Frederic Domergue

Biosynthesis of Very-Long-Chain Polyunsaturated Fatty Acids in Transgenic Oilseeds: Constraints on Their Accumulation

ω6- and ω3-polyunsaturated C20 fatty acids represent important components of the human diet. A more regular consumption and an accordingly sustainable source of these compounds are highly desirable. In contrast with the very high levels to which industrial fatty acids have to be enriched in plant oils for competitive use as chemical feedstocks, much lower percentages of very-long-chain polyunsaturated fatty acids (VLCPUFA) in edible plant oils would satisfy nutritional requirements. Seed-specific expression in transgenic tobacco (Nicotiana tabacum) and linseed (Linum usitatissimum) of cDNAs encoding fatty acyl-desaturases and elongases, absent from all agronomically important plants, resulted in the very high accumulation of Δ6-desaturated C18 fatty acids and up to 5% of C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acid. Detailed lipid analyses of developing seeds from transgenic plants were interpretated as indicating that, after desaturation on phosphatidylcholine, Δ6-desaturated products are immediately channeled to the triacylglycerols and effectively bypass the acyl-CoA pool. Thus, the lack of available Δ6-desaturated acyl-CoA substrates in the acyl-CoA pool limits the synthesis of elongated C20 fatty acids and disrupts the alternating sequence of lipid-linked desaturations and acyl-CoA dependent elongations. As well as the successful production of VLCPUFA in transgenic oilseeds and the identification of constraints on their accumulation, our results indicate alternative strategies to circumvent this bottleneck.

Metabolic engineering of fatty acids for breeding of new oilseed crops: strategies, problems and first results

Abbreviations: ACP = acyl carrier protein. – ACS = acyl-CoA synthase. – ARA = arachidonic acid. – CLA = conjugated linoleic acid. – CPT = CDP-choline :1,2-diacylglycerol cholinephosphotransferase. – DAGAT = acyl-CoA :1,2-diacylglycerol acyltransferase. – DHA = docosahexaenoic acid. – EPA = eicosapentaenoic acid. – ER = endoplasmic reticulum. – FAE = fatty acid elongase. – FAR = acyl-CoA reductase. – GLA = γ-linolenic acid. – GPAT = acyl-CoA : glycerol-3-phosphate acyltransferase. – HEAR = high-erucic acid rapeseed. – KAS = β-ketoacyl-ACP synthase. – KCS = β-ketoacyl-CoA synthase. – LPAAT = acyl-CoA : lysophosphatidic acid acyltransferase. – LPCAT = acyl-CoA : lysophosphatidylcholine acyltransferase. – MCF = medium-chain fatty acids. – mRNAi = antisense messenger ribonucleic acid containing inverted-repeat sequences. – PC = phosphatidylcholine. – PDAT = phospholipid : 1,2-diacylglycerol acyltransferase. – PLA2 = phospholipase A2. – TAG = triacylglycerol. – VLCPUFA = very long-chain polyunsaturated fatty acids. – WS = wax synthase

In vivo characterization of the first acyl-CoA Δ6-desaturase from a member of the plant kingdom, the microalga Ostreococcus tauri

Genomic DNA of Ostreococcus tauri, a fully sequenced marine unicellular alga from the phytoplankton, was used to amplify a gene coding for a typical front-end desaturase involved in polyunsaturated fatty acid biosynthesis. Heterologous expression in Saccharomyces cerevisiae revealed very high desaturation activity with Delta6-regioselectivity. Short-time kinetic experiments showed that the desaturase product was detected in the acyl-CoA pool 5 min after addition of the exogenous substrate to the yeast medium and long before its appearance in the total fatty acids. When this desaturase was co-expressed with the acyl-CoA Delta6-elongase from Physcomitrella patens and the lipid-linked Delta5-desaturase from Phaeodactylum tricornutum, high proportions of arachidonic or eicosapentaenoic acid were obtained, because nearly all of the Delta6-desaturated products were elongated. Furthermore, the product/educt ratios calculated in each glycerolipid for the Delta6-desaturase or for the acyl-CoA Delta6-elongase were in about the same range, whereas this ratio showed a very uneven profile in the case of the lipid-linked Delta5-desaturase. Finally, a sequence-based comparison of all the functionally characterized Delta6-desaturases showed that this enzyme was not related to any previously described sequence. Altogether, our data suggest that this desaturase from O. tauri is an acyl-CoA Delta6-desaturase, the first one cloned from a photosynthetically active organism.

Purification of the Acyl-CoA Elongase Complex from Developing Rapeseed and Characterization of the 3-Ketoacyl-CoA Synthase and the 3-Hydroxyacyl-CoA Dehydratase

Oleoyl-CoA elongase catalyzes four successive reactions: condensation of malonyl-CoA to oleoyl-CoA, reduction, dehydration, and another reduction. Evidence supporting this mechanism and the multienzymatic nature of the elongation complex are reported. A particulate membrane fraction from rapeseed is able to elongate intermediates (R,S) 3-hydroxy-20∶0-CoA and (E) 2,3–20∶1-CoA to very long chain fatty acids in the presence of malonyl-CoA. Studies of the 3-ketoacyl-CoA synthase activities showed that maximal activity could be measured by using 15 to 30 μM 18∶1-CoA and 30 μM malonyl-CoA, and that 18∶0-CoA and 18∶1-CoA were the best substrates. Comparison of the condensation and the overall elongation activities indicated that condensation is the rate-limiting step of the elongation process. The 3-hydroxyacyl-CoA dehydratase activity was maximal in the presence of 75 μM Triton X-100 and 25 μg of proteins. Finally, the acyl-CoA elongase complex was solubilized and purified. During the purification process, the 3-hydroxyacyl-CoA dehydratase copurified with the elongase complex, strongly suggesting that this enzyme belongs to the elongase complex. The apparent molecular mass of 700 kDa determined for the elongase complex, and the fact that four different polypeptide bands were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified fraction, further suggest that the acyl-CoA elongase is a multienzymatic complex.

Fatty Acid Desaturases from the Diatom Phaeodactylum Tricornutum

Very long-chain polyunsaturated fatty acids (PUFAs) such as arachidonic acid (ARA, 20:4Δ5,8,11,14) or eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) have been shown to display many beneficial effects on human health. Today, the main commercial sources for these fatty acids are fish oil and micro-organisms. Since biotechnology may allow the production of specific fatty acids by genetic engineering of crops, the possibility of producing PUFAs in rapeseed or linseed has lead to the search for the genes encoding the enzyme activities involved in PUFA biosynthesis.