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• W1998596631 abstract "ω3-Very long chain polyunsaturated fatty acids (VLCPUFA) are essential for human development and brain function and, thus, are indispensable components of the human diet. The current main source of VLCPUFAs is represented by ocean fish stocks, which are in severe decline, and the development of alternative, sustainable sources of VLCPUFAs is urgently required. Our research aims at exploiting the powerful infrastructure available for the large scale culture of oilseed crops, such as rapeseed, to produce VLCPUFAs such as eicosapentaenoic acid in transgenic plants. VLCPUFA biosynthesis requires repeated desaturation and repeated elongation of long chain fatty acid substrates. In previous experiments the production of eicosapentaenoic acid in transgenic plants was found to be limited by an unexpected bottleneck represented by the acyl exchange between the site of desaturation, endoplasmic reticulum-associated phospholipids, and the site of elongation, the cytosolic acyl-CoA pool. Here we report on the establishment of a coordinated, exclusively acyl-CoA-dependent pathway, which avoids the rate-limiting transesterification steps between the acyl lipids and the acyl-CoA pool during VLCPUFA biosynthesis. The pathway is defined by previously uncharacterized enzymes, encoded by cDNAs isolated from the microalga Mantoniella squamata. The conceptual enzymatic pathway was established and characterized first in yeast to provide proof-of-concept data for its feasibility and subsequently in seeds of Arabidopsis thaliana. The comparison of the acyl-CoA-dependent pathway with the known lipid-linked pathway for VLCPUFA biosynthesis showed that the acyl-CoA-dependent pathway circumvents the bottleneck of switching the Δ6-desaturated fatty acids between lipids and acyl-CoA in Arabidopsis seeds. ω3-Very long chain polyunsaturated fatty acids (VLCPUFA) are essential for human development and brain function and, thus, are indispensable components of the human diet. The current main source of VLCPUFAs is represented by ocean fish stocks, which are in severe decline, and the development of alternative, sustainable sources of VLCPUFAs is urgently required. Our research aims at exploiting the powerful infrastructure available for the large scale culture of oilseed crops, such as rapeseed, to produce VLCPUFAs such as eicosapentaenoic acid in transgenic plants. VLCPUFA biosynthesis requires repeated desaturation and repeated elongation of long chain fatty acid substrates. In previous experiments the production of eicosapentaenoic acid in transgenic plants was found to be limited by an unexpected bottleneck represented by the acyl exchange between the site of desaturation, endoplasmic reticulum-associated phospholipids, and the site of elongation, the cytosolic acyl-CoA pool. Here we report on the establishment of a coordinated, exclusively acyl-CoA-dependent pathway, which avoids the rate-limiting transesterification steps between the acyl lipids and the acyl-CoA pool during VLCPUFA biosynthesis. The pathway is defined by previously uncharacterized enzymes, encoded by cDNAs isolated from the microalga Mantoniella squamata. The conceptual enzymatic pathway was established and characterized first in yeast to provide proof-of-concept data for its feasibility and subsequently in seeds of Arabidopsis thaliana. The comparison of the acyl-CoA-dependent pathway with the known lipid-linked pathway for VLCPUFA biosynthesis showed that the acyl-CoA-dependent pathway circumvents the bottleneck of switching the Δ6-desaturated fatty acids between lipids and acyl-CoA in Arabidopsis seeds. Human development and health depend in many respects on the availability of long chain multiply unsaturated fatty acids of 20 or 22 carbons in length that contain up to 6 methylene-flanked cis-double bonds. These fatty acids are classified under the designation very long chain polyunsaturated fatty acids (VLCPUFAs). 2The abbreviations used are:VLCPUFAvery long chain polyunsaturated fatty acid(s)ARAarachidonic acidCLdiphosphatidylglycerolEPAeicosapentaenoic acidFAMEfatty acid methyl esterLPCATacyl-CoA:lysophosphatidylcholine acyltransferasePCphosphatidylcholinePEphosphatidylethanolaminePIphosphatidylinositolPSphosphatidylserineTAGtriacylglycerolRACErapid amplification of cDNA endsMES2-(N-morpholino)ethanesulfonic acidocsoctopine synthase. 2The abbreviations used are:VLCPUFAvery long chain polyunsaturated fatty acid(s)ARAarachidonic acidCLdiphosphatidylglycerolEPAeicosapentaenoic acidFAMEfatty acid methyl esterLPCATacyl-CoA:lysophosphatidylcholine acyltransferasePCphosphatidylcholinePEphosphatidylethanolaminePIphosphatidylinositolPSphosphatidylserineTAGtriacylglycerolRACErapid amplification of cDNA endsMES2-(N-morpholino)ethanesulfonic acidocsoctopine synthase. Nutritionally important VLCPUFAs include arachidonic acid (ARA, 20:4Δ5,8,11,14), an ω6-fatty acid, and the ω3-fatty acids eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19). ω3-VLCPUFAs are of particular interest from a nutritional standpoint since the uptake of these fatty acids is considered to be low in Western diets (1Simopoulos A.P. Am. J. Clin. Nutr. 1991; 54: 438-463Crossref PubMed Scopus (1831) Google Scholar). ω3-VLCPUFAs have long been investigated for their importance during human fetal development and the formation and function of the central nervous system, brain, and retina. In addition to their structural functions in membranes, several medical studies indicate that ω3-fatty acids have anti-inflammatory properties and, therefore, might be useful in the management of inflammatory and autoimmune diseases such as cardiovascular disease, major depression, arthritis, inflammatory bowel disease, asthma, and psoriasis (2Simopoulos A.P. J. Am. Coll. Nutr. 2002; 21: 495-505Crossref PubMed Scopus (1479) Google Scholar). very long chain polyunsaturated fatty acid(s) arachidonic acid diphosphatidylglycerol eicosapentaenoic acid fatty acid methyl ester acyl-CoA:lysophosphatidylcholine acyltransferase phosphatidylcholine phosphatidylethanolamine phosphatidylinositol phosphatidylserine triacylglycerol rapid amplification of cDNA ends 2-(N-morpholino)ethanesulfonic acid octopine synthase. very long chain polyunsaturated fatty acid(s) arachidonic acid diphosphatidylglycerol eicosapentaenoic acid fatty acid methyl ester acyl-CoA:lysophosphatidylcholine acyltransferase phosphatidylcholine phosphatidylethanolamine phosphatidylinositol phosphatidylserine triacylglycerol rapid amplification of cDNA ends 2-(N-morpholino)ethanesulfonic acid octopine synthase. Whereas mammals, including humans, can convert the essential precursors ω6–18:2Δ9,12 or ω3–18:3Δ9,12,15 (α-linolenic acid) to VLCPUFAs, a considerable proportion of VLCPUFA has to be taken up directly as components of the diet (3Goyens P.L. Spilker M.E. Zock P.L. Katan M.B. Mensink R.P. Am. J. Clin. Nutr. 2006; 84: 44-53Crossref PubMed Scopus (279) Google Scholar). Naturally occurring producers and sources of ω3-VLCPUFAs are microorganisms, including marine bacteria and microalgae. These organisms represent the starting point of the aquatic food chain by which VLCPUFAs ultimately accumulate in fish oils. At the moment the main source of ω3-VLCPUFAs for human consumption are fatty ocean fish, such as salmon, mackerel, tuna, or herring. Unfortunately, the increased demand for fish and fish oils has led to a depletion of fish stocks in vast ocean areas worldwide. Thus, as an exclusive source of VLCPUFAs, fish cannot cover the needs of a growing world population (4Hites R.A. Foran J.A. Schwager S.J. Knuth B.A. Hamilton M.C. Carpenter D.O. Environ. Sci. Technol. 2004; 38: 4945-4949Crossref PubMed Scopus (251) Google Scholar). Altogether, the increasing interest to find alternative sources of ω3-VLCPUFAs has led to various attempts to produce ω3-VLCPUFAs via the biotechnological introduction of new biosynthetic pathways in plants, ultimately aiming for VLCPUFA-production in annual oilseeds, e.g. in rapeseed or linseed. VLCPUFA synthesis in transgenic plants established so far starts from the plant endogenous fatty acids ω6–18:2Δ9,12 or ω3–18:3Δ9,12,15 and requires two distinct catalytic activities, desaturases and elongases. Both activities work together in an alternating manner to establish the final product. Genes for the biosynthesis of VLCPUFAs have been isolated from organisms of different kingdoms (algae, amoeba, fungi, moss, and flowering plants) and have been characterized by heterologous expression in various model systems regarding the properties of the encoded enzymes. In recent years a range of tests was done to establish the biosynthesis of VLCPUFAs in plants by introducing a variety of different front-end desaturases and elongases (5Graham I.A. Larson T. Napier J.A. Curr. Opin. Biotechnol. 2007; 18: 142-147Crossref PubMed Scopus (82) Google Scholar, 6Napier J.A. Annu. Rev. Plant Biol. 2007; 58: 295-319Crossref PubMed Scopus (213) Google Scholar). Two previous approaches are of particular relevance for the study presented here. First, the so called alternative pathway for the biosynthesis of VLCPUFAs was successfully tested in Arabidopsis leaves (7Qi B. Fraser T. Mugford S. Dobson G. Sayanova O. Butler J. Napier J.A. Stobart A.K. Lazarus C.M. Nat. Biotechnol. 2004; 22: 739-745Crossref PubMed Scopus (370) Google Scholar). This pathway is based on the sequential action of a Δ9 elongase (from Isochrysis galbana) (8Qi B. Beaudoin F. Fraser T. Stobart A.K. Napier J.A. Lazarus C.M. FEBS Lett. 2002; 510: 159-165Crossref PubMed Scopus (115) Google Scholar), a Δ8 desaturase (from Euglena gracilis) (9Wallis J.G. Browse J. Arch. Biochem. Biophys. 1999; 365: 307-316Crossref PubMed Scopus (112) Google Scholar), and a Δ5 desaturase (from the fungus Mortierella alpina) (10Michaelson L.V. Lazarus C.M. Griffiths G. Napier J.A. Stobart A.K. J. Biol. Chem. 1998; 273: 19055-19059Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The Δ9 elongase converts CoA-bound 18:2Δ9,12 and/or α-18:3 into 20:2Δ11:14 and/or 20:3Δ11,14,17, which could be further converted to ARA and EPA via lipid-bound Δ8 and Δ5 desaturation, respectively. In this fashion a rate-limiting Δ6-elongation step was avoided, and EPA accumulation of up to 3% of the leaf total lipid content of Arabidopsis thaliana was achieved. It must be noted, however, that leaf tissue is not oleogenic and that the fatty acids are directly incorporated into membrane lipids rather than stored as oils as is the case in seeds and as would be desirable for VLCPUFA production. Moreover, a crucial problem was observed. The Δ9-elongation products (20:2Δ11,14 and 20:3Δ11,14,17) accumulated to very high levels in the acyl-CoA pool of the transgenic Arabidopsis plants (11Sayanova O. Haslam R. Qi B. Lazarus C.M. Napier J.A. FEBS Lett. 2006; 580: 1946-1952Crossref PubMed Scopus (36) Google Scholar), indicating an inefficient transfer of these non-native fatty acids out of the acyl-CoA pool. In the second approach the coexpression of Δ6 and Δ5 desaturases of the diatom Phaeodactylum tricornutum with a Δ6 elongase from the moss Physcomitrella patens in linseed showed that the enzymes carried out Δ5 and Δ6 desaturation on lipid-bound substrates in the plant with a positional specificity for the sn-2 position of phosphatidylcholine (PC), whereas the Δ6 elongase preferred acyl-CoA-species as substrates (12Abbadi A. Domergue F. Bauer J. Napier J.A. Welti R. Zähringer U. Cirpus P. Heinz E. Plant Cell. 2004; 16: 2734-2748Crossref PubMed Scopus (268) Google Scholar, 13Domergue F. Abbadi A. Ott C. Zank T.K. Zahringer U. Heinz E. J. Biol. Chem. 2003; 278: 35115-35126Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Comprehensive acyl-CoA and lipid analysis of the EPA-producing transgenic linseed plants demonstrated that production of VLCPUFAs in plants requires not only the interplay of desaturases and elongases but also the transfer of acyl groups from the PC pool into the CoA pool and vice versa. This transfer is very likely catalyzed by the enzymatic activity of an acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT). The LPCAT endogenous to linseed does not accept the newly formed Δ6-desaturated fatty acids, and thus, the Δ6-desaturated fatty acids accumulated to high levels. In brief, the back-and-forth trans-acylation of fatty acids between the PC and CoA pools by the action of LPCAT represents a rate-limiting factor for VLCPUFA production by this transgenic approach in linseed (12Abbadi A. Domergue F. Bauer J. Napier J.A. Welti R. Zähringer U. Cirpus P. Heinz E. Plant Cell. 2004; 16: 2734-2748Crossref PubMed Scopus (268) Google Scholar). In the research mentioned as well as in several other studies, e.g. Refs. 14Wu G. Truksa M. Datla N. Vrinten P. Bauer J. Zank T. Cirpus P. Heinz E. Qiu X. Nat. Biotechnol. 2005; 23: 1013-1017Crossref PubMed Scopus (262) Google Scholar and 15Kinney, A. J., Cahoon, E. B., Damude, H. G., Hitz, W. D., Liu, Z. B., and Kolar, C. W. (U. S., 2004) WO 2004/071467 A2Google Scholar, desaturases from various organisms were analyzed for their feasibility to produce VLCPUFAs. The accumulated evidence indicates that the front-end desaturases characterized from algae, fungi, and moss are lipid-dependent desaturases and accept glycerolipid-linked substrates. In contrast to these lipid-dependent desaturases, a number of mammalian front-end desaturases accept acyl-CoA species as substrates (16Tocher D.R. Leaver M.J. Hodgson P.A. Prog. Lipid Res. 1998; 37: 73-117Crossref PubMed Scopus (245) Google Scholar, 17Okayasu T. Nagao M. Ishibashi T. Imai Y. Arch. Biochem. Biophys. 1981; 206: 21-28Crossref PubMed Scopus (108) Google Scholar, 18Sprecher H. Chen Q. Yin F.Q. Lipids. 1999; 34: 153-156Crossref PubMed Google Scholar). Recently, one acyl-CoA-dependent Δ6 desaturase from the microalga Ostreococcus tauri was identified (19Domergue F. Abbadi A. Zähringer U. Moreau H. Heinz E. Biochem. J. 2005; 389: 483-490Crossref PubMed Scopus (100) Google Scholar). From fatty acid analyses performed on microalgae like O. tauri, however, it could be concluded that further desaturases with a preference for acyl-CoA substrates can be isolated from those organisms. Such plant-like acyl-CoA-dependent enzymes with the correct substrate specificities may allow bypassing the rate-limiting transport and exchange of intermediates of VLCPUFA synthesis between lipid-bound desaturation in the PC pool and elongation steps in the acyl-CoA pool. Many vascular plants synthesize 18:2Δ9,12 and 18:3Δ9,12,15 in their seed oils but do not produce or incorporate VLCPUFAs with chain lengths above 18 carbon atoms or more than three double bonds into triacylglycerol (TAG). To produce the ω3-fatty acid, ω3–20:5Δ5,8,11,14,17 (EPA), in seed oils, it is necessary to introduce one additional elongation and two desaturation steps. To avoid the accumulation of ω6 byproducts of desaturation, the enzymes selected should be specific for ω3 substrates. A second known constraint to circumvent is the rate-limiting shuttling of fatty acids between PC and CoA pools observed by Abbadi et al. (12Abbadi A. Domergue F. Bauer J. Napier J.A. Welti R. Zähringer U. Cirpus P. Heinz E. Plant Cell. 2004; 16: 2734-2748Crossref PubMed Scopus (268) Google Scholar). To contribute to a solution for these problems, we report on the isolation and characterization of acyl-CoA-dependent desaturases from the microalgae Mantoniella squamata and O. tauri. Using the new enzymes, an entirely acyl-CoA-dependent ω3-VLCPUFA biosynthetic pathway consisting of Δ6- and Δ5 desaturase enzymes was successfully established in Saccharomyces cerevisiae and in seeds of A. thaliana plants. This modified pathway may allow a more efficient flux during VLCPUFA biosynthesis and avoids the bottleneck after Δ6 desaturation described by Abbadi et al. (12Abbadi A. Domergue F. Bauer J. Napier J.A. Welti R. Zähringer U. Cirpus P. Heinz E. Plant Cell. 2004; 16: 2734-2748Crossref PubMed Scopus (268) Google Scholar). Materials—Restriction enzymes and DNA-modifying enzymes were obtained from MBI Fermentas. Standards of fatty acids as well as all other chemicals were from Sigma; methanol, n-hexane, and isopropanol (all high performance liquid chromatography grade) were from Baker. Fatty acids and acyl-CoAs were either obtained from Cayman Chemicals or Larodan. Basic molecular biological and biochemical techniques were performed as described (20Ausubel F.M. Brent R.E. Kingston D.D. Seidmann J.R. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Green Publishing Associates and John Wiley and Sons Inc., New York1993Google Scholar). Algae Material and Growth Conditions—M. squamata SAG 65.90 (21Manton I. Parke M. Butcher. J. Marine Biol. Ass. UK. 1960; 39: 275-278Crossref Scopus (93) Google Scholar) was obtained from the algae culture collection Göttingen (SAG, Germany). O. tauri strain OTTH0595-genome was obtained from the Roscoff Culture Collection. Non-axenic cultures were grown in batch cultures under long daylight (14 h) conditions with 45 μmol of photons m–2 s–1 in 200 ml of Brackish Water Medium (1/2 SWES) complemented with soil extract at 20 °C. cDNA Library Construction and Random Sequencing of the cDNA Library—Total RNA from 7-day-old M. squamata cultures were isolated using the RNAeasy kit (Qiagen) per the manufacturer’s instructions. Poly A+ mRNAs were prepared using oligo-dT cellulose (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor NY1989Google Scholar) (see above) and reverse-transcribed via a reverse transcription kit (Promega) and then used for the construction of the cDNA library with a Lambda ZAP Gold library construction kit (Stratagene). After in vivo mass excision of the cDNA library, plasmid recovery and transformation of Escherichia coli (Stratagene), plasmid DNA was prepared on a Qiagen DNA preparation robot (Qiagen) according to the manufacturer’s instructions and submitted to random sequencing by the chain termination method using the ABI PRISM Big Dye Termination Cycle Sequencing Ready Reaction kit (PerkinElmer Life Sciences). Analyses and annotations of the EST sequences between 100 and 500 base pairs resulted in a non-redundant EST data base. Data base screening of the cDNA library yielded two sequences that were annotated as putative desaturases. Isolation and Cloning of Desaturase Sequences—To obtain full-length cDNA sequences, the rapid amplification of cDNA ends (RACE) technique was used. Therefore, 5 μl of total RNA isolated from M. squamata were reverse-transcribed and ligated to adaptor-ligated double-stranded cDNA by using the Marathon cDNA amplification kit (BD Bioscience). Adaptor-ligated double-stranded cDNA was used as template for 5′- and 3′-RACE PCRs to obtain the missing 5′-prime and 3′-prime ends of the coding sequences for several desaturases. For RACE reactions, gene-specific primers were designed from EST sequence information. Primers used in 5′- and 3′-RACE were as follows: for MsΔ6 (MsI) as 5′-RACE primer, 5′-CATCCGGGCGGCAGCGTCATCTTCTAC-3′, and as 3′-RACE primer, 5′-GGAGAAGAGGTGGTGGATGACCTGG-3′; for MsΔ5 (MsII) as 5′-RACE primer, 5′-CCGAGTGAGGGGAGTACGTGGCGGG-3′, and as 3′-RACE primer, 5′-CACTCTCCGGCGGGCTCAACTACC-3′. A 50-μl standard reaction contained 1× Advantage2 DNA polymerase buffer, 1 μl of Advantage 2 DNA Polymerase (BD Bioscience), 0.2 mm concentrations of each dNTP, 0.5 μm 5′ or 3′ primer, 0.5 μm adaptor primer 1 (AP1), and 5 μl of adaptor-ligated double-stranded cDNA. RACE-PCR amplification was performed as follows: 30 s at 94 °C; 5 cycles of 5 s at 94 °C, 4 min at 72 °C; 5 cycles of 5 s at 94 °C, 4 min at 70 °C; 20 cycles of 5 s at 94 °C, 4 min at 68 °C. Amplified products were isolated from agarose gels, purified using a gel extraction kit (GE Healthcare), and subsequently cloned into pGEM-T (Promega). Cloned inserts were sequenced using the ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems). Cloning cDNA for Desaturases into Yeast Expression Vectors—From the 5′- and/or 3′-cDNA sequence data obtained by RACE-PCR, the putative translation initiation codons and stop codons were identified, and this sequence information was used to obtain full-length cDNA clones of the putative desaturases from M. squamata. Gene-specific primers were designed to the 5′- and the 3′-ends of the coding regions of the corresponding nucleotide sequences, introducing restriction sites for cloning into the yeast expression vectors and the yeast consensus sequence for enhanced translation in front of the start codons (23Donahue T.F. Cigan A.M. Methods Enzymol. 1990; 185: 366-372Crossref PubMed Scopus (30) Google Scholar). The open reading frames of the MsΔ6 (MsI) and MsΔ5 (MsII) and of OtΔ5 (OtII) were amplified and modified by PCR. The following pairs of primers were used (restrictions sites are in bold, translation initiation sequences are in italics, and start or stop codons are underlined): for MsΔ6 as forward primer, 5′-ATGCGCGGCCGCACATAATGTGTCCTCCCAAGGAAT-3′, and as reverse primer 5′-ATGCAGATCTCTAGTGAGCGTGCGCCTTC-3′; for MsΔ5 as forward primer, 5′-ATGCGCGGCCGCACATAATGCCCCCGCGCGAGACCA-3′, and as reverse primer, 5′-ATGCAGATCTTCACCCGATGGTTTGAAGG-3′; and for OtΔ5 as forward primer, 5′-ATGCGCGGCCGCACATAATGGGGACGACCGCGCGCGAC-3′, and as reverse primer, 5′-ATGCAGATCTTCATCCGACGGTTTGGAGGGACGG-3′. The amplified cDNAs were cloned into the pGEM-T vector (Promega) before being released and cloned into the yeast expression vector (pESC-LEU or pESC-TRP, respectively, Stratagene) using the restriction sites inserted by PCR, yielding pESC-LEU-MsΔ6 and pESC-TRP-MsΔ5. For comparison with the known front-end desaturases from P. tricornutum, PtΔ6 and PtΔ5, or with the known acyl-CoA-dependent front-end desaturase from O. tauri, OtΔ6, the P. tricornutum or O. tauri cDNA clones were cloned into yeast expression vectors as described for the M. squamata desaturases, yielding pESC-LEU-PtΔ6-PSE1 and pESC-TRP-PtΔ5 using the primers (restrictions sites are in bold; start or stop codons are underlined): for OtΔ6 as forward primer, 5′-ATGCGCGGCCGCACATAATGTGCGTGGAGACGGAAAAT-3′, and as reverse primer, 5′-ATGCAGATCTTTACGCCGTCTTTCCGGAGTGT-3′; for PtΔ6 as forward primer, 5′-ATGCGCGGCCGCACATAATGGGCAAAGGAGGGGACGC-3′, and as reverse primer, 5′-ATGCAGATCTTTACATGGCGGGTCCATGCG-3′; for PtΔ5 as forward primer, 5′-ATGCGCGGCCGCACATAATGGCTCCGGATGCGGATAAG-3′, and as reverse primer, 5′-ATGCAGATCTTTACGCCCGTCCGGTCAAGGG-3′. To obtain pESC-LEU-MsΔ6-PSE1, pESC-LEU-OtΔ6-PSE1, and pESC-LEU-PtΔ6-PSE1, the Δ6 elongase from P. patens, PSE1 (24Zank T.K. Zahringer U. Beckmann C. Pohnert G. Boland W. Holtorf H. Reski R. Lerchl J. Heinz E. Plant J. 2002; 31: 255-268Crossref PubMed Scopus (91) Google Scholar), was cloned into the yeast expression vector. Primers used to amplify PSE1: the forward primer was 5′-AGTCGGATCCTATGGAGGTCGTGGAGAGAT-3′; the reverse primer was 5′-ATGCGCTAGCTCACTCAGTTTTAGCTCCCTT-3′. The Δ5 elongase from O. tauri, OtELO2 (25Meyer A. Kirsch H. Domergue F. Abbadi A. Sperling P. Bauer J. Cirpus P. Zank T.K. Moreau H. Roscoe T.J. Zahringer U. Heinz E. J. Lipid Res. 2004; 45: 1899-1909Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), was cloned in the yeast expression vector pESC-URA. The primers used to amplify OtELO2 were as forward primer, 5′-ATGCGGATCCATGAGCGCCTCCGGTGCGCTGCTGCC-3′, and as reverse primer, 5′-ATGCGCTAGCTTAGTCAATTTTTCGAGATCG-3′. Cloning and Vector Construction for Production of Transgenic Arabidopsis Plants—Plasmids for plant transformation were constructed based on the vector pUC19. For the generation of the constructs, a triple cassette containing three seed-specific USP promoters (26Bäumlein H. Boerjan W. Nagy I. Bassuner R. Van Montagu M. Inze D. Wobus U. Mol. Gen. Genet. 1991; 225: 459-467Crossref PubMed Scopus (88) Google Scholar), three OCS terminators, and three different polylinkers between each promoter and terminator was first introduced to the vector pUC19 (Pharmacia Corp.), yielding the USP123OCS plasmid. The open reading frames of the different desaturases and elongases were modified by PCR to create appropriate restriction sites adjacent to the start and stop codons, cloned into the pGEM-T vector (Promega), and sequenced to confirm their accuracy. The primers used were (restrictions sites are in bold, and start or stop codons are underlined): MsΔ6, forward, 5′-ATGCGCGGCCGCACATAATGTGTCCTCCCAAGGAAT-3′, reverse, 5′ATGCTCTAGACTAGTGAGCGTGCGCCTTC-3′; MsΔ5, forward, 5′-ATGCCCATGGACATAATGCCCCCGCGCGAGACCACCAC-3′, reverse, 5′-ATGCACCGGTTCACCCGATGGTTTGAAGGC-3′; OtΔ6, forward, 5′-ATGCGCGGCCGCACATAATGTGCGTGGAGACGGAAAAT-3′, reverse, 5′-ATGCTCTAGATTACGCCGTCTTTCCGGAGTGT-3′; OtΔ5, forward, 5′-ATGCCCATGGACATAATGTGGACGCCCGCGCGCGA-3′, reverse, 5′-ATGCACCGGTTCATCCGACGGTTTGGAGGG-3′; PtΔ6, forward, 5′-ATGCGCGGCCGCACATAATGGGCAAA GGAGGGGACGC-3′, reverse, 5′-ATGCTCTAGATTACATGGCGGGTCCATCGCGTA-3′; PtΔ5, forward, 5′-ATGCCCATGGACATAATGGCTCCGGATGCGGATAAGC-3′, reverse, 5′-ATGCGCGATCGCTTACGCCCGTCCGGTCAA-3′, PSE1, forward, 5′-AGTCGGATCCTATGGAGGTCGTGGAGAGAT-3′, reverse, 5′-ATGCGCTAGCTCACTCAGTTTTAGCTCCCTT-3′. The open reading frames were then released using the restriction sites created by PCR and successively inserted into the same sites of the polylinkers of the USP123OCS plasmid. The resulting cassette, containing the three genes each under the control of the USP promoter, was released by digesting the USP123OCS plasmid with SbfI or SacI and cloned into the corresponding sites of the binary vector pCAMBIA3300 yielding the constructs triple-Ms, triple-Ot, and triple-Pt. The binary vector pCAMBIA3300 (CAMBIA) uses the bar gene with the cauliflower mosaic virus 35 S promoter as a selectable marker in plants. The binary plasmid constructs were transformed into chemically competent Agrobacterium tumefaciens cells (strain EH105). Expression in S. cerevisiae—S. cerevisiae cells strain INVSc1 (Invitrogen) were transformed as described (27Hornung E. Korfei M. Pernstich C. Struss A. Kindl H. Fulda M. Feussner I. Biochim. Biophys. Acta. 2005; 1686: 181-189Crossref PubMed Scopus (27) Google Scholar). For induction, expression cultures were grown for 48 h to 72 h at 22 °C in the presence of 2% (w/v) galactose supplemented with 150–350 μm concentrations of appropriate fatty acid substrate and in the presence of 1% Igepal CA 630 (Nonidet P-40) from Sigma-Aldrich. Cells were harvested by centrifugation at 1200 × g for 5 min, and the pellets were washed twice with H2O before being used for further analysis. The host strain transformed with the empty vector(s) was used as negative control in all experiments. Arabidopsis Transformation—A. thaliana ecotype Columbia (Col-0) plants were transformed by floral dipping (37Clough S.J. Bent A.F. Plant J. 1998; 16: 735-743Crossref PubMed Google Scholar). T2 seeds were collected from individual T1 plants resistant to ammonium glufosinate and analyzed individually by GC. Fatty Acid Analysis—Fatty acid methyl esters (FAMEs) were obtained by methylation of microalgae or yeast cell sediments with 0.5 m sulfuric acid in methanol containing 2% (v/v) dimethoxypropane at 80 °C for 1 h. FAMEs were extracted in 2 ml of n-hexane, dried under N2, and analyzed by gas chromatography (GC). FAMEs of single or pooled Arabidopsis seeds were prepared by transesterification with trimethylsulfonium hydroxide (28Butte W. Reimann H.H. Walle A.J. Clin. Chem. 1982; 28: 1778-1781Crossref PubMed Scopus (7) Google Scholar). FAMEs of TLC-separated individual lipids were obtained by transmethylation with 333 μl of toluol/methanol (1:2 v/v) and 167 μl of 0.5 m NaOCH3 at room temperature for 20 min. FAMEs were extracted in 500 μl of NaCl, 50 μl of HCl (37%), and 2 ml of n-hexane, dried under N2, and analyzed by GC. The GC analysis was performed with an Agilent GC 6890 system coupled with a flame ionization detector equipped with a capillary 122-2332 DB-23 column (30 m × 0.32 mm; 0.5 μm coating thickness; Agilent). Helium was used as carrier gas (1 ml min–1). Samples were injected at 220 °C. The temperature gradient was 150 °C for 1 min, 150 to 200 °C at 15 °C min–1, 200 to 250 °C at 2 °C min–1, and 250 °C for 10 min. Data were processed using the HP ChemStation Rev. A09.03. FAMEs were identified by comparison with appropriate reference substances. Lipid Analysis—For lipids analysis expression has been carried out with 50-ml cultures. Harvested cell pellets were homogenized in 5 ml of chloroform/methanol 1:2 (v/v), and lipids were extracted on a shaker for 4 h and then for 20 h with 5 ml of chloroform/methanol 2:1 (v/v) at 4 °C. The resulting organic phases were combined and dried under N2. The remaining lipids were dissolved in 1 ml of chloroform. Separation of lipid classes (neutral lipids and phospholipids) was achieved using a silica column (Bond Elut SI, 100 mg/ml; Varian). Lipid extracts were loaded on the silica column pre-equilibrated with chloroform and then fractionated into the lipid classes by elution as follows; neutral lipids with chloroform and phospholipids with methanol/glacial acetic acid (9:1. v/v). Isolation of individual components of the phospholipid class was achieved by thin layer chromatography using appropriate standards and with methanol/chloroform/glacial acetic acid (25:65:8, v/v) as developing solvent. Lipid Analysis of Arabidopsis Seeds—For lipid analysis, 10 mg of seeds were homogenized in 4 ml of chloroform/methanol/glacial acetic acid (2:1:0.1 v/v/v) and incubated for 24 h at 4 °C. Seed residues were pelleted (2 min, 3000 × g). The supernatant was collected, and the pelleted seed residues were incubated with 2 ml of n-hexane for 30 min at room temperature. The resulting organic phases were combined and dried under N2. The dried lipids were dissolved in 200 μl of chloroform. Separation of lipid classes (TAG and" @default.
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• W1998596631 title "Metabolic Engineering of ω3-Very Long Chain Polyunsaturated Fatty Acid Production by an Exclusively Acyl-CoA-dependent Pathway" @default.
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