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The synthesis and accumulation of long chain polyunsaturated fatty acids such as eicosapentaenoic acid has previously been demonstrated in the seeds of transgenic plants. However, the obtained levels are relatively low, indicating the need for further studies and the better definition of the interplay between endogenous lipid synthesis and the non-native transgene-encoded activities. In this study we have systematically compared three different transgenic configurations of the biosynthetic pathway for eicosapentaenoic acid, using lipidomic profiling to identify metabolic bottlenecks. We have also used genetic crossing to stack up to ten transgenes in Arabidopsis. These studies indicate several potential approaches to optimize the accumulation of target fatty acids in transgenic plants. Our data show the unexpected channeling of heterologous C20 polyunsaturated fatty acids into minor phospholipid species, and also the apparent negative metabolic regulation of phospholipid-dependent Δ6-desaturases. Collectively, this study confirms the benefits of iterative approaches to metabolic engineering of plant lipid synthesis.

Although plant plastidial omega3-desaturases are closely related to microsomal desaturases, heterologous expression in yeast of the Helianthus annuus FAD7 omega3-desaturase showed low activity in contrast to similar expression of microsomal FAD3 omega3-desaturases. However, the removal of the plastidial transit peptide and the incorporation of a KKNL motif to the C-terminus of HaFAD7 increased the activity by 10-fold compared to the native protein. N-terminal fusion of transmembrane-domains from either the yeast microsomal ELO3, (a type III signal anchor domain), or FAE1, an endoplasmic reticulum membrane anchoring domain, resulted in moderate increases in enzyme activity (5- and 7-fold, respectively), suggesting that the first, most hydrophobic transmembrane domain of HaFAD7 is sufficient to direct targeting to, and insertion into, the endoplasmic reticulum membrane. Furthermore, fusing a hemagglutinin (HA) epitope tag upstream of an endogenous C-terminal KEK motif resulted in a significant loss of activity compared to the un-tagged construct, indicating that the endogenous KEK C-terminal di-lysine motif is capable of directing in yeast the ER-retention of this normally plastidial-located protein. Western blotting analysis of constructs with internal HA epitope revealed that in whole cell extracts, with the exception of the one bound to C-terminal, it did not display a reduced level of protein accumulation. Whilst ferredoxin was shown to be required for HaFAD7 activity in yeast, it appears not necessary for protein stability and accumulation of this plastidial desaturase in the endoplasmic reticulum., (2010 Elsevier Ltd. All rights reserved.)

Ferredoxins are proteins that participate in photosynthesis and in other processes that require reducing equivalents, such as the reduction of nitrogen or fatty acid desaturation. Two classes of ferredoxins have been described in plants: light-regulated photosynthetic ferredoxins and heterotrophic ferredoxins whose activity is not influenced by light. Genes encoding the two forms of ferredoxin have been cloned and characterized in developing sunflower cotyledons. Here, these genes were overexpressed in Escherichia coli and they were purified by ion exchange and size exclusion chromatography to study their capacity to supply electrons to two different sunflower desaturases: soluble stearoyl-ACP desaturase from sunflower cotyledons, and membrane bound desaturase FAD7 expressed in yeast. In both cases photosynthetic ferredoxin was the form that promoted the strongest desaturase activity.

cDNA clones encoding cytochrome b(5) fusion desaturases were isolated from Primula cortusoides L. and Primula luteola Ruprecht, species previously shown to preferentially accumulate either n-6 or n-3 Delta6-desaturated fatty acids, respectively. Functional characterisation of these desaturases in yeast revealed that the recombinant Primula enzymes displayed substrate preferences, resulting in the predominant synthesis of either gamma-linolenic acid (n-6) or stearidonic acid (n-3). Independent expression of the two Primula desaturases in transgenic Arabidopsis thaliana confirmed these results, with gamma-linolenic acid and stearidonic acid accumulating in both leaf and seed tissues to different levels, depending on the substrate specificity of the desaturase. Targeted lipid analysis of transgenic Arabidopsis lines revealed the presence of Delta6-desaturated fatty acids in the acyl-CoA pools of leaf but not seed tissue. The implications for the transgenic synthesis of C(20) polyunsaturated fatty acids via the elongation of Delta6-desaturated fatty acids are discussed, as is the potential of using Primula desaturases in the synthesis of C(18) n-3 polyunsaturated fatty acids such as stearidonic acid.

Fatty acid desaturases (FAD) play an important role in plant lipid metabolism and they can be found in several subcellular compartments such as the plastids and endoplasmic reticulum. Lipids are critical components of the cell membrane and, as a consequence, they are fundamental for the proper growth and development of all living organisms. We have used sequences from the conserved regions of known omega-3-desaturases to design degenerated oligonucleotides and clone a cDNA encoding a plastidial omega-3 desaturase from sunflower (HaFAD7). From its presumed full-length sequence, we predict that Hafad7 encodes a protein of 443 amino acids with a molecular mass of 50.8 kDa, and that it contains a putative chloroplast transit peptide of 51 amino acids. The predicted hydrophobicity of the protein identifies four potential membrane-spanning regions and, according to the TargetP algorithm, the protein should be targeted to the plastid/chloroplast membrane. RT-PCR analysis of its expression shows the transcript is preferentially expressed in photosynthetically active tissues. Heterologous expression of this protein in the unicellular cyanobacterium Synechocystis sp. PCC 6803 confirmed that the protein produced from this cDNA has omega-3 desaturase activity.