**1. Introduction**

Long chain polyunsaturated fatty acids (LC-PUFA) such as 20:5n-3 (EPA) and 22:6n-3 (DHA) are important compounds for most marine metazoans for their growth, reproduction, and development. They are not able to synthetize them in sufficient quantities and thus have to acquire them from their diet. On the basis of the food web, protists are the

**Citation:** Remize, M.; Planchon, F.; Garnier, M.; Loh, A.N.; Le Grand, F.; Bideau, A.; Lambert, C.; Corvaisier, R.; Volety, A.; Soudant, P. A 13CO2 Enrichment Experiment to Study the Synthesis Pathways of Polyunsaturated Fatty Acids of the Haptophyte *Tisochrysis lutea*. *Mar. Drugs* **2022**, *20*, 22. https://doi.org/ 10.3390/md20010022

Academic Editor: Ipek Kurtboke

Received: 2 November 2021 Accepted: 14 December 2021 Published: 24 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

main producers of these fatty acids and present a key role in marine ecosystem functioning. 20:5n-3 and 22:6n-3 are also particularly important in human nutrition. They are known to have beneficial effects on cardiovascular diseases or diabetes. However, due to high demand for human nutrition and aquaculture of carnivore species, a shortage of these two compounds found in fish oil is predicted to occur by 2050 [1]. Despite their economic and ecologic interests, biological and ecological processes responsible for their synthesis are still under investigation. It is, then, of first concern to understand how 20:5n-3 and 22:6n-3 are produced at the basis of the food webs, and how global changes could affect their availability at higher trophic levels.

In phytoplankton and microzooplankton, fatty acids are synthetized via different metabolic pathways [2–4]. The most "conventional" pathway is the fatty acid synthase (FAS) pathway, followed by the elongation and front-end desaturation steps of the n-3 and n-6 pathways. Starting with the initial formation of acetyl-CoA and then malonyl-CoA in aerobic conditions, these pathways produce more complex fatty acids by progressive addition of two atoms of carbon (elongation steps) or desaturations of precursors such as 16:0 or 18:0 [5–7]. These two pathways can be connected by the so-called ω-3 desaturase (or methyl end desaturase) pathway. Within the n-3 and n-6 pathways, an alternative route of Δ8 desaturation can also bypass the Δ6 desaturation step and has already been identified in Haptophyte [8]. These routes allowed the synthesis of 20:5n-3 as well as 22:6n-3 (Figure 1).

**Figure 1.** Microalgae fatty acid synthesis pathways. Desaturases are noted with "ΔX" (yellow arrows) and "ωY-des (ΔX)" (blue arrows), where X refers to the location of carbon holding the newly formed double bond from the front end (or carboxyl end) and Y its position from the methyl end. Elo: elongase, FAS: fatty acid synthase.

An alternative O2 independent pathway, called the polyketide synthase (PKS) pathway, is responsible of long chain PUFA synthesis such as 20:5n-3 and 22:6n-3 [3,9,10]. It has been found in bacteria and protists such as thraustochytrids, dinophytes, and haptophytes [11–15]. The PKS pathway relies on the same four basic enzymatic reactions (condensation, reduction, dehydration, and reduction) as the FAS pathway. Opposed to the conventional pathway, the PKS pathway is less energy consuming, because it requires fewer reduction and dehydration steps than "conventional" pathways [3]. The metabolites used to form the carbon chain are simultaneously desaturated and elongated, creating long-chain PUFA [3,16,17].

Even if some microalgae species share all or part of O2-dependent n-3 and n-6 pathways and O2-independent PKS pathways, PUFA composition of primary producers varies greatly according to species. Diatoms synthetize more 20:5n-3 as well as C16 PUFA, while

dinophytes or haptophytes contain more 22:6n-3 or C18 PUFA. Other groups such as cyanobacteria or some chlorophytes classes are unable to build 20:5n-3 or 22:6n-3 or only in very low proportions (<1%) [18–21].

To improve knowledge of the synthesis routes and production of 20:5n-3 and 22:6n-3, studies have focused on identifying genes coding for the different elongase and desaturase [10,22–25]. Moreover, in recent years, the use of 13C substrate allowed monitoring the incorporation of labelled substrates into targeted organic macromolecules. Different metabolic intermediates or end products such as fatty acids [26–29] are monitored and quantified. This has already been applied to *E. coli* [30], yeast [29], and microalgae [31–35]. The development of technics such as gas chromatography coupled to mass spectrometry (GC-c-IRMS) consists of a noticeable improvement in the direct resolution of isotopic composition of organic macromolecules the so-called compounds specific isotope analysis (CSIA) including fatty acids [36–39].

The present study aims at investigating the synthesis pathways of essential PUFA 20:5n-3 and 22:6n-3 using stable isotope (13C) labelling experiment of the haptophyte *Tisochrysis lutea*. *T. lutea* is intensively used in aquaculture (hatchery) and industry [40]. The incorporation of 13C was monitored in 11 FA during 24 h at a high temporal resolution (each 0.5 to 2 h). Progressive accretion of the 13C-labelled CO2 into FA (from precursors to PUFA of interest) allowed us to constrain FAS, elongase/desaturase, and PKS involvement in 20:5n-3 and 22:6n-3 production by *Tisochrysis lutea*. In parallel to the monitoring of 13C incorporation into FA, growth, physiological status, and other cellular parameters (morphology, viability, esterase activity, and lipid content) were monitored by flow cytometry analysis.
