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The production of polyunsaturated fatty acids (PUFA) in Tisochrysis lutea was studied using the gradual incorporation of a 13C-enriched isotopic marker, 13CO2, for 24 h during the exponential growth of the algae. The 13C enrichment of eleven fatty acids was followed to understand the synthetic pathways the most likely to form the essential polyunsaturated fatty acids 20:5n-3 (EPA) and 22:6n-3 (DHA) in T. lutea. The fatty acids 16:0, 18:1n-9 + 18:3n-3, 18:2n-6, and 22:5n-6 were the most enriched in 13C. On the contrary, 18:4n-3 and 18:5n-3 were the least enriched in 13C after long chain polyunsaturated fatty acids such as 20:5n-3 or 22:5n-3. The algae appeared to use different routes in parallel to form its polyunsaturated fatty acids. The use of the PKS pathway was hypothesized for polyunsaturated fatty acids with n-6 configuration (such as 22:5n-6) but might also exist for n-3 PUFA (especially 20:5n-3). With regard to the conventional n-3 PUFA pathway, Δ6 desaturation of 18:3n-3 appeared to be the most limiting step for T. lutea, “stopping” at the synthesis of 18:4n-3 and 18:5n-3. These two fatty acids were hypothesized to not undergo any further reaction of elongation and desaturation after being formed and were therefore considered “end-products”. To circumvent this limiting synthetic route, Tisochrysis lutea seemed to have developed an alternative route via Δ8 desaturation to produce longer chain fatty acids such as 20:5n-3 and 22:5n-3. 22:6n-3 presented a lower enrichment and appeared to be produced by a combination of different pathways: the conventional n-3 PUFA pathway by desaturation of 22:5n-3, the alternative route of ω-3 desaturase using 22:5n-6 as precursor, and possibly the PKS pathway. In this study, PKS synthesis looked particularly effective for producing long chain polyunsaturated fatty acids. The rate of enrichment of these compounds hypothetically synthesized by PKS is remarkably fast, making undetectable the 13C incorporation into their precursors. Finally, we identified a protein cluster gathering PKS sequences of proteins that are hypothesized allowing n-3 PUFA synthesis.

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 (13CO2) 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 t30. A. minutum rapidly incorporated 13C into 22:6n-3, which ended up being the most 13C-enriched polyunsaturated fatty acid (PUFA) in this experiment, with a higher 13C atomic enrichment than 18:4n-3, 18:5n-3, 20:5n-3, and 22:5n-3. Overall, the 13C atomic enrichment (AE) was inversely proportional to number of carbons in n-3 PUFA. C18 PUFAs, 18:4n-3, and 18:5n-3, were indeed among the least 13C-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 C20-C22 PUFA, because the AEs of the n-3 C18 PUFAs were lower than those of the n-3 C20-C22 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 O2-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 13C 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.

The present study sought to characterize the synthesis pathways producing the essential polyunsaturated fatty acid (PUFA) 20:5n-3 (EPA). For this, the incorporation of 13C was experimentally monitored into 10 fatty acids (FA) during the growth of the diatom Chaetoceros muelleri for 24 h. Chaetoceros muelleri preferentially and quickly incorporated 13C into C18 PUFAs such as 18:2n-6 and 18:3n-6 as well as 16:0 and 16:1n-7, which were thus highly 13C-enriched. During the experiment, 20:5n-3 and 16:3n-4 were among the least-enriched fatty acids. The calculation of the enrichment percentage ratio of a fatty acid B over its suspected precursor A allowed us to suggest that the diatom produced 20:5n-3 (EPA) by a combination between the n-3 (via 18:4n-3) and n-6 (via 18:3n-6 and 20:4n-6) synthesis pathways as well as the alternative ω-3 desaturase pathway (via 20:4n-6). In addition, as FA from polar lipids were generally more enriched in 13C than FA from neutral lipids, particularly for 18:1n-9, 18:2n-6 and 18:3n-6, the existence of acyl-editing mechanisms and connectivity between polar and neutral lipid fatty acid pools were also hypothesized. Because 16:3n-4 and 20:5n-3 presented the same concentration and enrichment dynamics, a structural and metabolic link was proposed between these two PUFAs in C. muelleri.