## 4 Changing the Cultivation Conditions for Optimizing the Production of Bioactive Compounds
### Compartmentalization of EPA and DHA Synthesis  
Concerning the galactolipid synthesis and the incorporation of EPA, both prokaryotic and eukaryotic types have been shown to be the main end products of biosynthesis [281]. The prokaryotic molecular species, C20 fatty acids such as EPA and ARA were mainly incorporated into the \(sn\)-1 position of the galactolipids, while C16 fatty acids were preferentially esterified into the \(sn\)-2 one. Concerning the eukaryotic species, both positions were occupied by the C20 fatty acids [281]. Independently, whatever the molecular species, the cytoplasmic and chloroplastic lipids were involved in the metabolism of galactolipids. Cytoplasmic linoleicyl-PC has been proposed to be converted into either arachidonyl-PC in the omega-6 pathway or into EPA-PC, which would be used to be converted into diacylglycerolglycerols (DAGs) moieties and then transferred to chloroplasts. In the chloroplasts, the DAGs have been proposed to be galactosylated to the respective monogalactosyldiacylglycerolglycerol (MGDG) molecular species. In the last steps, a chloroplastic delta-17 desaturation would be involved in the conversion of ARA-MGDG into galactolipids rich in EPA. Studies conducted with an inhibitor of the delta-6 desaturation involved in the chloroplastic conversion of linoleic acid into \(\alpha\)-linolenic  
Figure 2: Proposed lipid synthesis pathway in the marine microalgae. Role of chloroplast and endoplasmic reticulum compartments in lipid synthesis and location of omega-3 fatty acid incorporation [265,277–280]. ACP, acyl carrier protein; BL, betaine lipid; DES, desaturase; DAG, diacylglycerolglycerol; DAGAT, diacylglycerolcerol acyltransferase; DGDG, digalactosyldiacylglycerolglycerol; DHA, docosahexaenoic acid; ELO, elongase; EPA, eicosapentaenoic acid; FAS, fatty acid synthase; MGDG, monogalactosyldiacylglycerolglycerol; PL, phospholipid; TAG, triacylglycerolglycerol; \(\Delta\)-Cx, unsaturated fatty acid. acid and using radiolabelled carbon source (acetate) or fatty acid (linoleic acid) permit to elucidate the biosynthesis of EPA. These studies have confirmed the role played by the PC as substrate for the first step in the omega-3 pathway metabolism [282]. The PC were more involved in the desaturation of C18 fatty acids while some species such as PE and betaine lipid (diacylglycerollyceryl trimethylhomoserine DGTS) are preferential substrates for desaturation of C20 fatty acids involved in the EPA synthesis [283]. Moreover, these polar lipids were supposed to be imported to the chloroplastic compartment, from endoplasmic reticulum. The role of EPA and DGTS were suggested to be donors of C20-riched DAG, ARA and EPA, from which these fatty acids were incorporated into the galactolipid classes such as the MGDG [283]. In _Nannochloropsis_, the synthesis of EPA outside the chloroplast has been proposed by the use of radiolabelled acetate and by kinetic studies in a mutant strain. In these studies, an alteration of the fatty acid composition of lipids was observed, in relation with the EPA synthesis [284]. In the marine green alga _Chlorella minutissima_, the betaine lipid DGTS was characterized by a very high EPA content representing 90% of total fatty acids. By contrast with other algae, the EPA was incorporated into the both _sn_-1 and _sn_-2 positions of the DGTS. Moreover, a close relationship between the DGTS and the PC amounts was observed [285]. In _Chlorella minutissima_, the DGTS level showed a fluctuation which was inversely correlated with the level of MGDG, the other major lipid in this alga. Moreover, the fatty acid composition of the latter was very similar to that of DGTS and, taken together, these findings also support the proposed role of DGTS as a donor of EPA for chloroplast lipids in some microcalga species [285]. A mutant of _Porphyridium cruentum_, which was selected on the basis of impaired growth in function of temperatures, has been used to study lipid metabolism [286].