**5. Conclusions**

The synthesis of long-chain PUFA in *Tisochrysis lutea* appeared to involve multiple pathways (Figures 8–10). First, the assumption of the use of PKS pathway for 22:5n-6 (DPA-6) was attested regarding the fast enrichment observed for this FA as well as the absence of detectable intermediates more or equally enriched. PKS pathway appeared to

be particularly efficient in *T. lutea* and induced a strong incorporation of the 13C-marker. However, the possibility of use of the conventional n-6 PUFA pathway should not be excluded, as 18:2n-6 presented a similar level of enrichment as 22:5n-6. It would only endorse that following desaturation and elongation steps to form 22:5n-6 were particularly dynamic and thus did not allow the accumulation of the 13C-label into n-6 intermediates. Within n-3 PUFA pathway, the Δ6-desaturase route seemed slower than the n-6 pathway in *T. lutea* in producing the two C18 polyunsaturated fatty acids 18:4n-3 and 18:5n-3. We assumed 18:4n-3 and 18:5n-3 were unlikely synthesis intermediates of 20:5n-3 and 22:6n-3, as their enrichments were lower than the latter. Although 22:6n-3 was present in higher proportion than 22:5n-6, it was not enriched as fast, possibly because its synthesis may be more complex. Indeed, 22:6n-3 could be synthesized by *Tisochrysis lutea* via a combination multiples pathway: from 22:5n-6 via ω-3 desaturase pathway, from desaturation and elongation of 20:5n-3 and 22:5n-3, and via PKS pathway. Further studies are needed to better constrain the plausible routes taken by this prymnesiophyte to produce long chain PUFA.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/md20010022/s1, Table S1: Cellular parameters of *Tisochrysis lutea* (morphology (FSC and SSC), viability (FL1-SYTOX), and chlorophyll content (FL3) using flow cytometry analysis (mean ± SD of the 3 balloons). Values for SYTOX are in%, values for FL3/SSC/FSC in arbitrary unit (a.u), Table S2: Concentrations in <sup>μ</sup>g·L−<sup>1</sup> of all identified and quantified FA in neutral and polar lipid fractions according to sampling time, Table S3: Concentrations in <sup>μ</sup>molC·L−<sup>1</sup> of all identified and quantified FA in neutral and polar lipid fractions according to sampling time, Table S4: Concentrations in% of all identified and quantified FA in neutral and polar lipid fractions according to sampling time. Table S5: Mean ratio of atomic enrichment (AE) for pairs of FA (FAA vs. FAB) in the neutral lipids (NL) (mean ± SD, *n* = 9 sampling dates t8 to t24) for the two enriched balloons (Tl1, Tl2, Tl = *Tisochrysis lutea*). If the ratio is equal to or close to 1, A and B are assumed at equilibrium, and B is synthesized quickly from A; if the ratio is below 1, the transformation of B from A is possible but slow. Finally, if the ratio is above 1, A is not a main precursor of B, which has to be synthesized by a different pathway. Table S6: List of the potential candidate protein sequences involved in *Tisochrysis lutea* PKS synthesis pathway. The suspected function of each protein has been assumed using the NCBI conserved domain database (CDD) (Marchler-Bauer et al., 2017) by identifying the role of each domain recognized in the sequence. In columns KS/KR/DH/ER are written the number of domain corresponding to these functions in the studied sequences. ACS: acetyl-CoA synthetase, A\_NRPS: adenylation domain of the non-ribosomal peptide synthetase (NRPS), Croto: crotonase/enoyl-CoA hydratase, EntF: enterobactin non-ribosomal peptide synthetase or thioesterase domain of Type I PKS, FAAL: fatty acyl-AMP ligase, GrsT: alpha/beta hydrolase, HM: hydroxymethylglutaryl-CoA synthase, MT: methyltransferase, PP: phosphopantetheine-binding (="swinging arm"), Sulf: sulfotransferase, Thio: thioesterase. Figure S1: Proportions (%) of NL vs. PL (A) and proportions (%) of fifteen fatty acids in the NL fraction in average over the 24 h (B). Figure S2: Atomic enrichment of 11 main fatty acids in the polar lipid (NL) fraction during a 24h 13C labelling experiment. Tl: *Tisochrysis lutea*. Reference [68] is cited in the supplementary materials.

**Author Contributions:** Conceptualization, M.R., F.P. and P.S.; data curation, M.R.; Formal analysis, F.P.; funding acquisition, M.R., A.N.L., A.V. and P.S.; investigation, M.R., F.P., F.L.G., C.L., M.G. and P.S.; methodology, M.R., F.P., A.N.L., F.L.G., A.B., M.G. and P.S.; project administration, M.R. and P.S.; resources, R.C.; software, M.R., F.L.G. and A.B.; supervision, M.R., F.P., A.N.L. and P.S.; validation, M.R. and C.L.; visualization, M.R.; writing—original draft, M.R.; writing—review and editing, M.R., F.P., A.N.L., F.L.G., A.B., C.L., M.G, R.C., A.V. and P.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Université de Bretagne Occidentale (UBO) and the Center for Marine Sciences (CMS) at the University of North Carolina Wilmington (UNCW), the Interdisciplinary School for the Blue Planet (ISblue), and the Walter-Zellidja grant of the Académie Française.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** We would like to thank Philippe Miner, Nelly Le Goic, Margaux Mathieu-Resuge, Elodie Fleury, Adeline Bidault, Korydwen Terrasson, and Corentin Baudet for their help during sampling and Oanez Lebeau for her support during the isotopic analyses.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
