrans-ferase (AAPT).10 CPT can contribute to TAGbiosynthesis through a reversible conversionof PC to DAG (Figure 3).12 Afterwards, theresulting DAG can be converted into TAG byDGAT or PDAT enzymes. It has been hypothe-sized that the continuous reversible transfer ofDAG into PC may control, in part, the PUFAcontent of the seed oil,101 making CPT the keyenzyme regulating the route by which PUFAsare available for incorporation into TAG mole-cules.22,101,102

In addition, a recent publication demon-strated that a class of phospholipase enzyme(phospholipase D) plays an important role inthe conversion of PC into TAG.103 The attenua-tion by RNA interference of a soybean phos-pholipase D was responsible for higher levelsof di18: 2 (dilinoleoyl)-PC and PE in seedscompared to the wild type lines. The increasedpolyunsaturation was at the expense of PC andPE species containing monounsaturated orsaturated fatty acids. By contrast, a decrease inthe unsaturation of the TAG fraction of the soy-bean seeds was observed, suggesting thatphospholipase D suppression slows the con-version of PC into TAG.103

Regulation of oil biosynthesis inplantsThe accumulation at high levels of com-

pounds for nutrient storage is a characteristicevent of seed development which is regulatedby a common, at least in part, genetic programthat takes place during the seed maturationphase.104-108 The regulation of oil synthesisoccurs at multiple levels.109 Several of theenzymes involved in the synthesis, accumula-tion and degradation of neutral lipids havebeen identified and a great redundancy formost of the neutral lipid metabolic enzymeswas observed. The proteins involved in neutrallipid metabolism are well conserved acrossspecies, exhibiting remarkable homology toeach other.50

Synthesis and accumulation of storage com-

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pounds are regulated by numerous transcrip-tion factors (TFs) in an intricate networkinvolving genetic programs, and hormonal andmetabolic signals.110-112 Some of these TFs areknown as master regulators based on theirapparent capacity to regulate the action ofother TFs.18 The most important master regu-lators of seed maturation and storage accumu-lation include the LEAFY COTYLEDON genes(LEC1 and LEC2), FUSCA3 (FUS3), ABSCISICACID INSENTIVE3 (ABI3) and WRINKLED1(WRI1).18,111

Among the known transcriptional factorsthat regulate genes related to storage com-pounds, it was demonstrated that WRI1 activi-ty was responsible for the regulation of oilaccumulation by promoting the carbon fluxthrough the glycolysis pathway.113-116

WRI1 encodes a transcription factor of theAPETALA2-ethylene responsive element-bind-ing protein (AP2-EREBP) family.114 This TF isresponsible for specifying the regulatoryaction of other TFs, such as LEC2 and possiblyLEC1, during the fatty acid biosynthetic net-work.110,117

Studies in Arabidopsis demonstrated thatWRI1 up-regulates glycolytic gene expressionrequired for the conversion of sucrose to TAGbiosynthesis precursors.112,114 The correspon-ding wri1 mutant is deficient in oil biosynthe-sis and shows a reduction of 80% in seed oilcontent.14,115 The carbohydrate metabolismregulation in wri1 appears to be affectedbecause the activities of a number of key gly-colytic enzymes were highly reduced in thatmutant.18

Other transcription factors are also involvedwith the regulation of oil metabolism in devel-oping oil seeds.14 It was shown that seed-spe-cific overexpression of Arabidopsis LEC1 indeveloping seedlings of Arabidopsis, using anestradiol-inducible vector, up-regulates thetranscription level of known enzyme-codinggenes of fatty acid biosynthesis by about60%.118 The same study showed that theamount of fatty acids was 4.7 times higher intransgenic than wild-type seedlings and thatthe function of LEC1 in fatty acid biosynthesisregulation was partially dependent on ABI3,FUS3 and WRI1.18,118

FUS3 also seems to play an important role inoil accumulation in Arabidopsis.Transcriptomic analysis has revealed that theabundance of FUS3 transcript increasestogether with transcripts involved with fattyacid pathway.113 In addition, the inducibleexpression of Arabidopsis FUS3 resulted inrapid induction of gene expression associatedwith FA biosynthesis in seedlings. The sameresults were also observed in Arabidopsis pro-toplasts transiently expressing FUS3.18

Soybean DNA binding proteins with onezinc-finger motif, or Dof-type TFs, have also

been shown to have effects on oil accumula-tion. Overexpression of GmDof4 and GmDof11leads to an increased expression level of thegenes encoding the β-subunit of acetyl CoAcarboxylase (ACCase) and long-chain acyl-CoAsynthetase, respectively, both encodingenzymes involved with fatty acid biosynthesis.In addition, the transgenic lines with the high-est levels of GmDof4 or GmDof11 expressionalso present a lipid content increase rangingfrom 11 to 24%.18,119

However, besides the fatty acid biosynthesisregulation promoted by TFs, additional levelsof control certainly involve allosteric enzymeregulation, for example, at the level ofACCase15,120 or plastid pyruvate kinase,14,121,122

important precursors of TAG biosynthesis.

Oil bodiesSeveral organisms store lipids in subcellular

particles as food reserves, which will be usedthrough a period of active metabolism. Theselipid particles can be found in seeds, pollens,flowers, roots, stems of flowering plants,spores and vegetative organs of non-floweringplants and algae. These structures are also rep-resented in some animal cells, besides fungiand Euglena. However, seed subcellular stor-age lipid particles, have been studied moreextensively.123

Seeds of most plant species store TAGs asfood reserves for germination and post-germi-

native growth.123 The intracellular storage ofneutral lipids occurs in specialized compart-ments called lipid particles, lipid droplets or oilbodies.50

Oil bodies are relatively simple sphericalorganelles with approximately 1 µm in diame-ter that arise from the ER, the site of TAG syn-thesis, and are surrounded by a phospholipidmonolayer membrane embedded with proteinscalled oleosins.51,124,125 Oleosins in the seeds ofdiverse species are small proteins of about 15to 26 Kilodaltons123,126 that are usually presentas two or more highly conserved isoforms.51,127

According to the best accepted oil body bio-genesis model, proteins involved in neutrallipid metabolism accumulate in certainregions of the ER. Enzymes involved in TAGsformation are found among these polypep-tides.50 TAGs are synthesized in the ER and aresequestered, due to their hydrophobicity,between the two layers of the ER membrane.123

Because newly formed neutral lipids areunable to integrate into bilayer membranesthey cluster and accumulate in the hydropho-bic region between the two leaflets of the ERmembrane (Figure 4A and B). During ongoingTAG synthesis the droplet grows and forms abud (Figure 4C). After reaching a certain size,the budding particle, which has a TAG matrixsurrounded by a layer of phospholipids andoleosins, is released into the cytosol as amature oil body (Figure 4D).50,123

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Figure 4. Oil body biosynthesis. (A-B) Accumulation of triacylglycerols (TAGs) occursbetween the two layers of endoplasmic reticulum membrane (ER). (C) During ongoingsynthesis of TAGs the droplet grows and forms a bud. (D) After reaching a certain size,the budding particle is released into the cytosol as a mature oil body. Adapted fromAthenstaedt and Daum.50

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Lipid biosynthesis in algae

Eukaryotic algae represent a very diverseorganism group that is considered a key com-ponent for the most diverse ecosystems. Theyaccount for over half of the primary productionat the base of food chains.128

Their variable morphology and habitatsmean that eukaryotic algae contain a diversecomposition of acyl lipids and unusual fattyacids that are not found in other phyla.128 It iswidely accepted that the excellent capability ofalgae to adapt to very different environmentalconditions results from the fact that they cansynthesize a number of unusual compounds,responsible for their unusual pattern of cellu-lar lipids, as well as their ability to modify effi-ciently the lipid metabolism in response toenvironmental conditional changes.129,130

Indeed, some authors suggested that thecapacity of some algae to store VLC-PUFAs inTAG could be a reserve, which would allow theorganisms to adapt to any further rapidchanges in their environment, such as highlight intensity, UV radiation and low tempera-ture.131

Based on their lipid diversity, several thou-sands of algae and cyanobacterial species havebeen screened for high lipid content, resultingin the isolation and characterization of severalhundred oleaginous species. These speciescan be found among diverse taxonomic groups,and the total lipid content may vary noticeablyamong individual species or strains, withinand between taxonomic groups.132

Comparison of lipid metabolism inalgae and higher plantsLipid metabolism, specially the pathways to

fatty acids and TAG biosynthesis, is less under-stood in algae than in higher plants. It is gen-erally accepted that the basic pathways of fattyacid and TAG biosynthesis in algae are directlyanalogous to those demonstrated in higherplants based on the sequence homology andsome shared biochemical characteristics of anumber of genes and/or enzymes involved inlipid metabolism.132

However, there is some evidence of differ-ences in algae lipid metabolism. Unlike higherplants, where individual classes of lipids maybe synthesized and localized in a specific cell,tissue or organs (seeds or fruits), the completepathway from carbon dioxide fixation to TAGsynthesis and sequestration takes place withina single algal cell.132 After being synthesized,the accumulation of algal TAGs occurs indensely packed lipid bodies located in the cyto-plasm of the algal cell, although the lipid bodyformation and accumulation might also occurin the inter-thylakoid space of the chloroplastin some green algae species, such as

Dunaliella bardawil.133 Higher plants cannotsynthesize significant amounts of VLC-PUFAsabove C18, whereas many algae, especiallymarine species, possess the ability to synthe-size and accumulate large quantities of VLC-PUFAs, such as EPA, DHA and ARA.134 In addi-tion, it is hypothesized that in algae, the TAGbiosynthesis pathway may play a more activerole in the stress response, in addition to func-tioning as carbon and energy storage underenvironmental stress conditions.132

Fatty acid biosynthesis in algaeAlgae synthesize fatty acids as building

blocks for the formation of various types oflipids. Similar to higher plants, the most com-monly synthesized fatty acids have chainlengths ranging from C16 to C18 (Table 1).136

In general, saturated and mono-unsaturatedfatty acids are predominant in most algaeexamined. Indeed, the major saturated fatty

acid is palmitic acid, while oleic acid is muchless abundant than in higher plants.132

However, some algae and cyanobacteria areable to synthesize, as predominant FA species,medium-chain fatty acids (C10, C12 and C14),whereas others are able to produce VLC-FA(>C20). For instance, 27-50% of all fatty acidsfound in the filamentous cyanobacteriumTrichodesmium erythraeum are composed by aC10 fatty acid and up to nearly 70% of all fattyacids in the golden alga Prymnesium parvumare composed of C14 fatty acid.137 The distribu-tion of individual fatty acids is quite distinctand tightly regulated. This control relates pri-marily to the function of acyl lipids in themembrane composition.128

Another distinguishing feature of somealgae is the large amounts of VLC- PUFAs(>18C) as their major fatty acid components(Figure 5, Table 1), which resulted from sever-al desaturation/elongation steps, promoted by

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Figure 5. Very-long-chain polyunsaturated fatty acid (VLC-PUFA) biosynthesis ineukaryotic algae. Pentagons and triangles represent desaturases and elongases enzymesinvolved in algal VLC-PUFA biosynthesis, respectively. Adapted from Harwood andGuschina.128

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large classes of desaturase and/or elongaseenzymes (Figure 5). This is particularly truefor marine species. Examples include thegreen alga Parietochloris incise,131 the diatomPhaeodactylum tricornutum and the dinofla-gellate Crypthecodinium cohnii138 where theVLC-PUFAs ARA, EPA or DHA constituted themajor fatty acid species representing about33.6-42.5%, approximately 30% and 30-50% ofthe total fatty acid composition of these threespecies, respectively. In this scenario, the pres-ence of 20% or more of three kinds of fattyacids in algae is attracting much economicalinterest because of the role of algae at thebeginning of food chains and the perceivedneed for VLC-PUFAs in healthy diets.128

In addition, and alternatively to PUFAs, cer-tain algae species may contain other unusuallipids in their oil composition, includingchlorosulpholipids139 and halogenated fattyacids.140

Triacylglycerols accumulation inalgaeTAG biosynthesis in algae may occur via the

direct glycerol pathway (Kennedy pathway).141

The fatty acids that are produced in the chloro-plast are sequentially transferred from CoA topositions 1 and 2 of G3P, by GPAT and LPAATenzymes, respectively, forming PA.136 PA isthen dephosphorylated by a specific action ofPAP enzyme releasing DAG. In the final step ofTAG synthesis, a third fatty acid is transferredto the vacant position 3 of DAG, by the actionof DGAT. As in plants, the acyltransferasesinvolved in the TAG synthesis process seem toexhibit preferences for specific acyl-CoA, andthus may play an important role in determin-ing the final acyl composition of TAG mole-cules.132

Beyond the Kennedy pathway, the existencewas suggested of an acyl-CoA-independentsynthesis of TAG in algae, through PDAT andCPT activities, similar to that observed in plantand yeast. This pathway could play an impor-tant role in the regulation of the membranelipid composition in response to various envi-ronmental and growth conditions, since undervarious stress conditions, algae usually under-go rapid degradation of the photosyntheticmembrane with concomitant occurrence andaccumulation of cytosolic TAG-enriched lipidbodies.132

Despite the occurrence and the levels ofalgal TAG production appear to bespecies/strain-specific, oleaginous algae pro-duce only small quantities of TAG under opti-mal growth or favorable environmental condi-tions.142 Under optimal conditions of growth,algae are able to synthesize fatty acids mainlyfor esterification into glycerol-based mem-brane lipids, which represent about 5-20% oftheir dry cell weight (DCW). The membrane

lipid fatty acid composition includes medium-chain (C10-C14), long-chain (C16-18) andvery-long-chain (>C20) fatty acid deriva-tives.143

In contrast, under unfavorable environmen-tal or stress conditions, many algae speciespromote a shift in lipid metabolism from mem-brane lipid synthesis to the synthesis and stor-age of neutral lipids, especially in the form ofTAGs. The de novo biosynthesis and conver-sion of certain membrane polar lipids intoTAGs may contribute to the overall increase inTAG content. As a result, TAGs may account foras much as 80% of the total lipid content in thecell. As an example of the shift in the lipidmetabolism, many microalgae have the abilityto produce substantial amounts (20-50% DCW)of TAGs as lipid storage under photo-oxidativestress or other adverse environmental condi-tions.144

The major chemical stimuli to TAG accumu-lation in algae are nutrient starvation, salinityand growth-medium pH. On the other hand,the major physical stimuli are temperature andlight intensity. In addition to chemical andphysical factors, growth phase and/or aging ofthe culture also affects the content and fattyacid composition of TAG molecules.132

A significant variation in the algal lipid con-tent and fatty acid profile in response to differ-ent growth conditions was observed.145-147 InPavlova lutheri, a marine Pavlovophyceae, itwas reported that the proportions of PUFAs,especially EPA, were significantly higher underlow light. By contrast, content of saturatedfatty acids and DHA were significantly higherunder strong light.148 In addition, theseauthors also demonstrated that the growth andlipid composition presented a higher sensibili-ty to variations in light intensity than in car-bon source.Another example of light effect in lipid and

fatty acid composition includes Chlorella

zofingiensis, a green algae that can grow wellphotoautotrophically as well as heterotrophi-cally. It presented a 900% increase in lipid yieldin heterotrophic cells compared with photoau-totrophic cell culture. Moreover, about 80% oftotal lipid content was represented by neutrallipids in heterotrophic cells, with 88.7% beingTAGs. On the other hand, photoautotrophiccells accumulated mainly membrane lipids(glycolipids and phospholipids).149

Guihéneuf and co-workers150 investigatedthe effect of UV radiation (UV-R) on the lipidcomposition of two marine microalgae,Pavlova lutheri and Odontella aurita. Theresults indicated that the exposure to UV-Rtreatment led to a decrease in the proportionsof PUFAs especially into structural lipids (gly-colipids and phospholipids) in P. lutheri,whereas in O. aurita, exposure to UV-R did notchange the fatty acid composition and lipidfractions of the cells, suggesting that thisspecies is more resistant and seems to be ableto partially acclimate to UV-R.150

Aplications of Triacylglycerolsproduced by plants and algae

BiodieselBiodiesel is a clean-burning fuel derived

from vegetable oils or animal fats, which hasbeen used as alternative to diesel fuel.151 TAGsare the main components of the vegetable oilsand animal fats that are used for biodiesel pro-duction.152

TAGs are the most similar chemical to fossiloil and, therefore, is the best potential replace-ment for the chemical industry. In fact, fossiloil is derived from ancient lipid-rich organicmaterial, such as spores and planktonic algaethat were sedimented and transformed under

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Table 1. Major fatty acid and polyunsaturated fatty acid (PUFA) in algae.

Algal Class Major Fatty Acid Major PUFAs

Bacillariophyceae C16:0 and C16:1 C20:5 and C22:6Chlorophyceae C16:0 and C18:1 C18:2 and C18:3Euglenophyceae C16:0 and C18:1 C18:2 and C18:3Chrysophyceae C16:0 and C16:1 and C18:1 C20:5, C22:5 and C22:6Chryotophyceae C16:0 and C20:1 C18:3, C18:4 and C20:5Eustigmatophyceae C16:0 and C18:1 C20:3 and C20:4Prasinophyceae C16:0 and C18:1 C18:3 and C20:5Dinophyceae C16:0 C18:5 and C22:6Prymnesiophyceae C16:0 and C16:1 and C18:1 C18:2, C18:3 and C22:6Rhodophyceae C16:0 C18:2 and C20:5Xanthophyceae C14:0, C16:0 and C16:1 C 16:3 and C20:5Cianobacteria C16:0 and C16:1 and C18:1 C16:0, C18:2 and C18:3C14, Myristic acid; C16:0, Palmitic acid; C:16:1, Palmitoleic acid; C16:3, Hexadecatrienoic acid; C18:1, Oleic acid; C18:2, Linoleic acid; C18:3,Linolenic acid; C18:4, Parinaric acid; C18:5, Octadecatetraenoic acid; C20:1, Eicosenoic acid; C20:3, Dihomo-gamma-linolenic acid; C20:4,Arachidonic acid; C20:5, Eicosapentaenoic acid; C22:5, Docosapentaenoic acid; C22:6, Docosahexaenoic acid. Adapted from Cobelas. 135

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high pressure and temperature over millionsof years.153 TAGs consist of several differentfatty acids which present different physicaland chemical properties. Therefore, the com-position of these fatty acids will be the mostimportant parameters influencing the corre-sponding properties of the biodiesel derivedfrom TAGs.152

The advantages of the use of biodieselinclude its higher oxygenated state comparedto the conventional diesel, which leads to lowercarbon monoxide (CO) production andreduced emission of particulate matter, andthe few or lack of sulfur or aromatic com-pounds in its composition. These compoundsare present in the conventional diesel and con-tribute to sulfur oxide and sulfuric acid forma-tion, while aromatic compounds also increaseparticulate emissions and are considered car-cinogens.14 Furthermore, the use of biodieselconfers additional advantages, including ahigher flashpoint (allows safer handling andstorage), faster biodegradation (particularlyadvantageous in environmentally sensitiveareas where fuel leakage poses great hazards)and greater lubricity.14 However, the biggestenvironmental advantage of using biodiesel isthat it is a renewable energy source, since thereduced hydrocarbon chains of biodiesel arederived from solar energy: plants and algaecapture light energy during photosynthesis,converting carbon dioxide and water to thesugars, from which TAGs are derived.154

As a result of their rapid growth and sub-stantial production of TAGs, algae could beemployed as cell factories to produce oils andother lipids for biofuels and other biomateri-als, such as biodiesel, methane, hydrogen,ethanol, among others.132 In this context, bio-fuel production using microalgae offers sever-al advantages in relation to oilseed crops: a)the high growth rate and the high photosyn-thetic conversion efficiency of microalgaemakes it possible to satisfy the demand for bio-fuels using limited land resources; b) the cul-tivation of microalgae consumes less waterthan land crops; c) microalgae thrive insaline/brackish water/coastal seawater forwhich there are few competing demands; d)microalgae are able to synthesize and to accu-mulate large quantities of neutral lipids, main-ly TAGs; e) microalgae can utilize nutrientssuch as nitrogen and phosphorus from a vari-ety of wastewater sources, providing addition-al benefits through wastewater bio-remedia-tion; and f) are capable of tolerating high CO2

content in gas streams allowing high-efficien-cy of CO2 mitigation155,156 For these reasons,microalgae are capable of producing more oilper unit area of land, compared to terrestrialoilseed crops.157

On the other hand, one of the major draw-backs of microalgae for biofuel production is

the low biomass concentration in the microal-gal culture due to the limit of light penetration,which in combination with the small size ofalgal cells makes the harvest of algal biomass-es relatively costly.155

The main limitation for the replacement ofconventional diesel by biodiesel is thatbiodiesel still represents a small percentage oftotal diesel consumption, despite the largeincrease in its use. Two main factors have con-tributed to the limited adoption of biodiesel:problems with the fuel characteristics ofbiodiesel, namely poor cold-temperature prop-erties, higher rates of oxidation and increasedemission of nitrogen oxides (NOx) relative tothe conventional diesel and the interrelatedfactors of cost and supply limitations, since thetotal world plant oil production, for example,would only satisfy approximately 80% of USAdiesel demand.14

Genetic manipulation of fatty acidcontent Modification of oil composition could con-

tribute to the production of nutritionally andindustrially desirable oils in crop plants andalgae.64 The increasing global demand for oilshas intensified the research efforts to geneti-cally modify the organism to boost the oilyield.69 However, this requires not only a pre-cise manipulation of fatty acid but also TAGsynthesis in such a way that a specific synthe-sized fatty acid will be effectively incorporatedinto each position of TAG molecule.61

The natural diversity observed in seed andalgae TAGs indicates that there should be nobarriers to produce exotic FAs in domesticatedoilseed crops and algae, and also provides adeep and potentially useful gene pool forgenetic manipulation.10

Several unusual fatty acids described in thisreview, due to their unique chemical proper-ties, have important industrial applications.However, to make these economically attrac-tive, conventional oilseed crops will have to begenetically engineered to produce oils with asingle predominant unusual fatty acid in its oilcomposition. The existence of wild species,such as castor, which contains oil with 90%hydroxylated fatty acid, suggests that this is afeasible task.11 Thus, attractive targets forplant genetic engineering for altered TAG com-position are the temperate oilseed crops, suchas soybean, rapeseed, flax, and sunflower.10

Today, the generation of transgenic cropplants engineered to accumulate high levels ofspecific unusual fatty acids is a topic of enor-mous interest. However, the inability to specif-ically target unusual fatty acids to seed TAGs,and their excessive accumulation in mem-brane lipids, might disrupt seed membraneintegrity and impair seed development or ger-mination. To overcome this problem it is

essential to prevent the accumulation ofunusual fatty acids in seed membrane lipidsthrough engineering projects aimed at gener-ating viable, high-yielding transgenic plants byintroducing genes involved in the exclusion ofunusual fatty acids from membranes.11

Oilseeds provide an attractive platform forthe production of high-value fatty acids thatcan replace non-sustainable petroleum specialchemicals, such as diesel.67 However, in thecase of the production of fatty acids in trans-genic plants, the conversion of plant oils intobiodiesel and chemical feedstocks competeswith their use as food. Therefore, it isextremely important to ensure that these newindustrial productions do not affect the supplyof food at a time when world food require-ments are increasing rapidly.14,110

On the other hand, the production of PUFAsby marine and freshwater microalgae is thesubject of intensive research and increasingcommercial attention.128 In this scenario,metabolic engineering through genetic manip-ulation might represent a promising strategyfor the overproduction of algal oils.129

The focus of attention is the isolation anduse of novel fatty acid metabolic genes, espe-cially elongases and desaturases (Figure 5)that are responsible for PUFAs synthesis, fromdifferent species of algae and their transfer toplants in order to modify crops to create usefulnew products through genetic engeneeringtools.26 One example of this tendency is themetabolic engineering of omega-3 long-chainpolyunsaturated fatty acids in plants using anacyl-CoA D6-desaturase from the marinemicroalgae Micromonas pusilla, which origi-nated an artificial pathway that produced 26%EPA in Nicotiana benthamiana leaves.158 Theauthors also demonstrated that this enzymeappears to function as an acyl-CoA desaturasethat has preference for ω3 substrates, both inArabidopsis and in yeast transgenic lines.In this context, the identification and func-

tional characterization of enzymes involved inbiosynthesis of long-chain PUFAs in algae hasan important biotechnological significance forthe production of VLC-PUFAs in transgenicoilseed crops.159,161

In addition, it is believed that the most cost-ly downstream processing steps in fuel produc-tion using microalgal is that most microalgaewill not grow to a density higher than a fewgrams of biomass per liter of water. Whilethere are several possible low-cost solutions toconcentrate the biomass, these methods areslow and the resulting biomass may stillrequire further dewatering. One possible solu-tion is to manipulate the biology of microalgalcells to allow the secretion of fuels or feed-stocks directly into the growth medium,through the manipulation of lipid secretorypathway.156

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In conclusion, the complete clarification ofenzyme activities responsible for controllingthe flux of fatty acids to TAG will make a deci-sive contribution to the correct transfer ofunusual fatty acids into storage oils and cangenerate new tools for genetic oil manipula-tion.39,153 In this scenario, TFs regulatoryaction can ultimately affect several reactionsin biochemical pathways that contribute to theproduction and accumulation of storage com-pounds. Thus, modification of the expressionof genes encoding TFs represents anotherinteresting strategy that can be adopted inorder to increase the accumulation of desir-able oils in target organisms.18

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