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

Abstract

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.

Keywords: 22:6n-3; Alexandrium minutum; DHA; Dinophyte; PKS pathway; PUFA synthesis; encystment.

Figure 1

Experimental design of the enrichment experiment. The ¹³CO₂ is supplied to the culture depending on its pH. To sample the algae, pliers are used to close/open the tubes/ways needed to first put the balloon under pressure and then allow sampling and finally rinse the tubes after sampling.

Figure 2

Mean cell concentration (mean ± SD) of the 2 enriched balloons of A. minutum (a) and the corresponding bacteria concentration during the 54 h of the experiment (b).

Figure 3

Particulate organic carbon concentration (a) and Total Fatty Acid (TFA) concentration (b). The blue line corresponds to a significant increasing trend between t₀ and t₅₄ (Alm1, Alm2, A. minutum cultures in balloons 1 and 2).

Figure 4

Atomic enrichment of the dissolved inorganic carbon (DIC) (a). Atomic enrichment of particulate organic carbon (POC) (b) (Alm1, Alm2, A. minutum cultures in balloons 1 and 2).

Figure 5

Proportions of the 12 main fatty acids of A. minutum (mean ± SD of the two enriched balloons over the studied time period between t₀ and t₅₄) (a) Mean proportion of neutral lipids (NL) and polar lipids (PL) during the experiment. (b) Variation in NL proportion with time for the two enriched balloons (c).

Figure 6

Atomic enrichment for the 11 main fatty acids (mean ± SD for the two enriched balloons). 14:0 was not considered as it was not involved in the studied fatty acids synthesis pathways to produce n-3 PUFAs. t₀ is not shown here because A. minutum was not enriched at that time and t_2.5 was kept as the reference. The dashed line represents the DIC atomic enrichment (AE_DIC) at t₅₄.

Figure 7

Hypothesized polyketide synthase (PKS) pathways in A. minutum to synthesize 20:5n-3 and 22:6n-3. Numbers in boxes correspond to the final AE value (at t₅₄), white corresponds to neutral lipids and black to polar lipids. The PKS intermediates written in grey are not detected during the study, as the enzyme proteins might not release them until the final product is synthesized [19]. The circles represent the enzymes involved in these pathways: KS: 3-ketoacyl synthase; KR: 3-ketoacyl-ACP-reductase; DH: dehydrase; 2.2I/2.3I: 2-trans, 2-cis, or 2-trans, 3-cis isomerase; ER: enoyl-reductase. The numbers at the top of each pathways represent the suspected order of 20:5n-3 and 22:6n-3 synthesis according to the ¹³C-enrichment dynamics observed in this study.

Figure 8

Hypothesized pathways to produce C₁₈ FA in A. minutum. Numbers in boxes correspond to the final AE value (white for neutral lipids and black for polar lipids). The triangles symbolize the desaturases (front-end in yellow and methyl-end in purple), the circles the enzymes involved in the PKS pathway (KS: 3-ketoacyl synthase; KR: 3-ketoacyl-ACP-reductase; DH: dehydrase; 2.2I: 2-trans, 2-cis isomerase; 2.3I: 2-trans, 3-cis isomerase; ER: enoyl reductase). The ways with dashed arrows appear to be unlikely or cannot be proven with the enrichment dynamics. NL and PL written on the routes allowing the synthesis of 18:5n-3 indicate the fraction considered for each pathway.