A 13CO2 Enrichment Experiment to Study the Synthesis Pathways of Polyunsaturated Fatty Acids of the Haptophyte Tisochrysis lutea.

ABSTRACT

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 205n-3 (EPA) and 226n-3 (DHA) in T. lutea. The fatty acids 160, 181n-9 + 183n-3, 182n-6, and 225n-6 were the most enriched in 13C. On the contrary, 184n-3 and 185n-3 were the least enriched in 13C after long chain polyunsaturated fatty acids such as 205n-3 or 225n-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 225n-6) but might also exist for n-3 PUFA (especially 205n-3). With regard to the conventional n-3 PUFA pathway, Δ6 desaturation of 183n-3 appeared to be the most limiting step for T. lutea, "stopping" at the synthesis of 184n-3 and 185n-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 205n-3 and 225n-3. 226n-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 225n-3, the alternative route of ω-3 desaturase using 225n-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.