Critical Reviews in Biotechnology, 26:83–93, 2006Copyright ©c Taylor & Francis Group, LLCISSN: 0738-8551 print / 1549-7801 onlineDOI: 10.1080/07388550600697479

Polyunsaturated Fatty Acids: BiotechnologyDnyaneshwar Warude andKalpana JoshiInterdisciplinary School ofHealth Sciences, University ofPune, Pune, India

Abhay HarsulkarInteractive Research School forHealth Affairs, BharatiVidyapeeth Deemed University,Dhankawadi, Pune, India

ABSTRACT Polyunsaturated fatty acids like EPA and DHA have attracted agreat attention due to their beneficial effects on human health. At present,fish oil is the major source of EPA and DHA. Various alternative sources arebeing explored to get these essential fatty acids. Genes encoding enzymes in-volved in the biosyntheses of PUFAs have been identified, cloned and geneprospecting becomes a novel method for enhanced PUFA production. Desat-urase and elongase genes have important biotechnological appeal from geneticengineering point of view. This review highlights the research and results onsuch enzymes.

KEYWORDS polyunsaturated fatty acids, eicosapentanoic acid, docosahexaenoic acid,desaturase, elongase.

INTRODUCTIONPolyunsaturated fatty acids (PUFAs) are essential components of human

health. They differ from other fatty acids in that they contain more than onedouble bond. Linoleic Acid (LA) and α-Linolenic Acid (ALA) are the parent Es-sential Fatty Acids (EFAs) of Omega-6 and Omega-3 families, respectively. Mostseed oils contain LA while fish oil, canola oil, flaxseed oil, and green leafy veg-etables are the major sources of Omega-3 fatty acids. LA and ALA are precursorfatty acids that are elongated to form Long Chain Poly Unsaturated Fatty Acids(LCPUFAs) like Arachidonic Acid (AA), Eicosapentanoic Acid (EPA) and Do-cosahexaenoic Acid (DHA). These LCPUFAs are associated with various physio-logical and pathophysiological processes, thereby affecting human health. Theyare essential components of the cell membrane and constitute over 30 percent offatty acids in brain.15 In retina, DHA accounts for more than 60 percent of thetotal fatty acids.23 Clinical studies show that DHA is essential for the growth anddevelopment of the brain in infants and for maintenance of normal brain func-tion in adults.42 Cell growth and division, platelet aggregation, inflammatoryresponses, hemorrhage, vasoconstriction and vasodilation and immune func-tions are associated with PUFAs. Studies have shown their role in preventionand treatment of coronary heart disease, hypertension, type 2 diabetes, arthritis,cancer and other inflammatory and autoimmune disorders.70 Demand of PUFAsis constantly increasing while the sources producing them are depleting. Variousalternative sources are being explored for yielding these essential elements ofhealth. Desaturases and elongases are key enzymes in biosynthesis of PUFAsand occur in most living cells. They produce precursors for the production of

Address correspondence to KalpanaJoshi, to Interdisciplinary School ofHealth Sciences, University of Pune,Pune-411007, India. E-mail:kalpana@unipune.ernet.in

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eicosanoids in mammals, pheromones in insects andjasmonates in plants. Most of these compounds actas hormones and in turn govern important biologi-cal activities. Recent advances in molecular biologyand biotechnology have contributed considerably toPUFA research for bioprospecting and enhanced PUFAproduction.

BIOSYNTHESIS OF PUFAsBiosynthetic pathways leading to formation of EPA

and DHA have been investigated during the last fewyears. Precursors required for biosynthesis of these fattyacids, i.e. LA and ALA, cannot be synthesized in mam-malian tissues and hence are essential dietary compo-nents. Higher plants are the major source of these EFAs.Fatty acid biosynthesis in plants takes place in plas-tids. These organelles are widely thought to have orig-inated from photosynthetic bacterial symbionts andhence fatty acid metabolism in plants closely resem-bles that of bacteria.71 A repeated series of reactionsincorporate acetyl moieties of acyl-CoA, resulting inchains of length 16 or 18 carbons. The enzymes in-volved in the synthesis are acetyl-CoA carboxylase andfatty acid synthase. Stearic acid, a C18 saturated fattyacid, gets converted to oleic acid, a mono unsaturatedfatty acid. The condensation is catalyzed by enzyme�9 desaturase. �12 desaturase converts oleic acid intoLA, which further gets converted to ALA by the actionof �15desaturase. The conventional aerobic pathway,which operates in most (PUFA-synthesizing) eukaryoticorganisms, starts with �6 desaturation of both 18:2 n-6and 18:3 n-3, resulting in the synthesis of γ -linolenic(GLA; 18:3 �6,9,12) and octadecatetraenoic (OTA; 18:4�6,9,12,15) acids, respectively. This first desaturationstep is followed by �6-specific C2 elongation to 20: 3�8,11,14 and 20:4 �8,11,14,17 and further �5-desatura-tion to produce AA and EPA. From this point, thebiosynthesis of DHA may follow two pathways. Thefirst one is a linear pathway, involving C2 elongationof EPA to C22: 5 �,7,10,13,16,19 which is desaturatedby �4-specific desaturase to yield DHA. Another path-way, known as “Sprecher” pathway, is independent of�4-desaturation. It involves two consecutive C2 elon-gation cycles to yield 24:5 �,7,10,13,16,19 followedby �6- desaturation and one cycle of C2-shorteningvia ß-oxidation in the peroxisome to yield DHA.68

Recent identification of a Thraustochytrium �4 desat-

urase indicates that �4desaturation is indeed involvedin DHA synthesis (Figure 1). An alternative pathwayfor the biosynthesis of C20 PUFAs has been demon-strated in organisms that appear to lack �6desaturase ac-tivity. The protist Tetrahymena pyriformis, Acanthamoebasps. and Euglena gracilis synthesize PUFAs by this mech-anism. First step in this route is elongation of C18fatty acids, LA and ALA, to eicosadienoic (20:2 �11,14)n-6 and eicosatrienoic (20:3 �11,14,17) n-3 fatty acids,respectively. In turn, these C20 products are desatu-rated by an �8-desaturase to produce 20:3 �8,11,14 n-6and 20:4 �8,11,14,17 n-3 PUFAs that are the interme-diates of the conventional pathway. The products of�8-desaturation are then subjected to further desat-uration at the �5 position to produce AA and EPAand may be elongated with subsequent �4-desaturationto DHA. This so called “�8-desaturation” pathwayhas also been found in rat and human testis4 andin glioma and breast cancer cell lines7 as well as be-ing hypothesized to explain the synthesis of AA infelines, which appears to lack a �6desaturase activ-ity. A �8desaturase has been cloned from Euglenaand shown to be structurally related to the othercytochrome- b5 fusion desaturases (e.g. �6-, �5- and�4-) involved in PUFA synthesis.49 Recently, anotherpathway for DHA biosynthesis-the anaerobic polyke-tide synthase pathway has been reported to occur insome of the marine bacteria and primitive eukaryoteslike thraustochytrid protist Schizochytrium, a member ofthe Thraustochytriidae.57

GENETIC ENGINEERINGDesaturases and elongases are the critical enzymes

involved in the biogenesis of most of the PUFAs. Dur-ing the last decade, several genes responsible for thebiosynthesis of n-6 and n-3 PUFAs have been clonedfrom various organisms like algae, fungi, mosses, higherplants and mammals (Table 1).

DESATURASESDesaturases are iron-containing enzymes that intro-

duce a double bond in a specific position in long-chainfatty acids. There are two types of desaturases: omegaand delta desaturases, both of which are membrane-bound proteins. They create a double bond at afixed position counted from the carbonyl end of the

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FIGURE 1 Biosynthetic pathways of PUFAs.

fatty acids, aerobically. This reaction requires molec-ular oxygen, NAD(P)H, an electron transport system(ferredoxin- NADPH reductase and ferredoxin, or cy-tochrome b5 reductase and cytochrome b5) and a ter-minal desaturase. All the desaturases are acyl desat-urases containing a N-terminal cytochrome b5 domain,which serves as electron donor for their activity.72 Thesemembrane-bound desaturases can be divided into twosubgroups: Acyl lipid desaturases and acyl- coenzymeA (CoA) desaturases. The first group of enzymes is

located in the membranes of thylakoid, plant endo-plasmic reticulum (ER) and plastid. Acyl-lipid desat-urases in cyanobacteria and plant plastid can desatu-rate stearic (18:0) and oleic (18:1 n-9) acyl groups inmonogalactosyl diacylglycerol (cyanobacteria and plantplastid) and in phosphatidylglycerol (plant plastid),whereas plant ER desaturases mostly use fatty acids inphosphatidylcholine.80 The other subgroups, acyl-CoAdesaturases, are present in ER membrane and use fattyacyl-CoAs as substrates. These are present in animals

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TABLE 1 Genes coding for desaturases and elongases from various sources

Enzymes Type of organism Species

∆9 desaturase YeastInsect

ProtozoanCyanobacteria

Plants

Saccharomyces cerevisiae75

Drosophila melanogaster88

Trichoplusia ni40

Tetrahymena thermophila47

Anabaena variabilis63

Synechocystis sp.63

Arabidopsis19

Rose18

∆12 desaturase Plants

Cyanobacteria

InsectsMushroom

Arabidopsis thaliana52

Pomegranate seeds29

Parsley,36 olive,26

soybean,77 flax,10 cotton24

Synechocystis sp. Synechococcus sp.64

Anabaena variabilis64

Cockroach9 cricket16

Lentinula edodes62

∆15 desaturase Plants Arabidopsis thaliana89

Brassica napus5

Soyabean46

Castor83

∆6-desaturase MammalsNematodePlants

Mosses

Fungi

Human11

Caenorhabditis elegans48

Borago officinalis67

Echium20

Ceratodon purpureus72

Physcomitrella patens22

Mortierella alpina65

∆5 desaturase MammalsNematodeFungi

Algae

Human12

Caenorhabditis elegans85

Mortierella alpina52

Pythium irregulare27

Thraustochytrium sp.59

∆4-desaturase BacteriaPlantEuglenaAlgae

Thraustochytrium sp.58

Brassica juncea58

E. gracilis43

Pavlova lutheri54

Isochrysis sp.82

Bifunctional ∆5/∆6 desaturase Fish Danio rerio25

C18-20 n-3 desaturase Nematode Caenorhabditis elegans73

C20 ∆8-desaturase Protist (green algae) Euglena gracilis84

∆6-elongases NematodeMossesFungi

Caenorhabditis elegans8

Physcomitrella patens90

Mortierella alpina53

PUFA-elongase Mammals Human39

∆9-elongase Algae Isochrysis galbana55

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including insects and nematodes as well as fungi. Allthe mammalian desaturases that have been identifiedare acyl-CoA desaturases. Several reports on cloningand characterization of desaturase genes are available.In comparison, the work on the desaturase proteins isseverely trailing, mainly because of problems in pu-rifying them. Except �9, other desaturase are mem-brane bound and pose technical problems in solubiliz-ing them. Structural characterization of the desaturaseproteins is therefore poorly understood.

∆9 DesaturasesMono-unsaturated fatty acids (MUFAs) are synthe-

sized from saturated fatty acids by �9 desaturases.Mammalian �9 desaturase is referred to as stearoyl-CoA desaturase (SCD). Its gene was identified fromthe amino acid sequences78,79 and analysis showed thatthe enzyme contains three regions of conserved His-box motifs. Site-directed mutagenesis has shown thatall of these conserved His residues are essential forthe enzyme activity.69 The gene encoding for the en-zyme, named OLE1, was cloned first from the buddingyeast Saccharomyces cerevisiae.75 Later, it was proved thatthe OLE1 gene can be functionally replaced by the ratstearyl-CoA desaturase.74 �9desaturases in animals, in-cluding insects, nematodes and vertebrates, share com-mon features. Further, it has been proved that additionof unsaturated fatty acids to the growth medium sup-pressed the expression of OLE1 gene in some yeasts butnot in others. In S. cerevisiae low temperature stress wasfound to affect expression of the OLE1 gene. Genescoding for stearate �9 acyl-lipid desaturases have beenisolated from two cyanobacteria, Anabaena variabilisand Synechocystis sp. PCC 6803. The deduced aminoacid sequences of these enzymes are similar, in part,to those of stearoyl-CoA desaturases of rat, mouse andS. cerevisiae, but not to those of acyl-lipid desaturasesof higher plants.63 �9 desaturases have also been iso-lated from the insect Drosophila melanogaster,88 cabbagelooper moth, Trichoplusia ni,40 protozoan Tetrahymenathermophila47 showing sequence homology with well–defined �9 desaturases from rat, yeast and other insectsand vertebrates. Two genes specific to stearic acid de-saturation, FAT-6 and FAT-7, have been isolated fromCaenorhabditis elegans.86 Attempts have been made toexpress both wild type and engineered forms of rat SCDin human transformed kidney cells using recombinantadenovirus vector.46 Engineering �9 16:0-acyl carrier

protein (ACP) desaturase specificity based on combi-natorial saturation mutagenesis and logical redesign ofthe castor �9 18:0-ACP desaturase has also been tried.This approach helps in reengineering the enzyme toachieve substrate specificity with retention of the de-sired stability.87 Genes coding for �9 desaturases fromplants have also been characterized. Arabidopsis desat-urases (ADS) genes, ADS1 and ADS2,19 and desaturasefrom the petals of rose flowers [18] encode proteins ho-mologous to �9acyl-lipid desaturases of cyanobacteriaand acyl-CoA desaturases of yeast and mammals. Coldtemperature up-regulates ADS2 expression and down-regulates the ADS1 expression. Plant tissues expressing amammalian stearoyl-CoA �9 desaturase were reportedto accumulate �9 hexadecanoic acid (16:1), normallyvery minor in most plant tissues.45

∆12 DesaturasesThe major enzyme responsible for the synthesis

of LA from oleic acid is the oleate desaturase ofthe ER, and its activity is controlled by �12 fattyacid desaturase (FAD2) gene. This enzyme is thoughtto be an integral membrane protein that accepts1-acyl-2-oleoyl-sn-glycero-3-phosphocholine as a sub-strate and requires NADH, NADH: cyt b5 oxidore-ductase, cyt b5 and oxygen for activity.21 The FAD2gene has been cloned from various plants such asArabidopsis thaliana,52 pomegranate seeds,29 olive,26

flax,10 cotton,32 parsley and lower organisms. Func-tional expression of extraplastidial FAD2 in S. cere-visiae resulted in accumulation of linoleic acid.14 Mi-crosomal membrane preparations from the maturingcotyledons of common borage (Borago officinalis) ex-hibit �12 and �6 desaturase activities, which resultin the synthesis of linoleate and gamma-linolenate,respectively.24 Seed-specific isoforms of FAD2 are iso-lated from soyabean, which differ at only 24 aminoacid residues. Expression studies in yeast revealed thatthe FAD2-1A isoform is more unstable than FAD2-1B,particularly when cultures were maintained at elevatedgrowth temperatures.77 A novel �12 desaturase fromanimals, which converts oleic acid (18:1n-9) to linoleicacid (18:2n-6), was characterized in the house cricket,Acheta domesticus. Enzyme activity was located in themicrosomal fraction of whole insect homogenates.16

The cockroach, Periplaneta americana, can convert oleicacid to linoleic acid by a microsomal �12 desaturase.9

Cyanobacterial genes for enzymes that desaturate fatty

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acids at the �12 position, designated desA, were iso-lated from various species of cyanobacteria, namelySynechocystis, Synechococcus, Anabaena variabilis andSynechocystis.64 Also, A �12 desaturase from the ediblemushroom, Lentinula edodes, consisting of 1308 bp andcoding for 435 amino acids, has been isolated.62

Specific activity study of the �12 desaturase systemof Lipomyces starkeyi was carried out. The study demon-strated that this enzyme complex, functioning opti-mally between pH 7 and 8, has low thermal stability.Ca2+ may cause an aggregation of the microsomes, andHg2+ completely inhibits the activity, whereas Mg2+,Mn2+, and Zn2+ are activators. The �12 desaturase sys-tem is relatively specific towards oleic acid, though iso-mers of this fatty acid also have an action, either assubstrates or as competitive inhibitors on the activityof the system. The study of the effect of the exogenousoleoyl-CoA and elaidoyl-CoA on the specific activityof the �12 desaturase system showed a preference to-ward oleoyl-CoA.41 Temperature-induced membrane-lipid adaptation was studied in Acanthamoeba castellanii.There appears to be a rapid induction of �12 desat-urase activity in A. castellanii after a downward shift ingrowth temperature.33 Introduction of structurally de-fined peptide elicitor (Pep25 ) gene in suitable vector offungal origin has been shown to cause rapid and largechanges in the levels of various desaturated fatty acids.The peptide elicitor, upon, treatment of cultured pars-ley (Petroselinum crispum L.) cells, elicits two dienoic fattyacids, hexadecadienoic and linoleic, which are not de-tectable in control cells and together constitute up to 12percent of the total fatty acids in the transformed cells.�12 FAD mRNA accumulated rapidly and transientlyin elicitor-treated parsley cells, protoplasts, and leaves.36

∆15 DesaturasesALA is an important component of plant membrane

lipids as well as storage triacylglycerol. This fatty acidis synthesized by desaturation of glycerolipid-linkedlinoleic acid (18:2) by a �15 desaturase. There are twodistinct pathways of 18:3 fatty acid synthesis in plants,one operating in plastids and the other in the ER. Con-sequently, the �15 desaturase exists in two different iso-forms located in two different organelles. The genesencoding the ER isoform of the �15desaturase fromBrassica napus,5 safflower and Arabidopsis thaliana,89 aswell as the plastidic isoform from A. thaliana, soybean31

and castor,83 have been isolated.

∆6 Desaturases�6 desaturases (D6D) are another group of en-

zymes that catalyse the synthesis of PUFAs. First D6Dwas cloned from Synechocystis using gain-of-cloningmethod.60 Subsequently, other D6Ds were cloned fromCaenorhabditis elegans,48 Mucor rouxii,50 M. alpina,65

humans,11 Rhizopus arrhizus,91 mice and rats,3 and fromplant species like Echium,20 Borage officinalis67 using a se-quence homologous to the Synechocystis D6D, or otherdesaturases. D6D is classified as a front-end desaturasebecause it introduces a double bond between the pre-existing double bond and the carboxyl (front) end of thefatty acid. Sequence analysis of deduced amino acidshas shown that D6Ds contain the amino-terminal cy-tochrome b5 domain carrying heme-binding motifs.66

The Borage and spirulina D6Ds are acyl-lipid desat-urases, that use linoleate in phosphatidylcholine as asubstrate,38,76 whereas D6Ds in other species are acyl-CoA desaturases. Gamma-linolenic acid (GLA), a nutri-tionally important fatty acid in human and animal di-ets, is not produced in many nutritive crops. However,some plants, including most of the oil seed crops, pro-duce significant quantities of linoleic acid, a fatty acidthat could be converted to GLA by the enzyme delta6-desaturase if it were present. Expression of cyanobac-terial �6 desaturase gene in transgenic tobacco61 andborage �6 desaturase cDNA in tomato (Lycopersicon es-culentum L.)13 have been shown to produce significantamounts of GLA. Also, attempts to produce GLA inBrassica juncea by expressing delta 6 desaturase fromPythium irregulare were successful.28

∆5 DesaturasesThese are another group of front-end desaturases

present in animals, catalyzing the biosynthesis of highlyunsaturated fatty acids. After desaturation and elon-gation by D6D and elongase, respectively, D5Ds in-troduce another double bond at the �5 position of20-carbon fatty acids 20:3 n-6 and 20:4 n-3. D5Dgenes have been cloned from several animals includinghumans,12 rats,93 C. elegans85 and Liverwort Marchan-tia polymorpha.34 The human D5D gene encodes 444amino acids (the same number as the human D6D)and possesses 61% amino acid identity and 75% sim-ilarity to the human D6D. The predicted amino acidsequence of D5D contains all of the structural char-acteristics present in D6Ds. Recently, Hastings et al.25

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cloned and characterized a zebra fish (Danio retio) desat-urase that is homologous to both D6D and D5D. Thededuced amino acid sequence of this enzyme shared64% and 58% identity with human D6D and D5D, re-spectively. This enzyme was able to convert not onlyLA and ALA to 18:3 n-6 and 18:4 n-3, but also pro-duced AA and EPA when the enzyme was expressedin yeast. This result indicates that the zebra fish de-saturase possesses both D6D and D5D activities. D5Dfrom Pythium irregulare was expressed in Saccharomycescerevisiae and also in certain oil seed crops. Introduc-tion of this gene into B. juncea and flax, under the con-trol of seed-specific promoters, resulted in productionof �5unsaturated polymethylene-interrupted FA repre-senting more than 10% of the total FA in the seeds.The transgenic enzyme could also desaturate the exoge-nously supplied homo-gamma-linolenic acid to AA.27

Arabidopsis thaliana was transformed sequentially withgenes encoding a �9-specific elongating activity fromIsochrysis galbana, a �8-desaturase from Euglena gracilisand a �5-desaturase from Mortierella alpina resulting inproduction of two LCPUFAs, AA and EPA, in substan-tial quantities.56,92

∆4 DesaturasesVarious reports suggest the involvement of the ac-

tion of a �4-fatty acid desaturase in biosynthesis ofDHA. Genes encoding for the enzyme have been iso-lated and sequenced from various sources. Qui et al.58

have identified a cDNA coding for �4 fatty acid desat-urase from Thraustochytrium sp., which when expressedin S. cerevisiae introduced a double bond at position4 of 22:5(n − 3) and 22:4(n − 6) fatty acids, result-ing in the production of DHA and docosapentanoicacid. The enzyme, when expressed in Brassica juncea,under the control of a constitutive promoter, desatu-rated the exogenously supplied substrate 22:5(n − 3),resulting in the production of DHA in vegetative tis-sues. The gene from E. gracilis has also been clonedand it has been shown that the enzyme has strict�4-regioselectivity and requires the presence of a delta7-double bond in the substrate. Also, positional analysisof phosphatidylcholine revealed that the proportion ofthe �4-desaturated products was up to 20 times higherin the sn-2 position than in the sn-1 position.43 Pavlovalutheri and Isochrysis, marine microalgae, are also a richsource of LCPUFAs. Using an expressed sequence tagapproach, cDNAs specific to �4 desaturase have beenisolated.54,82

ELONGASESEnzymes that increase chain length are essential for

biosynthesis of long chain polyunsaturated fatty acids.In n-3 and n-6 biosynthetic pathways �6 elongaseconverts 18:4 �6,9,12,15 to 20:4 �8,11,14,17 and GLA to20:3 �8,11,14, respectively. Genes for this condensingenzyme have been cloned from different organ-isms like moss Physcomitrella patens,90 Isochrysis galbana,a marine prymnesiophyte microalgae,55 the ne-matode Caenorhabditis elegans,8 liverwort Marchantiapolymorpha L35 and zebra fish, Danio rerio.2 It isinteresting to note that substrate specificity of the�6 elongase with respect to n-6 and n-3 pathwayvaries in different organisms. The elongase, PSE1 fromP. patens, is more specific to the n-6 pathway ratherthan n-3, while genes coding for delta 6 elongase fromC. elegans and I. galbana catalyze elongation in bothpathways with similar magnitude. Elongase gene fromzebra fish gives elongases that show specificity for allcondensation reactions, viz. �5, �6, �7.

Fatty acid elongation can be divided into four dif-ferent reactions: condensation of malonyl-CoA witha long-chain acyl-primer to form beta-ketoacyl- CoA,reduction to b-hydroxyacyl-CoA, dehydration to trans-2-enoyl-CoA, and reduction of the trans double bondresulting in the elongated acyl-CoA.17 The microsomalelongation system consists of four distinct enzymes:beta-ketoacyl-CoA synthase, a beta-ketoacyl-CoA re-ductase, a beta-hydroxyacyl-CoA dehydratase, and anenoyl-CoA reductase catalyzing only a single step of thewhole sequence. Furthermore, several elongases occurwithin a plant, differing in their expression. It is thoughtthat the substrate specificities and catalytic activitiesof elongase complexes are controlled by beta-ketoacyl-CoA synthases (KCS), which are responsible for theinitial condensation reaction,44 whereas different con-densing enzymes apparently share the same set of re-ductases and dehydratases.37 During the last decade,several genes coding for ketoacyl-CoA synthases havebeen cloned from different plant species. These en-zymes, named KCS or FAE, are specific for saturatedand monounsaturated fatty acids that are used for thebiosynthesis of waxes and seed storage lipids. Interest-ingly, they do not share any sequence similarity with theELO genes from S. cerevisiae, which code for the corre-sponding condensing enzymes of the yeast fatty acidelongation systems. ELO1 is involved in the elonga-tion of saturated and monounsaturated medium-chainfatty acids (C14-C16), whereas ELO2 and ELO3 are

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required for the elongation of saturated long-chain fattyacids leading to C24 or C26, respectively.51,81 The rea-son for this obviously convergent development in thefirst step of fatty acid elongation is unclear and for along time no biochemical data was available that couldprovide direct evidence for the function of an ELO geneproduct acting as a condensing enzyme. Only recently,Moon et al.45 have shown that a beta-ketoacyl-CoA isindeed the first intermediate resulting from the activityof a recombinant ELO-like enzyme from mouse. Thecloned fungal and animal genes that are involved inthe elongation of polyunsaturated fatty acids (PUFAs)share homology with the yeast ELO sequences. For thefirst time the elongases sequences from moss P. patenshave shown homology to the yeast ELO sequences.

BIOTECHNOLOGY PROSPECTIVEBiotechnology has been widely adopted in agricul-

ture and explored for production of essential nutri-ents. Desaturase genes are being cloned in model plantslike tobacco and Arabidopsis to study accumulationof PUFA. Higher plants can produce long chain fattyacids with carbon number up to 18 only. GLA is themost unsaturated fatty acid produced by the oil seedplants. Some lower organisms like bacteria, protozoaand cold-water fishes have the complete machinery tosynthesize long chain PUFAs like AA, EPA and DHA.Mammals also possess the enzyme systems for biosyn-thesis of most of the vital PUFAs, including AA, EPAand DHA from the precursors (LA and ALA) obtainedthrough diet. Presently, fish oils and other seafood arethe major source of EPA and DHA. Obnoxious odor,heavy metal contamination and concerns of vegetari-ans limit the use of fish oil. This has stimulated greatinterest in oil seed plant research. Existence of differentpathways for PUFAs biosynthesis offers a wide range ofalternatives to plant biotechnologists in their quest toproduce desired fatty acids in transgenic oilseed crops.Long-term goals to produce AA, EPA and DHA intransgenic oilseed crops have led to the cloning andexpression of most of the genes coding for the de-saturases and elongases involved in LCPUFA biosyn-thesis. The first report towards this was expression ofborage �6desaturase in transgenic tobacco and Ara-bidopsis plants.67 Co-expression of the M. alpina �6-and �12desaturases in canola resulted in the accumu-lation of up to 50% of GLA.30 A step forward in as-sembling PUFAs in oilseed crops was co- expression of

�9desaturase from Isochrysis galbana, �8desaturase fromEuglena gracilis and �5desaturase from Mortierella alpinain arabidopsis that resulted in accumulation of AA andEPA.6 Also, seed-specific expression of cDNAs encod-ing fatty-acyl desaturases and elongases in transgenictobacco (Nicotiana tabacum) and linseed (Linum usitatis-simum) resulted in accumulation of �6desaturated C18fatty acids and up to 5% C20 PUFAs, including AAand EPA.1 Use of Genetically Modified (GM) plants forsynthesis of omega-3 fatty acids provides a sustainablesource of these important dietary elements. GM cropshave become a focus of controversy due to questions offood and environmental safety. Despite this, in the pastfew years the global area of commercially grown GMcrops has increased more than 30-fold to over 52 millionhectares. The number of countries involved has morethan doubled. Especially in developing countries, theGM crop area is anticipated to increase rapidly in thecoming years. Eventually, targeted efforts to produceEPA and DHA in oil seeds like sunflower, safflower andlinseed are highly desirable to bring omega-3 effectivelyinto the food chain.

ACKNOWLEDGEMENTWe would like to thank Dr. S. P. Mahadik for in-

troducing us to omega-3 fatty acid research, Dr P. K.Ranjekar and Dr. Bhushan Patwardhan for their con-stant encouragement, and Prof. J. Thomas Brenna forcritically reading the review. Research grant to AH fromDepartment of Biotechnology, Government of India, isacknowledged.

REFERENCES[1] Abbadi, A., Domergue, F., Bauer, J., Napier, J. A., Welti, R., Zahringer,

U., Cirpus, P., and Heinz, E. 2004. Biosynthesis of very long chainpolyunsaturated fatty acids in transgenic oilseeds: Constraints ontheir accumulation. Plant Cell. 6(10): 2734–2748.

[2] Agaba, M., Tocher, D. R., Dickson, C. A., Dick, Jr., and Teale, A. J.2004. Zebra fish cDNA Encoding multifunctional fatty acid elongaseinvolved in production of eicosapentaenoic (20:5n-3) and docosa-hexaenoic (22:6n-3) acids. Mar. Biotechnol. 65: 42–47.

[3] Aki, T., Shimada, Y., Inagaki, K., Higashimoto, H., and Kawamoto,S. 1999. Molecular cloning and functional characterization of ratdelta-6 fatty acid desaturase. Biochem. Biophys. Res. Com. 255:575–579.

[4] Albert, D. H., Rhamy, R. K., and Coniglio, J. G. 1979. Desaturationof eicosa-11, 14-dienoic acid in human testes. Lipids. 14: 498–500.

[5] Arondel, V., Lemieux, B., Hwang, I., Gibson, S., Goodman, H. M.,and Somerville, C. R. 1992. Map-based cloning of a gene controllingomega-3 fatty acid desaturation in Arabidopsis. Science. 258: 1353–1355.

[6] Baoxiu, Qi, Tom, F., Sam, M., Gary, D., Justine, B., Napier J. A.,Keith, S., and Colin, M. L. 2004. Production of very long chain

Dnyaneshwar Warude et al. 90

Cri

tical

Rev

iew

s in

Bio

tech

nolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y N

orth

Car

olin

a St

ate

Uni

vers

ity o

n 09

/07/

13Fo

r pe

rson

al u

se o

nly.

polyunsaturated omega-3 and omega-6 fatty acids in plants. Na-ture Biotech. 22: 739–745.

[7] Bardon, S., Le, M. T., and Alessandri, J. M. 1996. Metabolic conver-sion and growth effects of n-6 and n-3 polyunsaturated fatty acidsin the T47D breast cancer cell line. Cancer Lett. 99: 51–58.

[8] Beaudoin, F., Michaelson, L. V., Hey, S. J., Lewis, M. J., Shewry, P. R.,Sayanova, O., and Napier, J. A. 2000. Heterologous reconstitution inyeast of the polyunsaturated fatty acid biosynthetic pathway. Proc.Natl. Acad. Sci. USA. 97: 6421–6426.

[9] Borgeson, C. E., Renobales, M., and Blomquist, G. J. 1990. Char-acterization of the delta 12 desaturase in the American cockroach,Periplaneta americana: the nature of the substrate. Biochim BiophysActa. 1047(2): 135–140.

[10] Bourlaye, F., Scott, D., and Sylvie, C. 2004. Cloning of fatty acidbiosynthetic genes β-ketoacyl CoA synthase, fatty acid elongase,stearoyl-ACP desaturase, and fatty acid desaturase and analysis ofexpression in the early developmental stages of flax (Linum usitatis-simum L.) seeds. Plant Sci. 166(6): 1487–1496

[11] Cho, H. P., Nakamura, M. T., and Clarke, S. D. 1999. Cloning, ex-pression, and nutritional regulation of the mammalian delta-6 de-saturase. J. Biol. Chem. 274: 471–477.

[12] Cho, H.P., Nakamura, M., and Clarke, S. D. 1999. Cloning, expres-sion, and fatty acid regulation of the human delta-5 desaturase. J.Biol. Chem. 274: 37335–37339.

[13] Cook, D., Grierson, D., Jones, C., Wallace, A., West, G., and Tucker,G. 2002. Modification of fatty acid composition in tomato (Lycop-ersicon esculentum) by expression of a borage delta 6-desaturase.Mol Biotechnol. 21(2): 123–128.

[14] Covello, P. S., and Reed, D. W. 1996. Functional expression of theextraplastidial Arabidopsis thaliana oleate desaturase gene (FAD2)in Saccharomyces cerevisiae. Plant Physiol. 111(1): 223–226.

[15] Crawford, M. A., Costeloe, K., Ghebremeskel, K., Phylactos, A.,Skirvin, L., and Stacey, F. 1997. Are deficits of arachidonic and do-cosahexaenoic acids responsible for the neural and vascular compli-cations of preterm babies? Am. J. Clin. Nutr. 66: 1032–1041.

[16] Cripps, C., Borgeson, C., Blomquist, G. J., and Renobales, M. 1990.The delta 12-desaturase from the house cricket, Acheta domesticus(Orthoptera: Gryllidae): characterization and form of the substrate.Arch Biochem Biophys. 278(1): 46–51.

[17] Fehling, E., and Mukherjee, K. D. 1991. Acyl-CoA elongase from ahigher plant (Lunaria annua): metabolic intermediates of very long-chain acyl-CoA products and substrate specificity. Biochim. Biophys.Acta. 1082: 239-246.

[18] Fukuchi-Mizutani, M., Savin, K., Cornish, E., Tanaka, Y., Ashikari, T.,Kusumi, T., and Murata, N. 1995.Senescence-induced expression ofa homologue of delta 9 desaturase in rose petals. Plant Mol. Biol.29(4): 627–635.

[19] Fukuchi-Mizutani, M., Tasaka, Y., Tanaka, Y., Ashikari, T., Kusumi,T., and Murata, N. 1998. Characterization of delta 9 acyl-lipid de-saturase homologues from Arabidopsis thaliana. Plant Cell Physiol.39(2): 247–253.

[20] Garcia-Maroto, F., Garrido-Cardenas, J. A., Rodriguez-Ruiz, J.,Vilches-Ferron, M., Adam, A. C., Polaina, J., and Alonso, D. L. 2002.Cloning and molecular characterization of the delta6-desaturasefrom two echium plant species: production of GLA by heterologousexpression in yeast and tobacco. Lipids. 37(4): 417–426.

[21] Gennity, J. M., and Stumpf, P. K. 1985.Studies of the delta 12 de-saturase of Carthamus tinctorius L. Arch Biochem. Biophys. 239(2):444–454.

[22] Girke, T., Schmidt, H., Zahringer, U., Reski, R., and Heinz, E. 1998.Identification of a novel delta 6 acyl-group desaturase by targetedgene disruption in Physcomitrella patens. Plant J. 15: 39–48.

[23] Giusto, N. M., Pasquare, S. J., Salvador, G. A., Castagnet, P. I., Roque,M. E., and Ilincheta de Boschero, M. G. 2000. Lipid metabolism invertebrate retinal rod outer segments. Prog. Lipid Res. 39: 315–391.

[24] Griffiths, G., Stobart, A. K., and Stymne, S. 1988. Delta 6- anddelta 12-desaturase activities and phosphatidic acid formation in

microsomal preparations from the developing cotyledons of com-mon borage (Borago officinalis). Biochem J. 252(3): 641–647.

[25] Hastings, N., Agaba, M., Tocher, D. R., Leaver, M. J., and Dick, J. R.2001. A vertebrate fatty acid desaturase with delta 5 and delta 6activities, Proc. Natl. Acad.Sci. USA. 98: 14304–14309.

[26] Hernandez, M. L., Mancha, M., and Martinez-Rivas, J. M. 2005.Molecular cloning and characterization of genes encoding twomicrosomal oleate desaturases (FAD2) from olive. Phytochem,66(12):1417–1426.

[27] Hong, H., Datla, N., MacKenzie, S. L., and Qiu, X. 2002. Isolation andcharacterization of a delta 5 FA desaturase from Pythium irregulareby heterologous expression in Saccharomyces cerevisiae and oilseedcrops. Lipids. 37(9): 863–868.

[28] Hong, H., Datla, N., Reed, D. W., Covello, P. S., MacKenzie, S. L.,and Qiu, X. 2002. High-level production of gamma-linolenic acid inBrassica juncea using a delta 6 desaturase from Pythium irregulare.Plant Physiol. 129(1): 354–362.

[29] Hornung, E., Pernstich, C., and Feussner, I. 2002. Formation ofconjugated Delta11 Delta13-double bonds by Delta12-linoleic acid(1,4)-acyl-lipid-desaturase in pomegranate seeds. Eur. J. Biochem.269(19): 4852–4859.

[30] Huang, Y. S., Mukerji, P., Das, T., and Knutzon, D. S. 2001. Trans-genic production of long-chain olyunsaturated fatty acids. WorldRev. Nutr.Diet. 88: 243–248.

[31] Iba, K., Gibson, S., Nishiuchi, T., Fuse, T., Nishimura, M., Arondel, V.,Hugly, S., and Somerville, C. R. 1993. A gene encoding a chloroplastomega-3 fatty acid desaturase complements alterations in fatty aciddesaturation and chloroplast copy number of the fad 7 mutant ofArubidopsis tkaliana. J. Biol. Chem. 268: 24099–24105.

[32] Irma, L. P., Wisatre, K., Mongkol, N., John, E. K., Kent, D. C., andRobert, M. P. 2001. Molecular cloning and functional expression ofthe gene for a cotton Delta-12 fatty acid desaturase (FAD2). Biochim-ica et Biophysica Acta (BBA)—Gene Structure and Expression 1, 522(2): 122-129.

[33] Jones, A. L., Hann, A, C., Harwood, J. L., and Lloyd, D.1993.Temperature-induced membrane-lipid adaptation in Acan-thamoeba castellanii. Biochem J. 290: 273–278.

[34] Kajikawa, M., Yamato, K. T., Kohzu, Y., Nojiri, M., Sakuradani, E.,Shimizu, S., Sakai, Y., Fukuzawa, H., and Ohyama, K. 2004. Isolationand characterization of delta (6)-desaturase, an ELO-like enzymeand delta (5)-desaturase from the liverwort Marchantia Polymorphaand production of arachidonic and eicosapentaenoic acids in themethylotrophic yeast Pichia pastoris. Plant Mol. Biol. 54(3): 335–352.

[35] Kajikawa, M., Yamato, K. T., Sakai, Y., Fukuzawa, H., and Ohyama,K. 2006. Isolation and functional characterization of fatty aciddelta5-elongase gene from the liverwort Marchantia polymorphaL. FEBS lett. 580(1): 149–154.

[36] Kirsch, C., Hahlbrock, K., and Somssich, I. E. 1997. Rapid and tran-sient induction of a parsley microsomal delta 12 fatty acid desaturasemRNA by fungal elicitor. Plant Physiol. 115(1): 283–289.

[37] Kohlwein, S. D., Eder, S., Oh, C. S., Martin, C. E., Gable, K., Bacikova,D., and Dunn, T. 2001. Tsc13p is required for fatty acid elongationand localizes to a novel structure at the nuclear-vacuolar interfacein Saccharomyces cerevisiae. Mol. Cell Biol. 21: 109–125.

[38] Kurdrid, P., Subudhi, S., Hongsthong, A., and Ruegjitchatchawalya,M. 2005. Tanticharoen, M. Functional expression of Spirulina-delta6desaturase gene in yeast, Saccharomyces cerevisiae. Mol. Biol. Rep.32(4): 215–226.

[39] Leonard, A. E., Kelder, B., Bobik, E. G., Chuang, L. T., Lewis, C. J.,Kopchick, J. J., Mukerji, P., and Huang, Y. S. 2002. Identificationand expression of mammalian long-chain PUFA elongation enzymes.Lipids. 37: 733–740.

[40] Liu, W., Ma, P. W., Marsella-Herrick, P., Rosenfield, C. L., Knipple,D. C., and Roelofs, W. 1999. Cloning and functional expression ofa cDNA encoding a metabolic acyl-CoA delta 9-desaturase of thecabbage looper moth, Trichoplusia ni. Insect Biochem. Mol. Biol.29(5): 435–443.

91 Polyunsaturated Fatty Acids: Biotechnology

Cri

tical

Rev

iew

s in

Bio

tech

nolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y N

orth

Car

olin

a St

ate

Uni

vers

ity o

n 09

/07/

13Fo

r pe

rson

al u

se o

nly.

[41] Lomascolo, A., Dubreucq, E., and Galzy, P. 1996. Study of the delta12-desaturase system of Lipomyces starkeyi. Lipids. 31(3): 253–259.

[42] Martinetz, M. 1992. Tissue levels of polyunsaturated fatty acids dur-ing early human development. J. Pediatr. 120: S129-S138.

[43] Meyer, A., Cirpus, P., Ott, C., Schlecker, R., Zahringer, U., and Heinz,E. 2003. Biosynthesis of docosahexaenoic acid in Euglena gracilis:biochemical and molecular evidence for the involvement of a Delta4-fatty acyl group desaturase. Biochem. 42(32): 9779–9788.

[44] Millar, A. A., and Kunst, L. 1997. Very-long-chain fatty acid biosyn-thesis is controlled through the expression and specificity of thecondensing enzyme. Plant J. 12: 121–131.

[45] Moon, H., Hazebroek, J., and Hildebrand, D. F. 2000. Changesin fatty acid composition in plant tissues expressing a mammaliandelta9 desaturase. Lipids. 35(5): 471–479.

[46] Mziaut, H., Korza, G., Benraiss, A., and Ozols, J. 2002. Selectivemutagenesis of lysyl residues leads to a stable and active form ofdelta 9 stearoyl-CoA desaturase. Biochim. Biophys. Acta. 1583(1):45–52.

[47] Nakashima, S., Zhao, Y., and Nozawa, Y. 1996. Molecular cloningof delta 9 fatty acid desaturase from the protozoan Tetrahymenathermophila and its mRNA expression during thermal membraneadaptation. Biochem J. 317: 29–34.

[48] Napier, J. A., Hey, S. J., Lacey, D. J., and Shewry, P. R. 1998. Iden-tification of a Caenorhabditis elegans delta-6-fattyacid-desaturaseby heterologous expression in Saccharomyces cerevisiae. Biochem.J. 330: 611–614.

[49] Napier, J. A., Michaelson, L. V., and Sayanova, O. 2003. The roleof cytochrome b5 fusion desaturases in the synthesis of polyunsat-urated fatty acids. Prostaglandins Leukot. Essent. Fatty Acids. 68:135–143.

[50] Na-Ranong, S., Laoteng, K., Kittakoop, P., Tantichareon, M., andCheevadhanarak, S. 2005. Substrate specificity and preference ofDelta 6-desaturase of Mucor rouxii. FEBS Lett. 579(12): 2744–2748.

[51] Oh, C. S., Toke, D. A., Mandala, S., and Martin, C. E. 1997. ELO2and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene,function in fatty acid elongation and are required for sphingolipidformation. J. Biol. Chem. 272: 17376–17384.

[52] Okuley, J., Lightner, J., Feldmann, K., Yadav, N., Lark, E., and Browse,J. 1994. Arabidopsis FAD gene encodes the enzyme that is essentialfor polyunsaturated lipid synthesis. Plant cell. 6: 147–158.

[53] Parker-Barnes, J. F., Das, T., Bobik, E., Leonard, A. E., Thurmond, J.M., Chaung, L. T., Huang, Y. S., and Mukerji, P. 2000. Identificationand characterization of an enzyme involved in the elongation of n-6and n-3 polyunsaturated fatty acids. Proc. Natl. Acad. Sci. USA 97:8284–8289.

[54] Pereira, S. L., Leonard, A. E., Huang, Y. S., Chuang, L. T., and Mukerji,P. 2004. Identification of two novel microalgal enzymes involved inthe conversion of the omega3-fatty acid, eicosapentaenoic acid, intodocosahexaenoic acid. Biochem J. 384: 357–366.

[55] Qi, B., Beaudoin, F., Fraser, T., Stobart, A. K., Napier, J. A., andLazarus, C. M. 2002. Identification of a cDNA encoding a novelC18-Delta(9) polyunsaturated fatty acid-specific elongating activityfrom the docosahexaenoic acid (DHA)-producing microalga, Isochry-sis galbana. FEBS Lett. 510(3): 159–165.

[56] Qi, B., Fraser, T., Mugford, S., Dobson, G., Sayanova, O., Butler, J.,Napier, J. A., Stobart, A. K., and Lazarus, C. M. 2004. Production ofvery long chain polyunsaturated omega-3 and omega-6 fatty acidsin plants. Nat. Biotechnol. 22(6): 739–745.

[57] Qiu, X. 2003. Biosynthesis of docosahexaenoic acid (DHA, 22:6-4, 7,10,13,16,19): two distinct pathways. Prostaglandins. LeukotEssent Fatty Acids. 68(2): 181–186.

[58] Qiu, X., Hong, H., and MacKenzie, S. 2001. Identification of adelta-4 Fatty Acid Desaturase from Thraustochytrium sp. involvedin the biosynthesis of docosahexanoic acid by heterologous expres-sion in Saccharomyces cerevisiae and Brassica juncea. J. Biol. Chem.273(34): 31561–31566.

[59] Qiu, X., Hong, H., and MacKenzie, S. L. 2001. Identification of adelta 4 fatty acid desaturase from Thraustochytrium sp. involved in

the biosynthesis of docosahexanoic acid by heterologous expressionin Saccharomyces cerevisiae and Brassica juncea. J. Biol. Chem. 276:31561–31566.

[60] Reddy, A. S., Nuccio, M. L., Gross, L. M., and Thomas, T. L.1993. Isolation of a delta 6-desaturase gene from the cyanobac-terium Synechocystis sp. strain PCC 6803 by gain-of-function ex-pression in Anabaena sp. strain PCC 7120. Plant Mol. Biol. 27: 293–300.

[61] Reddy, A. S., Thomas, T. L. 1996. Expression of a cyanobacterialdelta 6-desaturase gene results in gamma-linolenic acid productionin transgenic plants. Nat. Biotechnol. 14(5): 639–642.

[62] Sakai, H., and Kajiwara, S. 2005. Cloning and functional charac-terization of a Delta12 fatty acid desaturase gene from the basid-iomycete Lentinula edodes. Mol. Genet Genomics. 273(4): 336–341.

[63] Sakamoto, T., Wada, H., Nishida, I., Ohmori, M., and Murata,N. 1994. Delta 9 acyl-lipid desaturases of cyanobacteria. Molecu-lar cloning and substrate specificities in terms of fatty acids, sn-positions, and polar head groups. J. Biol. Chem. 269(41): 25576–25580.

[64] Sakamoto, T., Wada, H., Nishida, I., Ohmori, M., and Murata, N.1994. Identification of conserved domains in the delta 12 desat-urases of cyanobacteria. Plant Mol Biol. 24(4): 643–650.

[65] Sakuradani, E., Kobayashi, M., and Shimizu, S. 1999. Delta 6 de-saturase from arachidonic acid producing Mortierella: gene cloningand its heterologous expression in a fungus. Aspergillus. Gene. 238:445–453.

[66] Sayanova, O., Shewry, P. R., and Napier, J. A. 1999. Histidine-41 ofthe cytochrome b5 domain of the borage delta 6 fatty acid desat-urase is essential for enzyme activity. Plant Physiol. 121: 641–646.

[67] Sayanova, O., Smith, M. A., Lapinskas, P., Stobart, A. K., and Dob-son, G. 1997. Expression of a borage desaturase cDNA containingan N-terminal cytochrome b5 domain results in the accumulation ofhigh levels of delta 6-desaturated fatty acids in transgenic tobacco.Proc. Natl.Acad. Sci. USA. 94: 4211–4216.

[68] Sayanova, O. V., and Napier, J. A. 2004. Eicosapentaenoic acid:biosynthetic routes and the potential for synthesis in transgenicplants. Phytochem. 65: 147–158.

[69] Shanklin, J., Whittle, E., and Fox, B. G. 1994. Eight histidine residuesare catalytically essential in a membrane-associated iron enzyme,stearoyl-CoA desaturase, and are conserved in alkane hydroxylaseand xylene monoxygenase. Biochem. 33: 12787–12794.

[70] Simopoulos, A. P. 1999. Essential fatty acids in health and chronicdisease. Am. J. Clin. Nutr. 70(3): 560–569.

[71] Somerville, C., Browse, J., Jaworski, J. G., and Ohlrogge, J. B. 2000.Lipids, In: Biochemistry and Molecular Biology of Plants. Buchanan,B. B., Gruissem, W., Jones, R. L.,eds., New Delhi: IK International465.

[72] Sperling, P., Ternes, P., Zank, T. K., and Heinz, E. 2003. The evolutionof desaturases. Prostaglandins Leukot. Essent. Fatty Acids. 68: 73–95.

[73] Spychalla, J. P., Kinney, A. K., and Browse, J. 1997. Identification ofan animal o-3 fatty acid desaturase by heterologous expression inArabidopsis. Proc. Natl. Acad. Sci. USA. 94: 1142–1147.

[74] Stukey, J. C., MacDonough, V. M., and Martin, C. E. 1990. TheOLE1 gene of Saccharomyces cerevisiae encodes the delta 9 fattyacid desaturase and can be functionally replaced by rat stearyl-CoAfatty acid desaturase gene. J. Biol. Chem. 265: 20144-20149.

[75] Stukey, J. E., McDonough, V. M., and Martin, C. E. 1989. Isolationand characterization of OLE1, a gene affecting fatty acid desatu-ration from Saccharomyces cerevisiae. J. Biol. Chem. 264: 16537–16544.

[76] Stymne, S., and Stobart, A. K. 1986. Biosynthesis of gamma-linolenic acid in cotyledons and microsomal preparations of the de-veloping seeds of common borage (Borago officinalis). Biochem. J.240: 385–393.

[77] Tang, G. Q., Novitzky W. P., Carol Griffin, H., Huber, S. C., and Dewey,R. E. 2005. Oleate desaturase enzymes of soybean: evidence of

Dnyaneshwar Warude et al. 92

Cri

tical

Rev

iew

s in

Bio

tech

nolo

gy D

ownl

oade

d fr

om in

form

ahea

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re.c

om b

y N

orth

Car

olin

a St

ate

Uni

vers

ity o

n 09

/07/

13Fo

r pe

rson

al u

se o

nly.

regulation through differential stability and phosphorylation. PlantJ. 44(3): 433–446.

[78] Thiede, M. A., and Strittmatter, P. 1985.The induction and charac-terization of rat liver stearyl-CoA desaturase mRNA. J. Biol.Chem.260: 14459–14463.

[79] Thiede, M. A., Ozols, J., and Strittmatter, P. 1986. Construction andsequence of cDNA for rat liver stearyl coenzyme A desaturase. J.Biol. Chem. 261: 13230–13235.

[80] Tocher, D. R., Leaver, M. J., and Hodgson, P. A. 1998. Recent ad-vances in the biochemistry and molecular biology of fatty acyl de-saturases. Prog. Lipid Res. 37: 73–117.

[81] Toke, D. A., and Martin, C. E. 1996. Isolation and characterization ofa gene affecting fatty acid elongation in Saccharomyces cerevisiae.J. Biol. Chem. 271: 18413–18422.

[82] Tonon, T., Harvey, D., Larson, T. R., and Graham, I. A. 2003. Iden-tification of a very long chain polyunsaturated fatty acid Delta4-desaturase from the microalga Pavlova lutheri. FEBS Lett. 553(3):440–444.

[83] Van de Loo, F. J., and Somerville, C. R. 1994. Plastid 0-3 fatty aciddesaturase cDNA from Ricinus communis. Plant Physiol. 105: 443–444.

[84] Wallis, J. G., and Browse, J. 1999. The delta 8-desaturase of Euglenagracilis: an alternate pathway for synthesis of 20-carbon polyunsat-urated fatty acids. Arch. Biochem. Biophys. 365: 307–316.

[85] Watts, J. L., and Browse, J. 1999. Isolation and characterization ofa delta 5-fatty acid desaturase from Caenorhabditis elegans. Arch.Biochem. Biophys. 362:175–182.

[86] Watts, J. L., and Browse, J. 2000. A palmitoyl-CoA-specific delta9fatty acid desaturase from Caenorhabditis elegans. Biochem. Bio-phys. Res. Commun. 272(1): 263–9.

[87] Whittle, E., and Shanklin, J. 2001. Engineering delta 9-16:0-acylcarrier protein (ACP) desaturase specificity based on combina-torial saturation mutagenesis and logical redesign of the cas-tor delta 9–18:0-ACP desaturase. J. Biol. Chem. 276(24): 21500–21505.

[88] Wicker-Thomas, C., Henriet, C., and Dallerac, R. 1997. Par-tial characterization of a fatty acid desaturase gene inDrosophila melanogaster. Insect Biochem. Mol. Biol. 27(11): 963–972.

[89] Yadav, N. S., Wierzbicki, A., Aegerter, M., Caster, C. S., Perez-Girau,L., Kinney, A. J., Hitz, W. D., Booth, J.R. Jr., Schweiger, B., Stecca,K.L., Allen, S.M., Blackwell, M., Reiter, R. S., Carlson, T. J., Russell,S. H., Feldmann, K. A., Pierce, J., and Browse, J. 1993. Cloning ofhigher plant 0-3 fatty acid desaturases. Plant Physiol. 103: 467–476.

[90] Zank, T. K., Zahringer, U., Beckmann, C., Pohnert, G., Boland, W.,Holtorf, H., Reski, R., Lerchl, J., and Heinz, E. 2002. Cloning andfunctional characterisation of an enzyme involved in the elongationof Delta 6-polyunsaturated fatty acids from the moss Physcomitrellapatens. Plant J. 31(3): 255–268.

[91] Zhang, Q., Li, M., Ma, H., Sun, Y., and Xing, L. 2004. Identificationand characterization of a novel Delta 6-fatty acid desaturase genefrom Rhizopus arrhizus. FEBS Lett. 556(1-3): 81–85.

[92] Zhu, M., Liu, Z., Yu, L. J., Zhu, L., and Cheng, H. 2005. Isolation andfunctional identification of delta 5 desaturase gene from Mortierellaalpina. Ye chuan Xue Bao. 32(9): 986–992.

[93] Zolfaghari, R., Cifelli, C. J., Banta, M. D., and Ross, A. C. 2001.Fatty acid delta (5)-desaturase mRNA is regulated by dietary vitaminA and exogenous retinoic acid in liver of adult rats. Arch. Biochem.Biophys. 391: 8–15.

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