With regard to the DGATs we
With regard to the DGATs, we have found that the majority of algal species encode at least one DGAT1, although it appears to be absent from Ostreococcus and Micromonas, as is the case for yeast, S. cerevisiae. More surprising is the presence of multiple DGAT2 genes in algae, with the exception of C. merolae, which has one gene, and E. huxleyi, which has none (Table 2). If the latter observation is confirmed to be true, this will be a novel finding for eukaryotes. Nevertheless in all other algae surveyed, 2 or more DGAT2 genes were found. An interesting pattern that emerged from comparing the sequences of these multiple DGAT2s was that the genes in a particular algal species are more divergent from each other than they are between algal species. Green and red algae are thought to have arisen by the endosymbiosis of a cyanobacterium that gave rise to the chloroplast. All land plants arose from the charophyte branch of the green algal lineage. Diatoms, heterokonts and haptophytes are much more divergent from these basal algae, and from each other, although they all contain complex chloroplasts, which arose through secondary endosymbiosis of a red alga (Dorrell and Smith, 2011). We therefore expected that green algal DGATs should be most related in terms of sequence similarities to higher plants, followed by red algae and other algae clades. From the dendogram of DGAT2s, it is clear that this is not the case: the multiple DGAT2s within an algal species are highly divergent, more so than the DGAT2s between different higher plant species or between mammalian and fungal DGAT2s. The vast majority of algal DGAT2s seem to be distantly related to both higher plant and animal DGAT2s. Assuming that biochemical analysis confirms the enzymatic identity of these putative DGAT2s, we tentatively propose that the various putative DGAT2 isoforms found in modern algal groups could represent a very ancient gene ferulic acid event that occurred prior to the subsequent divergence of various eukaryotic lineages. These isoforms were then gradually lost in eukaryotic lineages that would form the basal groups of complex multicellular organisms until only one particular isoform was selected for prior to the speciation of multicellular organisms. The algal lineages that did not develop complex multicellularity retained the various DGAT2 isoforms, perhaps to enable the production of different TAGs within the same cell, which in multicellular organisms can take place via spatial separation of fatty acid biosynthesis in different cell types. A good way to validate this theory would be to look for DGAT2s in basal lineages of eukaryotes as well as multicellular brown algae to see if this pattern can be seen across the tree of life. While DGATs have long been thought to be associated with the ER membrane, recent reports have indicated that DGATs could also be localised to mitochondria-associated membranes in mice (Stone et al., 2009) and there is growing evidence that there may be an independent TAG biosynthesis pathway in the chloroplasts of C. reinhardtii, based on studies of sta6 mutants which are defective in starch biosynthesis (Fan et al., 2011, Goodson et al., 2011). This may mean that there are different DGAT splice forms or multiple DGAT isoforms with different subcellular targeting peptides. If these hypotheses turn out to be true, TAG biosynthesis in algae could prove much more complicated than currently thought, and taking this into account for future metabolic engineering efforts will be crucial. The capability to modify algal species to produce high levels of designer biofuels or high-value lipid compounds on-demand will be highly reliant on basic research into both the enzymes involved in the biosynthesis pathways, as well as transcription factors involved in controlling the expression of these genes. While the lack of fully sequenced algal genomes is currently seen as a major obstacle to research into potential industrial production algal strains, new methods are currently being developed to sidestep such research bottlenecks (Guarnieri et al., 2011). Refinement of this technique could potentially enable large throughput screening of productive algal strains combined with the ability to elucidate the details of TAG biosynthesis for each individual algal species.