Identification of Polyunsaturated Fatty Acids Synthesis Pathways in the Toxic Dinophyte Alexandrium minutum Using (13)C-Labelling

The synthetic pathways responsible for the production of the polyunsaturated fatty acids 22:6n-3 and 20:5n-3 were studied in the Dinophyte Alexandrium minutum. The purpose of this work was to follow the progressive incorporation of an isotopic label ((13)CO(2)) into 11 fatty acids to better understand the fatty acid synthesis pathways in A. minutum. The Dinophyte growth was monitored for 54 h using high-frequency sampling. A. minutum presented a growth in two phases. A lag phase was observed during the first 30 h of development and had been associated with the probable temporary encystment of Dinophyte cells. An exponential growth phase was then observed after t(30). A. minutum rapidly incorporated (13)C into 22:6n-3, which ended up being the most (13)C-enriched polyunsaturated fatty acid (PUFA) in this experiment, with a higher (13)C atomic enrichment than 18:4n-3, 18:5n-3, 20:5n-3, and 22:5n-3. Overall, the (13)C atomic enrichment (AE) was inversely proportional to number of carbons in n-3 PUFA. C(18) PUFAs, 18:4n-3, and 18:5n-3, were indeed among the least (13)C-enriched FAs during this experiment. They were assumed to be produced by the n-3 PUFA pathway. However, they could not be further elongated or desaturated to produce n-3 C(20)-C(22) PUFA, because the AEs of the n-3 C(18) PUFAs were lower than those of the n-3 C(20)-C(22) PUFAs. Thus, the especially high atomic enrichment of 22:6n-3 (55.8% and 54.9% in neutral lipids (NLs) and polar lipids (PLs), respectively) led us to hypothesize that this major PUFA was synthesized by an O(2)-independent Polyketide Synthase (PKS) pathway. Another parallel PKS, independent of the one leading to 22:6n-3, was also supposed to produce 20:5n-3. The inverse order of the (13)C atomic enrichment for n-3 PUFAs was also suspected to be related to the possible β-oxidation of long-chain n-3 PUFAs occurring during A. minutum encystment.