Figure 7. Competition between n-3 PUFA substrates for desaturation by Fads2. Recombinant S. cerevisiae expressing Fads2 were grown in the presence of 25 mM of 24:5n-3 and 0–400 mM ALA. Fatty acids were extracted from the recombinant S. cerevisiae and the amount of each fatty acid was expressed as a percentage of the total amount of all fatty acids. The proportion of substrate fatty acid converted to desaturation product was calculated as [product/(product + substrate)]6100. The results are the means 6 S.D. of triplicate incubations. Values with different symbols are significantly different from each other. doi:10.1371/journal.pone.0029662.g007 as fish, chickens, rats and humans. Freshwater fish and salmonids which consume herbivorous or omnivorous diets are generally considered to be capable of synthesizing DHA from ALA. Interestingly, zebrafish and Atlantic salmon are the only non- mammalian vertebrates with a functionally characterised Elovl2 [21,22]. Searches of the ENSEMBL genomes suggest that the carnivorous fish species Tetraodon nigroviridis and Gasterosteus aculeatus and even omnivorous fish species Takifugu rubripes and Oryzais latipes do not contain Elovl2 homologs [22]. The lack of Elovl2 could impose an absolute block on their ability to synthesize DHA from ALA and accordingly Elovl2 may be a genetic marker for DHA synthetic capacity of a species. Role of Elongase Reactions in LCPUFA Synthesis Materials and Methods Materials All reagents were obtained from InvitrogenTM Australia Pty. Ltd. (Mount Waverley, Vic, Aust.) unless otherwise stated. The PUFA substrates and internal standard 17:0 were obtained from Nu-Chek Prep, Inc. (MN, USA), Cayman Chemical Company (Sapphire Bioscience, Australia) or Larodan Fine Chemicals (Malmo¨, Sweden). Cloning the Elovl2, Elovl5 and Fads2 cDNA Total RNA was extracted from the livers of Dark Agouti rats with the RNeasy kit (Qiagen, Australia). RT-PCR amplification of the Elovl2, Elovl5 and Fads2 open reading frames (ORFs) was performed using the primers in Table 1 and OneStep RT-PCR Enzyme Mix (Qiagen). The primers were used to generate restriction enzyme sites flanking the ORFs. The resulting cDNAs and the expression vector pYES2 were restriction enzyme treated and ligated using T4 DNA ligase (1.5 Weiss units) (Promega, WI, USA). Transformation of the resulting constructs, pYES2-Elovl2, pYES2-Elovl5 and pYES2-Fads2 into MAX EfficiencyH DH5aTM Competent E. coli cells was performed using heat-shock. Putative transformants were selected using 100 mg mL-1 ampicillin and PCR screening. Recombinant plasmids were purified and the presence of the gene insert in the correct orientation was confirmed via sequencing at the Institute of Medical and Veterinary Science (Adelaide, Australia). Heterologous expression of the Elovl2, Elovl5 and Fads2 ORFs in Saccharomyces cerevisiae The pYES2-Elovl2, pYES2-Elovl5 and pYES2-Fads2 constructs were used to transform Saccharomyces cerevisiae strain INVSc1 for the production of recombinant protein, using the S. c. EasyCompTM Transformation Kit. Successfully transformed yeast cells were selected on uracil dropout medium. Recombinant yeast cells were maintained in synthetic minimal defined medium for yeast without uracil containing 2% glucose. Upon fatty acid supplementation, glucose was replaced with 2% galactose for induction of gene expression. Figure 8. N-3 long-chain polyunsaturated fatty acid synthesis showing two-stages of D6-desaturase involvement and the individual elongase involvement. ? denotes that Elovl5 or Elovl2 can catalyse this reaction. doi:10.1371/journal.pone.0029662.g008 PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e29662