Use of radiolabeled substrates to determine the desaturase and elongase activities involved in eicosapentaenoic acid and docosahexaenoic acid biosynthesis in the marine microalga Pavlova lutheri

The marine flagellate Pavlova lutheri is a microalga known to be rich in long-chain polyunsaturated fatty acids (LC-PUFAs) and able to produce large amounts of n-3 fatty acids, such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). As no previous study had attempted to measure the metabolic step of fatty acid synthesis in this alga, we used radiolabeled precursors to explore the various desaturation and elongation steps involved in LC-PUFA biosynthesis pathways. The incorporation of C-14-labeled palmitic ([1-C-14] 16:0) and dihomo-gamma-linolenic ([1-C-14] 20:3n-6) acids as ammonium salts within the cells was monitored during incubation periods lasting 3, 10 or 24 h. Total lipids and each of the fatty acids were also monitored during these incubation periods. A decrease in the availability and/or accessibility of the radiolabeled substrates was observed over the incubation time. This decrease with incubation time observed using [1-C-14] 16:0 and [1-C-14] 20:3n-6 as substrates was used to monitor the conversion of C-14-labeled arachidonic acid ([1-C-14] 20:4n-6) into longer and more unsaturated fatty acids, such as 20:5n-3 and 22:6n-3, over shorter incubation times (1 and 3 h). A metabolic relationship between the n-6 and n-3 fatty acid series was demonstrated in P. lutheri by measuring the Delta 17-desaturation activity involved in the conversion of eicosatetraenoic acid to 20:5n-3. Our findings suggest that the biosynthesis pathway leading to n-3 LC-PUFA involves fatty acids of the n-6 family, which act as precursors in the biosynthesis of 20:5n-3 and 22:6n-3. This preliminary work provides a method for studying microalgal LC-PUFA biosynthesis pathways and desaturase and elongase activities in vivo using externally-radiolabeled fatty acid precursors as substrates. The use of the [1-C-14] 20:4n-6 substrate also highlighted the relationships between the n-6 and the n-3 fatty acid series (e.g. Delta 17-desaturation), and the final elongation and desaturation steps required for n-3 LC-PUFA formation in P. lutheri. (C) 2013 Elsevier Ltd. All rights reserved.