metabolic engineering of saccharomyces cerevisiae …acids used as substrates were purchased from...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2011, p. 1854–1861 Vol. 77, No. 50099-2240/11/$12.00 doi:10.1128/AEM.01935-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Metabolic Engineering of Saccharomyces cerevisiae for Production ofEicosapentaenoic Acid, Using a Novel �5-Desaturase from

Paramecium tetraurelia�

Sabina Tavares,1,2 Thomas Grotkjær,1 Thomas Obsen,1 Richard P. Haslam,3

Johnathan A. Napier,3 and Nina Gunnarsson1*Fluxome A/S, Gymnasievej 5, 3660 Stenløse, Denmark1; Technical University of Denmark, Center for Microbial Biotechnology,

Søltofts Plads, b. 221, 2800 Kongens Lyngby, Denmark2; and Rothamsted Research,Harpenden, Herts AL5 2JQ, United Kingdom3

Received 13 August 2010/Accepted 17 December 2010

Very-long-chain polyunsaturated fatty acids, such as arachidonic acid (ARA), eicosapentaenoic acid (EPA),and docosahexaenoic acid (DHA), have well-documented importance in human health and nutrition. Sustain-able production in robust host organisms that do not synthesize them naturally requires the coordinatedexpression of several heterologous desaturases and elongases. In the present study we show production of EPAin Saccharomyces cerevisiae using glucose as the sole carbon source through expression of five heterologous fattyacid desaturases and an elongase. Novel �5-desaturases from the ciliate protozoan Paramecium tetraurelia andfrom the microalgae Ostreococcus tauri and Ostreococcus lucimarinus were identified via a BLAST search,and their substrate preferences and desaturation efficiencies were assayed in a yeast strain producing the �6and �3 fatty acid substrates for �5-desaturation. The �5-desaturase from P. tetraurelia was up-to-2-fold moreefficient than the microalgal desaturases and was also more efficient than �5-desaturases from Mortierellaalpina and Leishmania major. In vivo investigation of acyl carrier substrate specificities showed that the�5-desaturases from P. tetraurelia, O. lucimarinus, O. tauri, and M. alpina are promiscuous toward the acylcarrier substrate but prefer phospholipid-bound substrates. In contrast, the �5-desaturase from L. majorshowed no activity on phospholipid-bound substrate and thus appears to be an exclusively acyl coenzymeA-dependent desaturase.

During the past two decades, very-long-chain polyunsatu-rated fatty acids (VLC-PUFAs) like arachidonic acid (ARA;20:4�6), eicosapentaenoic acid (EPA; 20:5�3), and docosa-hexaenoic acid (DHA; 22:6�3) have attracted the attention ofthe scientific community as well as the dietary supplement andfood industries due to their proven health benefits.

VLC-PUFAs are constituents of biological membranes, par-ticipate in cellular processes like cell signaling and hormonereceptor binding (29), and act as biosynthesis precursors foreicosanoids and other anti-inflammatory mediators. Consump-tion of VLC-PUFAs is associated with cardioprotective effects,prevention of diseases like Alzheimer’s disease, Parkinson’sdisease, and cancer, and is essential for correct development ofbrain and visual function in infants (3, 18, 29). Since the humanbody cannot synthesize VLC-PUFAs de novo in adequatequantities, dietary intake of these compounds is important.Fish and fish oils are the major nutritional dietary sources ofVLC-PUFAs, but concerns over the continual depletion ofwild fish stocks and contamination of the oceans lead to theobvious conclusion that alternative sustainable sources are ur-gently needed (24). VLC-PUFAs are present in a wide rangeof different organisms, from mammals, fungi, mosses, and bac-teria to lower plants, but it is within the microalgae that themost efficient producers of VLC-PUFA are found, especially

for the �3 fatty acids. Curiously, saprophytic and pathogenicforms of life, like the Oomycetes (e.g., species from Pythium[37] and Saprolegnia [22]), the Entomophthorales (Entomoph-thora [6] and Conidiobolus [13]), the Labyrinthulids (30), andthe protozoa (Leishmania and Trypanosoma [39]), also pro-duce VLC-PUFAs and apparently contain the complete path-way for PUFA synthesis.

Some industrial processes exploit these efficient PUFA pro-ducers as cell factories for more sustainable production ofthese compounds (25), although the culture of nonadaptednative organisms can often prove to be problematic and costly.Alternatively, the heterologous production of VLC-PUFAs indedicated hosts is also seen as a potential solution. The in-creasing number of reports describing the isolation of desatu-rases and elongases from a variety of organisms and theirsuccessful expression in several hosts like Saccharomyces cer-evisiae and plants (1, 16, 20, 28, 31, 36) confirm the availabilityof a genetic tool kit with which to metabolically engineer thesynthesis of these important fatty acids.

Synthesis of ARA and EPA can be achieved through asequence of desaturations and elongations, the final steps be-ing the introduction of a double bond between carbons 5 and6 in dihomo-�-linolenic acid (20:3�6) and eicosatetraenoicacid (20:4�3), respectively, by a �5-desaturase (Fig. 1). The�5-desaturases belong to the “front-end” desaturases family,since they introduce a double bond between an existing doublebond and the carboxyl terminal of the fatty acid chain.

The “front-end” desaturases contain a fused N-terminal cy-tochrome b5-like domain, which has been claimed to be essen-

* Corresponding author. Present address: Terranol A/S, SøltoftsPlads b. 223, 2800 Kongens Lyngby, Denmark. Phone: 45 47188400.Fax: 45 45884148. E-mail: [email protected].

� Published ahead of print on 30 December 2010.

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tial for catalytic activity, and three conserved histidine boxes(17, 33). Genes encoding �5-desaturase activities were firstisolated from the ARA-producing fungus Mortierella alpina (9,12) by using a PCR-based approach with primers designed forthose conserved histidine boxes, which had been previouslyobserved in �6-desaturases (14, 15, 26, 34). The same methodhas been successfully used to isolate most of the desaturasesdescribed in the literature. An alternative is the use of theconserved histidine boxes as well as other typical motifs inbioinformatics searches, which, combined with the increasingnumber of genome sequencing projects, provides a faster andmore direct approach toward identification and expression ofnovel genes. In addition, the facile and robust procedure ofwhole-gene synthesis and codon optimization opens the doorto a limited synthetic biology approach to pathway engineer-ing, meaning that multiple nonnative sequences encoding theVLC-PUFA biosynthetic pathway can be expressed in a suit-able “chassis” (a host such as yeast) to generate a transgenicorganism with novel functionality. Despite the benefits ofbioinformatics, our understanding of the underlying biochem-istry and regulation of this pathway is still incomplete.

Although detailed biochemical studies on purified front-enddesaturases and PUFA elongases are still missing, several re-search groups have studied their acyl carrier substrate speci-ficities in detail using in vivo systems (4, 5). In lower eu-karyotes, it is suggested that the elongation step in PUFAbiosynthesis takes place in the acyl coenzyme A (acyl-CoA)pool, whereas �5- and �6-desaturations occur mainly on phos-pholipid (PL)-linked substrates (4). This creates an acyl ex-change bottleneck (so-called “substrate dichotomy”) in thepathway, since the �6-desaturated products do not enter theacyl-CoA pool, and therefore elongation and subsequent �5-desaturation are not efficiently reconstituted. However, recentstudies have identified microalgal �6- and �5-desaturases

which accept CoA-bound substrates, and these findings suggestthat an acyl-CoA-dependent pathway might overcome this en-dogenous metabolic blockade (8). Similarly, it has been sug-gested that use of the alternative �9-elongation/�8-desatura-tion pathway (1) (which requires only a single acyl exchange)represents an additional solution to this problem.

In the present study we describe the identification of novel�5-desaturases from a ciliate protozoan, Paramecium tetraure-lia, and two microalgae, Ostreococcus tauri and Ostreococcuslucimarinus. The �5-desaturases were functionally expressed inan engineered yeast strain capable of producing the substrates20:3�6 and 20:4�3, resulting in the production of ARA andEPA. To our knowledge, this represents the first report of anEPA/ARA-producing Saccharomyces cerevisiae strain withoutthe need of fatty acid supplementation. In order to fully eval-uate the acyl carrier substrate specificities of these novel de-saturases, we studied the distribution over time of the fatty acidsubstrate and product of �5-desaturation in different lipid frac-tions and compared the results with previously known �5-desaturases from Mortierella alpina and Leishmania major.

MATERIALS AND METHODS

Materials. Restriction enzymes were obtained from New England BioLabs(United Kingdom). Polymerases were from Finnzymes (Finland), and the fattyacids used as substrates were purchased from Larodan Fine Chemicals (Sweden).Fatty acid methyl ester (FAME) standards were from Nu-Check-Prep. Fattyacyl-CoAs (16:0, 17:0, 18:0, 18:1, 18:2, 20:0, 22:0, and 24:0) were obtained fromAvanti Polar Lipids and Sigma-Aldrich. Unless otherwise stated, all other chem-icals were from Sigma. Synthetic genes were obtained from Genscript and codonoptimized for expression in S. cerevisiae.

Identification and cloning of �5-desaturases. A list of �5-desaturase (D5D)protein sequences with verified activities was used as query for a BLAST searchin the GenBank database. The outputs were combined into a unique list in whichredundant sequences were removed. In the subsequent analysis the sequenceswere ranked based on typical features of front-end desaturases, i.e., the presenceof the HPGG motif in the N terminus of the amino acid sequence, responsiblefor heme binding and catalytic activity; the existence and order of three con-served histidine boxes (HXXXH, HXXXHH, and QXXHHLFP); the presenceof the motif DPDI (and variations) between the second and third HIS boxes; thepercent identity with other �5-desaturases; and the length of the sequence.Moreover, the best hits were assessed based on the fatty acid composition of theoriginal organisms in order to confirm the presence of ARA, EPA, and therespective precursors in the fatty acid profiles. The selected sequences wereacquired as synthetic genes codon optimized for S. cerevisiae in vector pUC57(Table 1). The open reading frames (ORFs) were reamplified with primerscontaining NotI/EcoRI restriction sites and introducing the translation initiationsequence ACC immediately upstream of the start codon, and they were sub-cloned into a replicative pESC-URA-based expression vector under the controlof the constitutive S. cerevisiae TEF1 promoter. The constructs were designatedpSF28-OtD5D, pSF28-OlD5D, pSF28-Ptet1D5D, and pSF28-Ptet2D5D. In ad-dition to the putative desaturases, a described �5-desaturase from Leishmaniamajor (accession number XM_001680969) (10) was also acquired as a codon-optimized gene and expressed in the same system as the putative genes. Theconstruct was designated pSF28-LmD5D.

TABLE 1. �5-Desaturases used in this study

Source organism GenBankaccession no.

Paramecium tetraurelia 1..........................................................HQ678517Paramecium tetraurelia 2..........................................................HQ678518Ostreococcus lucimarinus .........................................................HQ678519Ostreococcus tauri .....................................................................HQ678520Leishmania major .....................................................................HQ678521Mortierella alpina.......................................................................GU593328

FIG. 1. Simplified representation of PUFA biosynthetic pathwaysfrom stearic acid (18:0) to ARA, EPA, and DHA.

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The �5-desaturase sequence MaD5D (GenBank accession number GU593328)was isolated from Mortierella alpina CBS 608.70 cDNA by PCR using the primers5�-ATGGGTACGGACCAAGGAAAAACC-3� and 5�-CTACTCTTCCTTGGGACGGAGTCC-3�, designed to match the �5-desaturase described previouslyin reference 12. The ORF was reamplified with primers containing restrictionsites and was subcloned for sequencing. Sequence verification of two clonesshowed that the isolated �5-desaturase gene contained slight variations com-pared to the MaD5D reported in reference 12 (96% identity of the proteinsequence). The verified sequence was then subcloned into a pESC-URA-basedexpression vector, under the control of the strong S. cerevisiae TEF1 promoter,and designated pSF28-MaD5D. The �5-desaturase sequences used in this studyare summarized in Table 1.

Strains. For activity assays with substrate supplementation and for time courseexperiments, S. cerevisiae strain CEN.PK 113-5D (MATa ura3-52) was trans-formed with plasmid DNA using the polyethylene glycol-lithium acetate method.Transformants were selected on 2% glucose agar plates without uracil (35).

In order to verify �5-desaturation activity in a yeast strain producing 20:3�6and 20:4�3, plasmid DNA was transformed into the proprietary S. cerevisiaestrain FS01699 (Fluxome A/S), which contains chromosomal integrations ofgenes encoding a �9-desaturase (D9D), a �12-desaturase (D12D), a �6-desatu-rase (D6D), a �6-elongase (D6E), and an �3-desaturase (FAD3) under thecontrol of strong yeast promoters (Table 2). Briefly, FS01699 was constructed asfollows: the MaD9D, MaD12D, and MaD6E genes were isolated from M. alpinacDNA as described above for the �5-desaturase, using amplification primersdesigned to match described M. alpina genes (references 41, 31, and 21, respec-tively). The SkFAD3 gene was isolated from Saccharomyces kluyveri genomicDNA using primers matching the �3-desaturase described in reference 20. ThePCR primers used for gene amplification were 5�-ATGGCAACTCCTCTTCCCCCCTCC-3�- and 5�-CTATTCGGCCTTGACGTGGTCAGTGC-3� for the �9-desaturase, 5�-AACCCTTTTTCAGGATGGCACC-3� and 5�-AAAGTTGTGTCCGGTAAATGCTTC-3� for the �12-desaturase, 5�-ATGGAGTCGATTGCGCCATTCC-3� and 5�-TTACTGCAACTTCCTTGCCTTCTCC-3� for the �6-elongase, and 5�-GGTCTCGAGCCACCATGTCTATTGAAACAGTCGG-3�and 5�-GGCCGCGGATCATTGACTGGAACCATCTT-3� for the �3-desatu-rase. The Ostreococcus tauri �6-desaturase (OtD6D) (5) was codon optimizedfor expression in S. cerevisiae (Backtranslation tool; Entelechon) and was assem-bled from synthetic oligonucleotides by PCR. Following subcloning and sequenc-ing of the genes, OtD6D, MaD9D, MaD12D, and SkFAD3 were sequentiallyintegrated together with constitutive yeast promoters at selected chromosomalsites through a PCR-based gene-targeting approach modified from that de-scribed in reference 7. The gene-targeting substrates were designed such that adirect repeat of the promoter sequence on either side of the insert enabledlooping out of the Kluyveromyces lactis URA3 selection marker through homol-ogous recombination between repeats; this event was selected for on 5-fluoro-orotic acid plates. MaD6E under the control of the S. cerevisiae PYK1 promoterwas integrated at the trp1-289 marker. Strain FS01699 additionally contains adeletion of the POT1 gene and a replacement of the FAS1 promoter with theADH1 promoter.

Expression of �5-desaturases in S. cerevisiae. For substrate feeding experi-ments, precultures were grown in 2% glucose minimal medium (40) at 30°C untilan optical density at 600 nm (OD600) of approximately 2 to 3 was reached. Glassinserts (Supelco) containing 0.5 ml of 2% glucose minimal medium with 1%(wt/vol) Tergitol solution (type NP-40; 70% in H2O) and a 0.5 mM concentrationof the appropriate fatty acid substrate dissolved in absolute ethanol were inoc-ulated to an initial OD600 of 0.2. The glass inserts (triplicates) were placed intoa deep multiwell plate, covered with sterile sealing tape, and incubated for 48 hat 30°C, tilted, with shaking at 230 rpm. For expression of �5-desaturases in20:3�6- and 20:4�3-producing S. cerevisiae, precultures grown as describedabove were inoculated in 100 ml 2% glucose minimal medium to an initial OD600

of 0.1 and cultivated for 48 h at 30°C with shaking at 150 rpm.

For the time course experiments, preinocula were grown for approximately24 h in 2% glucose minimal medium at 30°C. Strains were then reinoculated in100 ml 2% glucose minimal medium with 1% (wt/vol) Tergitol (type NP-40; 70%in H2O) to an initial OD600 of 0.1 and grown at 30°C until reaching late expo-nential phase (OD600, �3) in order to guarantee expression of the desaturasesand a sufficient amount of biomass for analysis. At this point (time 0 min),samples of approximately 30 mg (dry weight) of cells were harvested by centrif-ugation at 1,730 � g for 5 min and washed with an equal volume of water, andthe pellet was used for fatty acid analysis. For acyl-CoA analysis, samples of 1OD unit were harvested by a short-spin centrifugation of 20 s and dropped intoliquid nitrogen, with the exception of the 24-h samples, in which 8 OD units wereharvested. The kinetic experiment was started immediately after taking time zerosamples, by addition of 0.5 mM 20:3�6 to the cultures. Samples were taken after5 min, 1 h, 4 h, and 24 h in the presence of the exogenous substrate as describedabove for time zero.

Fatty acid and lipid analyses. Total fatty acid analysis of yeast cultures wasperformed according to the methods described in reference 19. FAMEs wereanalyzed on a gas chromatograph (GC; Agilent 7890A) coupled to a flameionization detector (FID). Samples were injected at 190°C into a DB-Wax col-umn (10-m by 0.1-mm inner diameter; 0.1-�m film thickness; J&W Scientifics),and immediately after injection the temperature was increased to 260°C at12°C/min. Free fatty acid 23:0 was used as the internal standard. The FAMEswere identified based on their relative retention times by comparison with stan-dards of LC- and VLC-PUFAs. Analysis of the different lipid classes (neutrallipids and phospholipids) was performed according to the methods described inreference 23. The lipid fractions were transmethylated, and FAMEs were ana-lyzed as described previously. Fatty acid yield (in mg FA/OD unit) was deter-mined by the amount of each fatty acid and the OD600 of the initial cell suspen-sion.

Acyl-CoA analysis. Yeast cells harvested from suspension and snap-frozen inliquid nitrogen were extracted according to methods described in refererence 11for subsequent quantitative analysis of fluorescent acyl-etheno-CoA derivativesby high-performance liquid chromatography. Analysis of acyl-CoA was per-formed using an Agilent 1100 LC system with a Phenomenex LUNA 150-mm by2-mm C18(2) column. The methodology and gradient conditions were describedpreviously (10, 32). The synthesis of acyl-CoA 18:4 was performed according tomethods described in reference 38.

RESULTS

Identification of putative �5-desaturases. A BLAST searchwas performed in GenBank using characterized �5-desaturaseprotein sequences as queries. Putative �5-desaturase sequencesfrom the unicellular ciliate Paramecium tetraurelia (Ptet1D5D)and from the microalgae Ostreococcus tauri (OtD5D) and Os-treococcus lucimarinus (OlD5D) were chosen for further anal-ysis (Table 1). The Ptet1D5D protein shared 30% identity withM. alpina D5D (accession number AF054824), 26% with Phae-odactylum tricornutum D5D (accession number AY082392),and 32% identity with Mantoniella squamata D5D (accessionnumber AM949596). O. tauri and O. lucimarinus sequencesshared 82% identity between each other, 31% with M. alpinaD5D, and 66% with M. squamata D5D.

Several of the �5-desaturases identified in our search werepreviously annotated and functionally characterized as such,contributing to validating the scoring method. The �5-de-

TABLE 2. Desaturase- and elongase-encoding genes integrated in strain FS01699

Genename Activity GenBank

accession no. Integration site Promoter

OtD6D �6-Desaturase (Ostreococcus tauri) HQ678522 DCI1 (�2,893) ADH1MaD9D �9-Desaturase (Mortierella alpina) GU593324 POX1 (�112,263) TDH3MaD12D �12-Desaturase (Mortierella alpina) GU593325 FOX2 (�12,709) TDH3MaD6E �6-Elongase (Mortierella alpina) GU593327 trp1-289 PYK1SkFAD3 �3-Desaturase (Saccharomyces kluyveri) GU593329 GPP1 (�70,791) HXT7

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saturases from L. major (LmD5D) (39) and M. alpina(MaD5D) (9, 12) were also included in the further analysis,the latter being previously described as a phospholipid-de-pendent desaturase (4).

Functional characterization of �5-desaturases in S. cerevi-siae. Synthetic genes encoding codon-optimized versions ofthe putative desaturases were expressed in S. cerevisiae in thepresence of exogenously supplied fatty acid substrates for �5-desaturation. Analysis of the fatty acid compositions of thestrains expressing Ptet1D5D, OtD5D, OlD5D, MaD5D, andLmD5D in the presence of 20:3�6 and 20:4�3 showed theappearance of new peaks, identified as ARA and EPA, respec-tively, confirming that the novel genes encode �5-desaturases(Fig. 2 shows representative chromatograms). The analysisshowed that P. tetraurelia D5D had the highest desaturationefficiency in the system used, followed by M. alpina D5D andO. tauri D5D, while O. lucimarinus D5D and L. major D5Dwere less active (Fig. 3).

FIG. 2. �5-Desaturase activities of Ptet1D5D. GC-FID chromatograms of fatty acids from yeast strains expressing Ptet1D5D (a and c) orharboring the empty plasmid pSF28 (b and d), cultivated with supplementation of 0.5 mM 20:3�6 (a and b) or 20:4�3 (c and d) fatty acids,respectively.

FIG. 3. Desaturation efficiencies of Ptet1D5D, OlD5D, OtD5D,LmD5D, and MaD5D. Yeast strains expressing Ptet1D5D, OlD5D,OtD5D, LmD5D, and MaD5D genes were cultivated in the presenceof 0.5 mM 20:3�6 or 20:4�3 fatty acids. Desaturation efficiencies{[(product)/(substrate product)] � 100} were calculated using thepercentages of substrates and products in total fatty acids. Each valuerepresents the mean standard deviation of five biological replicates.


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Functional characterization of �5-desaturases in a strainproducing 20:3�6 and 20:4�3. To evaluate activity in a com-pletely reconstituted ARA/EPA pathway, the �5-desaturaseswere expressed in an engineered S. cerevisiae strain producing20:3�6 and 20:4�3. The background strain coexpressed anacyl-CoA-dependent �6-desaturase from the microalga O.tauri; a �9-desaturase, a �12-desaturase, and a �6-elongasefrom M. alpina; and an �3-desaturase from S. kluyveri. Since inthis strain an acyl-CoA-dependent �6-desaturase delivers thedirect substrate for �6-elongation (avoiding the step of trans-ferring the �6-desaturated fatty acid from phospholipid toacyl-CoA), �6-elongation is not expected to be a bottleneck(5). Fatty acid analysis of the background strain expressing thevarious �5-desaturases (Table 3) confirmed that the elonga-tion step is highly efficient in this strain (substrate conversionswere in the range of 85 to 95% for both the �6 and the �3substrates) and showed that all the �5-desaturases were ableto convert the endogenously produced 20:3�6 and 20:4�3into ARA and EPA, respectively. Analysis of the desatura-tion efficiencies for the �5-desaturases (Fig. 4) showed thatPtet1D5D was the most efficient enzyme in both substrates,followed closely by MaD5D. OlD5D, OtD5D, and LmD5D

showed very similar conversions of 20:3�6, and OlD5D andOtD5D were more efficient toward the �3 substrate thanLmD5D.

A second �5-desaturase from Paramecium tetraurelia. In theinitial BLAST search, several putative front-end desaturasesequences from P. tetraurelia were found. Interestingly, eachsequence was accompanied by a nearly identical (ca. 95%amino acid sequence identity) second allele in the genome ofP. tetraurelia. The unicellular eukaryotic ciliate P. tetraureliahas in fact experienced a whole-genome duplication event (2),and this may explain the presence of a second, closely relatedsequence. Another particularity of P. tetraurelia is that TAAand TAG, stop codons in the standard genetic code, encodeglutamine (27), making it necessary to codon optimize genesfrom this organism before expression in hosts using theuniversal genetic code. After verifying �5-desaturase activ-ity for one of the alleles (Ptet1D5D), we sought to investi-gate whether the second, nearly identical allele (accessionnumber XM_001436681; Ptet2D5D) was also functional. Thecodon-optimized Ptet2D5D exhibited similar enzymatic activ-ity as Ptet1D5D, both when expressed in a 20:3�6/20:4�3-producing strain (Fig. 5) and when tested by exogenous sup-

TABLE 3. Fatty acid composition of strains endogenously producing 20:3�6 and 20:4�3 and additionally expressing Ptet1D5D, OlD5D,OtD5D, LmD5D, or MaD5Da

Fatty acid% of total FA (mean SD)

Ptet1D5D OlD5D OtD5D LmD5D MaD5D Control

16:0 15.77 1.78 19.57 1.88 23.25 3.26 17.12 2.28 18.38 0.66 15.36 4.3416:1�9 36.32 2.91 29.18 2.73 29.52 3.7 29.85 2.82 32.44 0.99 34,35 2,7118:0 4.14 0.52 3.72 0.51 3.56 0.17 3.91 0.57 3.8 0.48 4.41 0.4818:1�9/�7 30.35 2.36 24.69 2.03 19.38 2.17 20.17 2.98 21.6 0.86 24.04 2.7518:2�6 6.46 1.98 7.66 1.25 8.21 0.94 13.98 3.31 10.12 1.25 8.27 3.7118:3�6 0.08 0.01 0.1 0.09 0.18 0.02 0.09 0.08 0,16 0,02 0.06 0.0720:3�6 0.57 0.08 1.45 0.26 1.48 0.22 1.17 0.31 1.32 0.38 1.35 0.2120:4�6 0.21 0.05 0.17 0 0.19 0 0.17 0.02 0.46 0.26 0.03 0.0318:3�3 0.54 0.72 2.5 0.32 3.01 0.44 2.57 0.5 1.59 0.64 1.61 0.7718:4�3 0.07 0 0.18 0.19 0.2 0.06 0.1 0.09 0.13 0.12 0.08 0.1120:4�3 0.62 0.04 1.96 0.62 1.86 0.1 1.65 0.67 1.22 0.45 1.41 0.8820:5�3 0.34 0.04 0.42 0.1 0.49 0.05 0.3 0.15 0.5 0.29 0 0.01

a Controls were the same background strain harboring the empty plasmid pSF28. The values represent the means SD of three independent experiments.

FIG. 4. Desaturation efficiencies of Ptet1D5D, OtD5D, OlD5D,LmD5D, and MaD5D in a strain endogenously producing 20:3�6 and20:4�3. The �5-desaturases were coexpressed with MaD9D, MaD12D,OtD6D, and MaD6E genes. Desaturation efficiencies were calculatedas described in the legend of Fig. 3. Each value represents the mean standard deviation of three independent experiments.

FIG. 5. Desaturation efficiencies of Ptet1D5D and Ptet2D5D in astrain endogenously producing 20:3�6 and 20:4�3. The �5-desaturaseswere coexpressed with MaD9D, MaD12D, OtD6D, and MaD6Egenes. Desaturation efficiencies were calculated as described in thelegend of Fig. 3. Each value represents the mean standard deviationof three independent experiments.


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plementation of 20:3�6 and 20:4�3 fatty acids (data notshown). No activity was measured when 18:2�6, 18:3�3, or20:2�6 was supplied to the cultivation medium (data notshown).

In addition to Ptet1D5D and Ptet2D5D, other sequences similarto front-end desaturases but either shorter or longer than the average�5-desaturase were identified in the P. tetraurelia genome (acces-sion numbers XM_001450326, XM_001453029, XM_001425061,XM_001461896, XM_001434078, and XM_001425226). Thesesequences were also expressed in yeast and tested by feedingwith exogenous fatty acids, but no �5-, �6-, or �8-desaturaseactivity was detected in the presence of the appropriate sub-strates (data not shown).

Acyl carrier specificities of �5-desaturases. In order to in-vestigate the acyl carrier specificity of the �5-desaturases fromthis study, distribution of fatty acids in the main lipid fractionswas followed over a 24-h period after addition of exogenous20:3�6 to the cultures. Incorporation of the fatty acid substrate20:3�6 reflected an identical pattern in all the strains analyzed(Fig. 6a for Ptet1D5D). Already at 5 min after supplementa-tion, 20:3�6 could be detected in the total fatty acids (TFA),steryl esters (SE), triacylglycerols (TAG) (Fig. 6a), and acyl-CoA pool (Fig. 7). The amount of 20:3�6 in TFA, TAG, andSE pools increased during the 4 h after supplementation andremained approximately constant for up to 24 h in the first twopools, while in SE the amount of 20:3�6 decreased drasticallyfrom the 4-h to the 24-h sample. In contrast to the immediateincorporation into TAG and SE, 20:3�6 was detected in the PLand diacylglycerols (DAG) pools only 4 h after substrate ad-dition.

The desaturation product ARA was first observed after 1 hin the TFA of Ptet1D5D- and LmD5D-expressing strains, aswell as in the TAG pool of the latter, although in smalleramounts. ARA was not detected in the 5-min samples of anystrain, nor in the 1-h samples of OlD5D, OtD5D, or MaD5D(Fig. 8). After 4 h, the desaturation product was present in theTFA and TAG pools of all strains. Also at this time point,ARA was first detected in the PL fraction, but only in thePtet1D5D-expressing strains. This appearance of ARA in theTAG prior to its detection in the phospholipids suggests thatdesaturation takes place in the acyl-CoA pool and that the�5-desaturated product is transferred to the TAG via diacyl-glycerol acyltransferases. After 24 h the desaturation product

was present in the PL of all strains except the strain expressingLmD5D (Fig. 8). In fact, ARA was not detected in the phos-pholipids of this strain at any time point, indicating thatLmD5D does not accept the phospholipid-bound substrate. Inthe strains where ARA was found in the phospholipids, itspercentage relative to other fatty acids was always higher inthat pool than other lipids, indicating a preference for phos-pholipid-bound substrate. This was particularly pronouncedfor Ptet1D5D, where already in the 4-h sample, ARA consti-tuted 2.3% of fatty acids in PL and 0.6% of fatty acids in TAG(corresponding to 27% and 1.9% conversion efficiencies, re-spectively).

ARA could not be detected in the acyl-CoA pool of anystrain with the method used, except in the 24-h samples (Fig.7). It is, however, likely that ARA was present at levels below

FIG. 6. Distribution of 20:3�6 and total fatty acids in the lipid fractions of a Ptet1D5D-expressing yeast strain during a time course feedingexperiment. Exogenous fatty acid substrate (0.5 mM 20:3�6) was added to the yeast culture at time zero, and samples for lipid analysis werecollected at the indicated time points. (a) Absolute amount of 20:3�6 in each lipid fraction, expressed in mg/OD unit. (b) Absolute amount of totalfatty acids in each lipid fraction, expressed in mg/OD unit.

FIG. 7. Fatty acid profile of the acyl-CoA pool of a Ptet1D5D-expressing yeast strain during a time course feeding experiment. Theexperiment was carried out as described in the legend of Fig. 6. Sam-ples of 1 OD unit were collected at the indicated time points (for the24-h samples, aliquots of 8 OD units were collected), and the acyl-CoAfraction was analyzed. *, ARA peak in the 24-h sample.


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the detection limit in the acyl-CoA pool, where it was effi-ciently taken up and accumulated as TAG and SE, thus justpassing through the acyl-CoA pool at a high turnover rate.Interestingly, in contrast to 20:3�6, ARA was never detected inthe SE pool of any strain, which indicates that Are1p andAre2p acyltransferases cannot use that fatty acid as substrate.ARA also could not be detected in the DAG pool, indicatingthat it is not used for DAG synthesis by Gat1p, Gat2p, andSlc1p but is efficiently channeled to TAG by Dga1p activity.This is furthermore supported by the fact that ARA appears asTAG prior to other lipid pools.

DISCUSSION

Production of very-long-chain polyunsaturated fatty acids intransgenic organisms is a process still far from optimal. Het-erologous expression of desaturases and elongases usually re-sults in low PUFA yields in the host organism compared to thelevels observed in native PUFA producers. The overall effi-ciency of the PUFA pathway is likely to be influenced by lipiddynamics inside the cell, where interplay between desaturases,elongases, and acyltransferases may be determinants. For ex-ample, a higher flux in the pathway is to be expected when bothdesaturases and elongases have an identical acyl carrier pref-erence, using CoA-activated fatty acids as substrate (4, 8). Onthe other hand, competition for that same substrate by someacyltransferases present in the host may limit the flux throughthe pathway, since such competing reactions transfer the acyl-CoA substrate to other lipids which are not substrates forelongation and desaturation. In the present study, the time-dependent incorporation of the substrate 20:3�6 into differentlipids provides some hints on the activities of these acyltrans-ferases in S. cerevisiae. The exogenously supplied 20:3�6 ap-peared in the TAG and SE lipid fractions already at 5 min afteraddition of this fatty acid to the medium, while it was detectedin the phospholipids and DAG only after 4 h. This pattern of20:3�6 assimilation agrees well with previous findings thatexogenous free fatty acids are CoA activated as soon as theyenter yeast cells (4) and indicates that diacylglycerol and sterol

acyltransferases (Dga1p, Are1p, and Are2p) are very activewith the CoA-activated form of 20:3�6 as substrate. In con-trast, the later appearance of 20:3�6 in DAG and PL com-pared to TAG (and in smaller amounts) may indicate that theacyltransferases involved in DAG synthesis in S. cerevisiae(i.e., Gat1p, Gat2p, and Slc1p) are less active on CoA-activated 20:3�6 than the DAG-acyltransferases Dga1p,Are1p, and Are2p. Another possible explanation is that TAGis formed from preexisting DAG and that the replenishment ofDAG is achieved through the activity of phospholipase C onthe phospholipids, which initially decreased (Fig. 6b). In thecontext of a fully reconstituted PUFA pathway, the highlyactive native diacylglycerol and sterol acyltransferases maycompete with �5-desaturases for CoA-activated 20:3�6, andacyl-CoA-dependent �5-desaturases may therefore be less ef-ficient than phospholipid-dependent versions due to limitedsubstrate availability.

The overall most efficient desaturase in the present study,Ptet1D5D, was highly active on phospholipid-linked substratesbut also seemed to have the capacity to act on the acyl-CoAsubstrate. In contrast, LmD5D, which exclusively preferred theacyl-CoA substrate, was the least efficient enzyme, illustratingthat exclusive acyl-CoA substrate specificity is not an advan-tage per se. In the Ptet1D5D-expressing strain, �5-desaturationin the PL fraction was evident at the earliest possible samplingpoint, i.e., as soon as the 20:3�6 substrate was detected in PL.At this sampling point, ARA was predominantly found in PLand to a lesser extent in TAG. At the later sampling point,increased amounts of ARA were found both in the PL and inTAG. This may indicate that �5-desaturation occurs on acyl-CoA substrates followed by incorporation into TAG by acyl-transferases, or that PL-bound fatty acids are released andtaken up into TAG. Both of these models are supported by ourdata.

Analyses of lipid distribution in different pools performedafter 24 or 48 h of incubation have in the past been used todistinguish between lipid-linked and acyl-CoA-dependent�5-desaturases (4, 8). A predominant accumulation of de-saturation products in the phospholipids was considered tobe characteristic of lipid-linked �5-desaturases, whereas aroughly equal distribution between phospholipids and neu-tral lipids was associated with acyl-CoA-dependent �5-desatu-rases. In our results, we observed approximately equal distri-bution of ARA in the phospholipids and TAG of strainsexpressing Ptet1D5D, OlD5D, and OtD5D after 24 h of incu-bation, while in the MaD5D-expressing strain larger amountsof ARA accumulated in the phospholipids compared to theTAG. However, our results also show that initial desaturationin MaD5D, Ptet1D5D, OtD5D, and OlD5D occurs in theacyl-CoA until the substrate is available in the PL, subse-quently taking place there with higher conversion efficiency.Therefore, assessing acyl carrier specificity based solely onmeasurement at a single time point does not appear to befeasible. Altogether, this leads us to conclude that acyl carrierspecificity is not the only important feature of front-end de-saturases and we hypothesize that certain promiscuity towardthe acyl carrier used as substrate is beneficial for overall �5-desaturation efficiency.

FIG. 8. Absolute amounts of ARA in the lipid fractions of �5-desaturase-expressing strains during time course feeding experiments.Yeast strains expressing Ptet1D5D, OtD5D, OlD5D, LmD5D, orMaD5D were cultivated as described in the legend of Fig. 6, andsamples were taken at the indicated time points. No ARA was detectedin any of the lipid fractions at time points 0 or 5 min.


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ACKNOWLEDGMENTS

This research was financially supported by Fluxome A/S and by aPh.D. grant from the Danish Ministry of Science, Technology andInnovation. Rothamsted Research receives grant-aided support fromthe BBSRC (United Kingdom).

We thank J. M. Mouillon, M. Sendelius, and S. Bjørn for construc-tion of background strain FS01699 and plasmid pSF28 and S. Jacobsenfor technical assistance.

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