Despite the considerable research into LPAAT and DGAT enzymes [15, 20–24], the role of GPAT enzymes in lipid metabolism of Nannochloropsis is understudied. Here we have shown that GPAT overexpression can improve growth-associated NL accumulation in N. oceanica. NL content was increased in overexpression mutants only under replete conditions, and not after exposure to N-depletion. This suggests, that GPAT availability or regulation can be rate-limiting for NL synthesis during exponential growth, but not after exposure to nutrient stress. This is in accordance with the transcriptional upregulation of endogenous ER-localized GPAT in N. oceanica during N-depletion [14], which may increase GPAT availability or activity to levels that allow increased carbon flux towards TAGs. Our findings are in line with previous findings, showing increased TAG accumulation in GPAT-overexpressing transformants of other microalgal species [32, 37–41]. Our results show that growth-associated lipid accumulation in N. oceanica can be enhanced by expression of a single rate-limiting enzyme. Accordingly, lipid production processes in continuous operation might become feasible by optimizing this microalga through metabolic engineering. Continuous operation circumvents the negative impacts of nutrient starvation on biomass and lipid productivities, and can therefore be desirable compared to 2-step processes [42, 43].
Interestingly, overexpression of the endogenous GPAT NO03G04130 and heterologous expression of a GPAT gene from A. obliquus had similar effects on NL contents and productivities of transformants in our experiments (Tab. S1), suggesting that heterologous enzymes might be able to functionally complement or substitute endogenous counterparts in Nannochloropsis. This opens up avenues for sophisticated synthetic biology strategies in this organism, in order to manufacture microalgal strains with FA and lipid metabolism geared towards production of “designer lipids”. The differences that were observed between NoGPAT and AoGPAT mutants in terms of FA profile, photosynthetic performance, and PL content during N-depletion, may be related to differences in enzyme characteristics such as membrane integration, substrate preference, and kinetic parameters, or to different expression levels.
It was previously shown that overexpression of endogenous GPAT genes increased PUFA contents in the diatom Phaeodactylum tricornutum [37, 39]. Similarly, overexpression of NoGPAT in this study shifted the FA composition especially of neutral lipids to higher fractions of different long chain (LC) and very long chain PUFAs (VLC-PUFAs, Fig. 5). VLC-PUFAs such as C20:5 (EPA) are considered high-value compounds that are associated with health benefits in humans, and they are an essential component of aquaculture feed [4, 44, 45]. LC-PUFAs such as C18:3 further have potential for treatment and prevention of inflammatory disorders, cardiovascular disorders, cancer, and diabetes [46–48]. Therefore, the increased PUFA content of NoGPAT-M1 and M2 is intriguing (Tab. S1). The substantially increased fraction of C18:2, C18:3, C20:4 and C20:5 in NLs of these mutants suggests that increased NoGPAT activity stimulates PUFA synthesis. In eukaryotes, PUFAs are synthesized in the ER by enzymatic action of elongases and FA desaturases (FADs) that use different glycerolipid species as substrates [49]. In Nannochloropsis, an ER-localized pool of the glycerophospholipid phosphatidylcholine (PC) is likely the carrier for desaturation reactions of C18, whereas phosphatidylethanolamine (PE) and/or the betaine lipid diacylglyceryltrimethylhomoserine (DGTS) were suggested as carriers for C20 desaturation reactions [50,51]. In a model proposed by Han and colleagues [50], PUFA synthesis in N. oceanica begins with the desaturation of C18:1\({}^{\varDelta 9}\) bound to the sn-2 position of PC by action of \(\varDelta 12\)-FAD, producing C18:2\({}^{\varDelta \text{9,12}}\), which is subsequently desaturated by \(\varDelta 6\)-FAD, yielding C18:3\({}^{\varDelta \text{6,9},12}\). C18:3 is released from PC by phospholipase PLA2, and the free FA is activated to C18:3-CoA by LACS. \(\varDelta 6\)-FA elongase catalyzes the elongation of C18:3-CoA to C20:3-CoA, which is incorporated into DAG via the Kennedy pathway. DAG is a branching point for de novo synthesis of glycerolipids like PC, PE, DGTS and for TAG. The central role of DAG illustrates the significance of GPAT in lipid and PUFA metabolism, as GPAT initiates the de novo synthesis of DAG. An increased level of DAG synthesis through action of GPAT might increase synthesis of other ER-located glycerolipids such as PC and PE, and thereby the availability of substrate for FAD enzymes. In this context, a previous study has shown that PUFA synthesis in N. oceanica is not limited by abundance of \(\varDelta 12\)-FAD, which catalyzes the desaturation of PC-bound C18:1 to C18:2, exemplified by similar FA compositions of \(\varDelta 12\)-FAD overexpression transformants and the wild type under N-replete conditions [52]. This suggests that C18:1 desaturation could be increased either by improving the allocation of \(\varDelta 12\)-FA substrate (PC-bound C18:1) or by removal of its product C18:2. Accordingly, increased C18 desaturation in NoGPAT-M1 and M2 may be the result of either increased synthesis of PC-bound C18:1, or due to improved removal of C18:2/C18:3 from the PC pool. Therefore, further studies should focus on quantification of different lipid classes, analysis of the glycerolipid-specific FA composition and their stereochemical distribution, to elucidate which lipid classes are enriched for PUFAs in NoGPAT-M1 and M2. The increased abundance of PUFAs in NoGPAT-M1 and M2 may further be due to a preference of NoGPAT for these FA species, which could be investigated by heterologous expression studies in yeast, or by in vitro assays, although in vitro enzyme specificity does not necessarily reflect in vivo FA compositions [53]. Notably, C18:2, C18:3 and C20:4 were increased in both, NLs and PLs of NoGPAT-M1 and M2, but the fraction of C20:5 per TLs was unchanged (Fig. 5a, Tab. S1). Similarly, a recent study by Poliner and colleagues [54] has shown that simultaneous overexpression of \(\varDelta 5\), \(\varDelta 9\) and \(\varDelta 12\)-FAD in N. oceanica increased fractions of C18:2 and C20:4 in TLs by 125% and 73%, respectively, whereas C20:5 was increased by only 25% compared to the wild type. The authors hypothesize that this may be connected to a biological limit of the C20:5 fraction in PLs, which cannot be exceeded without compromising membrane functionality. Assuming this model, sequestration of C20:5 into NLs of NoGPAT-M1 and M2 may serve like a valve that prevents excessive incorporation of VLC-PUFAs into chloroplast membrane lipids.
It should further be noted that a growing body of evidence suggests a role of Kennedy pathway intermediates in intracellular signaling cascades [55,56]. Consequently, NoGPAT overexpression may results in differential expression of other FA or lipid metabolism-related genes, similarly to what was recently reported for a GPAT and LPAAT overexpression transformant of the diatom Phaeodactylum tricornutum [57]. Transcriptomic analyses of NoGPAT-M1 and M2 may help to unravel the mechanism behind the altered PUFA synthesis in these transformants.
AoGPAT-M1 and M2 showed significantly increased photosynthetic efficiency compared to all other strains (Fig. 3c). This was accompanied by increases in NL contents (Fig. 4a) and average cell size (Fig. 3d), whereas PL contents were decreased (Fig. 4b). Future studies should investigate a possible connection between the decreased PL content and the increased photosynthetic efficiency and NL contents of AoGPAT mutants. These kind of studies may include quantification of PL classes and detailed characterization of photosynthetic parameters. In this context, a recent study has shown that a Nannochloropsis gaditana mutant that was likely impaired in synthesis of the main photosynthetic PLs MGDG and DGDG, displayed an increased proton motif force across the thylakoid membrane and an increased TL content under N-replete, but not N-depleted conditions [58].