Incorporation of omega-3 polyunsaturated fatty acids into glycerolipids in microalga <i>Emiliania huxleyi</i> : radiochemical analysis of glycerolipid biosynthesis

Marine microalgae are primary producers of nutritionally important ω-3 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA, 22:6n-3), and octadecapentaenoic acid (OPA, 18:5n-3). However, how these fatty acids, after synthesis, are incorporated into glycerolipids in microalgae remains largely unknown. To investigate the assembly of fatty acids and glycerol into different glycerolipids, ¹⁴C-acetate and ¹⁴C-glycerol were used to trace their flux into glycolipids (GL) and phospholipids (PL) in the microalga <i>Emiliania huxleyi</i>. ¹⁴C-acetate labeling showed that freshly synthesized fatty acids were predominantly incorporated into phosphatidylethanolamine (PE) and phosphatidylcholine (PC) over monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), with the more efficient incorporation into the sn-2 position of PL. A similar flux pattern of glycerol backbone to glycerolipids was also observed in ¹⁴C-glycerol labeling. Simultaneous decrease of both acyl and backbone fluxes to PE alongside the corresponding increase in PC over the labeling courses revealed the conversion of PE to PC through head group methylation. Positional analysis of labeled glycerolipids showed that saturated fatty acids (SFA) were initially found exclusively at the sn-2 position of MGDG, while OPA, not DHA, emerged in MGDG with preference at the sn-1 position. In contrast, DHA, not OPA, was incorporated into PC with preference at the sn-2 position. These results indicate that the two ω-3 fatty acids are incorporated through completely separate pathways in <i>E. huxleyi</i>. The knowledge about the biosynthesis and assembly of ω-3 PUFAs at the stereospecific positions of glycerolipids is highly valuable for developing targeted strategies in the metabolic engineering of these fatty acids in microalgae.IMPORTANCEMarine microalgae are primary producers of nutritionally important ω-3 long-chain polyunsaturated fatty acids (PUFAs). However, how these fatty acids, after synthesis, are incorporated into glycerolipids in microalgae remains largely unknown. To investigate the assembly of fatty acids and glycerol into different glycerolipids, ¹⁴C-acetate and ¹⁴C-glycerol were used to trace their flux into glycolipids and phospholipids with two different protocols, steady-state labeling and pulse-chase labeling in <i>Emiliania huxleyi,</i> a cosmopolitan microalga that can produce a high level of docosahexaenoic acid (DHA, 22:6n-3) and octadecapentaenoic acid (OPA, 18:5n-3). Results indicate that the two ω-3 fatty acids are incorporated through completely separate pathways with high stereospecificity in <i>E. huxleyi</i>. Knowledge gained from the research is highly valuable for developing targeted strategies for the optimized production and metabolic engineering of these fatty acids in microalgae and heterologous systems.