Considering the divergence of multiple DGAT isoforms
Considering the divergence of multiple DGAT isoforms, we examined whether MiDGAT1 and MiDGAT2s utilized different fatty acids as the substrate to synthesize TAGs. GC-MS analysis indicated that C16:0 and C18:0 were the major components of TAGs in yeast cells, and all three MiDGATs displayed the same preference towards these fatty acids. This is different from some previous reports: in P. tricornutum, PtDGAT1 tended to incorporate saturated 16:0 and C18:0 fatty acids into TAG, whereas PtDGAT2 had a preference for unsaturated fatty acids , . In Ostreococcus tauri, a gene encoding OtDGAT2 was identified and the enzyme exhibited broad substrate specificity . In C. reinhardtii, CrDGAT2E (DGTT2) tended to incorporate varying acyl-CoA species into TAGs, probably preferring very-long-chain acyl groups. Meanwhile, CrDGAT2D (DGTT3) and 2A (DGTT4) preferred C16:0 and C18:1, respectively. CrDGAT2E (DGTT5) did not exhibit substrate specificity, implying it could be a nonactive pseudogene . Such distinct synthesis patterns suggested that different algae may have developed their own strategies. In M. incisa, DGAT1 and 2 families seem to function in the same cellular compartments, leading to the accumulation of similar TAG species. More detailed study will be carried out in future to verify this result, e.g. the examination of fatty 5-Methyl-CTP profiles in TAGs and algal cells, and fatty acid feeding experiments.
In conclusion, this is the first report describing DGATs in the microalga M. incisa. The identification and functional analysis of one MiDGAT1 and two MiDGAT2s may contribute to developing a basis for enhancing cellular TAG contents through genetic engineering. In future, based on the transcriptome database of M. incisa, more MiDGATs may be continually identified, whose roles in TAG synthesis await discovery.
Acknowledgments Authors acknowledge financial support from the National Natural Science Foundation of China (31402274, 31172389), the Special Project of Marine Renewable Energy from the State Oceanic Administration (SHME2011SW02), China Postdoctoral Science Foundation (2014M551381) and Shanghai Universities First-class Discipline Project of Marine Sciences.
Introduction For industrial uses, fatty acids with hydroxy, epoxy or unusual positions of desaturation are desired (Meier zu Beerentrup and Röbbelen, 1987). Castor oil containing up to 90% ricinoleic acid (12-hydroxyoleic acid) is obtained from Ricinus communis L. (Euphorbiaceae) and it is traded in quantity. Kleiman et al. (1965) in a survey of euphorb seed oils identified one species with a high content (60%) of vernolic acid (12,13-epoxyoleic acid): Euphorbia lagascae Spreng. Baumann et al. (1988) also listed Euphorbia lathyris L., the caper spurge, as a potential new industrial crop due to its high content of oleic acid (80–90%). Oilseed crops with unusual fatty acids can be developed either by domestication of wild species (Pascual-Villalobos et al., 1994) or by genetic transformation of existing ones (Murphy, 1993). Transforming the gene that is responsible for the production of the functional group (e.g. hydroxy or epoxy) into an established oilseed crop (e.g. rape or sunflower) is not sufficient to obtain a high expression and accumulation of the unusual fatty acid in the transgenic plant (Broun and Somerville, 1997). But, as reported by Kinney et al. (1998), a greater amount of such fatty acids could be achieved by storing them in triacylglycerols (TAG). The enzyme that catalyses the synthesis of TAG from diacylglycerol (DAG) is the acyl-CoA: 1,2-diacylglycerol acyltransferase (DGAT) and it is intimately associated with membranes. It has also been suggested that the selectivity of a DGAT of any particular crop for its native substrate is a limiting factor in the flexibility to incorporate other fatty acids (Wiberg et al., 1994, He et al., 2004b). Murphy (1993) indicated that DGAT appears to have a relatively low substrate specificity towards a variety of acyl-CoAs compared with other acyltransferases that are involved in earlier steps of lipid synthesis.