Generation of transgenic P. tricornutum strains overexpressing candidate DGAT2s.
The native codon sequences for P. tricornutum DGAT2A, DGAT2B and a T. pseudonana DGAT2 (TpDGAT2), codon-optimized for expression in P. tricornutum, were cloned into the pPhOS2 vector  and the resulting constructs (Pt_DGAT2A, Pt_DGAT2B and Pt_TpDGAT2) were then transformed into P. tricornutum via biolistics (as described in ). Multiple zeocin-resistant colonies (>10) obtained for each construct were confirmed by PCR and then used to inoculate cultures for further screening by gas chromatography coupled to ion flame detection (GC-FID) analysis of fatty acid methyl esters (FAMEs). From this screening, we obtained multiple independent transgenic lines for each construct (Additional file 1: Table S1). The FA profiles and lipid content of selected independent overexpression lines were then analysed and compared to that of WT. As previously observed  the main FAs in total lipid extracts of WT and transgenic cells in decreasing order were C16:1, EPA, C16:0 and C14:0 (Fig. 1a). Transgenic lines overexpressing Pt_DGAT2B and Tp_DGAT2 demonstrated slightly elevated DHA levels.
To visualise the impact of transgene expression, a Nile Red fluorescence assay was used to evaluate the ability of the candidate DGAT2 overexpressing lines to accumulate enhanced levels of neutral lipids. Previously, analysis of transcript levels of the DGAT2 genes under replete growth conditions showed that the expression level of PtDGAT2A and PtDGAT2B progressively increased from day two to day four, peaked and then decreased to a low level in the following days . Hence, transgenic strains overexpressing DGAT2s were cultivated for three days in replete F/2 medium to the early exponential (E) phase (2.5x106 cells). Nile Red staining of neutral lipids showed substantial differences between different DGAT2 clones and WT (Fig. 1b). All PtDGAT2B expressing clones showed a marked increase of TAG, while Pt_DGAT2A and Tp_DGAT2 cells did not accumulate oil levels compared to WT cells.
To confirm gene transcription and elevated expression of DGAT2 genes, quantitative real-time PCR (qRT-PCR) was carried out on selected transgenic lines. Based on the results of Nile Red staining, one of the most promising transgenic lines overexpressing each of the DGAT2 isoforms were characterized. Transgenic cultures were grown in F/2 medium and RNA extracted at the early E phase. Subsequent qRT-PCR analysis confirmed expression of Pt_DGAT2A_1, Pt_DGAT2B_5 and Tp_DGAT2_6 transcripts in the selected transgenic strains and demonstrated that the Pt_DGAT2B transgene is expressed at higher levels than the other two transgenes, Tp_DGAT2 and Pt_DGAT2A (Fig. 1c). Although the genes were under the control of the same promoter, the relative expression levels for the three DGAT2 isoforms were significantly different (p < 0.001, for expression of both DGAT2A and of DGAT2B genes). For cell lines overexpressing native PtDGAT2A and PtDGAT2B, transcripts were increased relative to WT by 2.3 and 3.0-fold, respectively. Based on the combined analysis of FA profiles, Nile Red staining and qRT-PCR analysis, the clone with the highest lipid content, Pt_DGAT2B_5, now designated DGAT2B, was taken forward for further detailed analysis.
Generation of transgenic P. tricornutum strains co-expressing PtDGAT2B and Δ5-elongase from the picoalga O. tauri.
To evaluate the effect of PtDGAT2B overexpression on the incorporation of omega-3 LC-PUFAs in TAG, coding sequences for the O. tauri Δ5-elongase (OtElo5) and PtDGAT2B were cloned in the previously described pPhOS2 vector , generating DGATElo construct. Ten individual zeocin-resistant clones were confirmed by PCR and screened for FA content (Additional file 2: Table S2). Four of the independent clones containing the highest levels of the new fatty acid, docosapentaenoic acid (DPA, 22:5n-3), the product of elongating activity of Δ5-elongase, and DHA were selected for further analysis. FAMEs analysis of the selected transgenic strains confirmed the presence of DPA in the range of 1.8-4.6% accompanied by increased DHA levels (up to 3.8-8.5% compared to 1.8% in WT) in the stationary (S) phase of growth (Fig. 2a). Compared to WT, transgenic clones demonstrated substantially altered fatty acid profiles, containing on average 2-fold higher levels of 16:3 and 16:4, while the levels of C18:2 and C18:3n-3 were slightly reduced. The rise in 16:3 and 16:4 unsaturated FA is most likely due to the increase in substrate levels (C16:1) available for further desaturation by stroma desaturases. The lipid content of these clones was further screened by Nile Red assay (Fig. 2b). The clone with the highest level of DHA and enhanced neutral lipid content, DGATElo_8 (designated DGATElo) was taken forward for further analysis.
Cell growth and transgene expression: the impact of PtDGAT2B overexpression under N-replete and N-deplete conditions.
Nutrient deprivation has been shown to induce TAG accumulation in microalgae . To address the role of PtDGAT2B in TAG synthesis we studied cell growth under N-replete and N-deplete conditions during the most active phase of oil accumulation (72 hrs). N-replete F/2 media was supplemented with additional P and N according to Abida et al., to ensure that these elements were not limiting during cell growth . N-deplete media contained F/2 enriched with added P and no N. The growth rates and lipid accumulation of transgenic lines were determined under both N-replete and N-depleted conditions at 24-hour intervals for a total of 72 hours. The analysis of the growth profiles showed that there were no differences in growth patterns between the transgenic lines and the WT under both conditions. The rate of cell division under both N-replete and N-deplete conditions was both linear and consistent for transgenic and WT cells. (Fig. 3a, b). Whereas the overall level of cell division differed substantially between the two nitrogen conditions (p < 0.01), decreasing in the N-deprived cultures. To confirm transcription of both the OtElo5 and PtDGAT2B genes during the experiment, RNA was extracted from triplicate cultures at 72 h of growth after resuspension in either N-replete or N-free media. Following cDNA synthesis qRT-PCR confirmed that overexpression of PtDGAT2B and transcription of the OtElo5 gene were occurring under experimental conditions (Fig. 3c, d). We have previously described the successful expression of OtElo5 under the endogenous fcpA promoter of highly expressed fcpA gene . However, fcp promoters may not be strong enough to overexpress introduced activities due to the presence of light-responsive cis-regulatory elements  and stability under low nutrient conditions. Seo et al., showed that the endogenous elongation factor 2 (EF2) promoter isolated from P. tricornutum drove the expression of a transgene 1.2-fold stronger than that driven by the fcp promoter in light conditions and was stable throughout light and dark cycles . However, recently we have shown that the expression of the Δ5-elongase (OtElo5) gene with an EF2 promoter resulted in comparable levels of EPA and DHA to that in transgenic strains in which the OtElo5 gene was under control of the fcpA promoter . In this study we tested both promotors’ efficacies in response to Nitrogen treatment in transgenic lines expressing OtElo5 (Additional file 3: Table S3). The results indicate that the fcpA promoter is suitable for transgene expression of DGAT2B in P. tricornutum when grown in different N conditions.
Effect of PtDGAT2B overexpression on fatty acid composition under N- replete and N-deplete conditions.
To assess the impact of PtDGAT2B heterologous expression on cell FA profiles, cells were grown in N- replete and N-deplete conditions and analysed at 24, 48 and 72 h time points respectively. The fatty acid profiles of selected independent overexpression lines DGAT2B and DGATElo were then compared to that of WT and transgenic Pt_OtElo5. Fatty acid composition (Mol%) was clearly affected by N treatment, for example DGAT2B cells accumulated slightly higher levels of DHA in comparison to WT in N-replete conditions (Fig. 4). Irrespective of N treatment, the highest DHA levels were observed in DGATElo cells. As expected, cells expressing OtElo5 (Pt_OtElo5 and DGATElo) contained decreased EPA compared to that of DGAT2B and WT at all time points due to the action of the Δ5-elongase (Fig. 4). The novel FA DPA was only present in the strains expressing OtElo5 (Pt_OtElo5 and DGATElo).
When total FAMEs were quantified (expressed as nmol mg-1), the main differences between strains were observed in the content of 16:0 and 16:1 (Additional file 4: Figure S1). As expected, [28, 31], N deprivation was accompanied by significant changes in the levels of 16:0 and 16:1 (p <0.001) and time (p <0.001). The abundance of 16:0 and 16:1 was higher in N-starved cultures and increased over time (p=0.002 and p =0.019 respectively) in all strains. Higher levels of 16:0 and 16:1 were observed in cells overexpressing PtDGAT2B (p=0.003 and p < 0.01 respectively), where the content of 16:0 and 16:1 in N-limited conditions at 72 h reached 442 nmol.mg-1 and 443 nmol.mg-1 respectively, corresponding to a 2- and 1.5-fold increase in comparison to the WT. An increase in the levels of C16 under N-limitation has been reported previously [28, 31] and may be due to the substrate specificity of DGAT2 genes responding to N deprivation. In this study we demonstrate that the protein encoded by PtDGAT2B has preference for C16 acyl groups. Overall, the accumulation of LC-PUFA (EPA, DPA and DHA) was largely unaffected either by N-depletion or time. However, in all experimental conditions, transgenic cells accumulated higher levels of DHA than WT, with most efficient accumulation observed in DGATElo. Interestingly, after 72 hours in N-depleted medium, DGATElo cells accumulated 2.8- fold higher levels of EPA and 2-fold higher levels of DPA than the single Pt_OtElo5 strain (73 nmol.mg-1 and 26 nmol.mg-1 respectively). These results indicate that PtDGAT2B plays an important role in FA accumulation under N-replete and N-deplete conditions. Although PtDGAT2B has a strong substrate preference for C16 FAs, it also demonstrates the broad substrate specificity of this acyltransferase, particularly towards LC-PUFAs, such as EPA, DPA and DHA.
Lipidomic analysis of WT and transgenic cells under N-replete and N-deplete conditions
To gain insight into the ability of PtDGAT2B to enhance TAG accumulation we performed a comprehensive analysis comparing glycerolipid profiles at different time points for WT and transgenic strains grown in N-replete and N-deprived conditions. Mass spectrometry (ESI-MS/MS) based lipidomic approaches have been used previously in our laboratory to characterise P. tricornutum lipid turnover and remodelling. The reliability of the analysis was demonstrated by the principle component analysis (PCA), which showed clear clustering of the strain and treatment replicates (Fig. 5). Cells were harvested for analysis at 24, 48 and 72 hours, corresponding to E and S phases of growth. Furthermore, the sampling period captured the impact of transgene expression and the application of nitrogen stress, which induces a significant reorganisation of cellular lipid metabolism. A multivariate statistical approach (PCA) was used to decrypt the significant differences observed in such a comprehensive analysis. The first two principal components account for 48.69% of variation within the data and are shown in Figure 5. Clear patterns are visible in this 2-dimensional representation of the data, with the first principal component separating out N- and N+ treatments and within the N- treatments distinguishing between the different time effects. There is separation of the data set over time under N-deplete conditions, but not N-replete, demonstrating the accumulation and remodelling of lipids in response to N deprivation. The second principal component shows separation of the different transgenic lines, with Pt_OtElo5 and DGATElo clustering together and DGAT2B and WT separating into a second group.
The relative contribution each lipid has on the direction of each principle component was determined. Additional file 5: Table S4 lists the 15 lipids with highest loadings associated with these dimensions. Under N-deplete conditions, the first principal component, a high TAG and low polar lipid response is clear. Specific TAG species with high abundance are 48:2 and 48:3 presumptively 16:0, 16:1- containing TAGs and the EPA- containing TAG 50:3. Other lipid species contributing to this dimension are the galactolipids mono- and digalactosyldiacylglycerol (MGDG and DGDG). The second principal component can be interpreted as an average of selected lipids, namely phosphatidylcholines (PC), Lyso-PCs (LPC) and diacylglycerylhydroxymethyltrimethyl-β-alanine (DGTA), thus high values of principle component two (associated with WT and DGAT strains) correspond to high abundance in these lipids. Compared to WT and DGAT2B, cells expressing the OtElo5 gene contain lipids (LPC, PC and DGTA) abundant in DPA and DHA. Several important observations can be made from the PCA analysis. When considering the full set of lipid species, cells overexpressing the PtDGAT2B gene cluster with WT although the lipid content is altered (Fig. 6, Additional file 4: Figure S1 and Additional File 6: Figure S2). Introduction of the OtElo5 gene separates out cell lines expressing it under N-replete and N-deplete conditions. Cells expressing the OtElo5 gene and overexpressing PtDGAT2B, individually or in combination, cluster together. These differences between the cell lines are driven by lipids containing DPA and DHA, e.g. DGTA 44:12 among others. As expected, the PCA plots show that the biggest impact on distribution is N stress and that this is driven by changes in abundance of TAG. Other lipid species contribute to changes in lipid profile, but they are largely minor components. These data suggest that in response to N stress, de novo biosynthesis of TAG primarily utilizing the ER-localized Kennedy pathway, rather than remodelling of membrane lipids, is the major driver in TAG accumulation. The importance of the Kennedy pathway in TAG biosynthesis in stress-induced diatoms has been reported before [2, 3]. This study gives a clear indication of the involvement of one of the major contributors to this pathway, PtDGAT2B, in TAG biosynthesis under N-replete conditions.
Impact of transgene expression on TAG accumulation under N-replete and N-deplete conditions
First, we examined the impact of PtDGAT2B overexpression on lipid accumulation in transgenic cells. Both the DGAT2B and DGATElo cells contained elevated TAG compared to WT and Pt_OtElo5 cells irrespective of N conditions at all-time points (Fig. 6 and Additional file 7: Table S5). The results demonstrated that N deprivation is not required for enhanced TAG accumulation in the PtDGAT2B overexpressing strains. Under N-replete conditions at 72 h the DGAT2B cells accumulated 22 nmol.mg-1 DW of TAG, which corresponded to a 3.5-fold increase in comparison to the WT. These results indicated that PtDGAT2B overexpression markedly improved lipid productivity in cells of P. tricornutum. Since N deprivation stimulates lipid production in various microalgae species, P. tricornutum WT and transgenic lines were grown and analysed in N-replete and N-deplete conditions. N depletion induced TAG accumulation in all cell types and resulted in substantially higher TAG than under replete conditions. We observed a ~ 2-fold increase in TAGs in comparison to the control in the DGATElo cells (with the highest level of 313.7 nmol.mg-1) and a 1.4-fold increase in DGAT2B and Pt_Elo5 cells (240 nmol.mg-1 and 243 nmol.mg-1 respectively) after 72 h.
To further examine lipid production in P. tricornutum cells, TAG accumulation in lipid bodies of the WT and transgenic cells was assessed qualitatively by BODIPY 505/515 staining and confocal microscopy (Additional file 6: Figure S2). Under N-replete conditions oil bodies in WT and Pt_OtElo5 cells remain almost unchanged over the time course, whereas PtDGAT2B overexpressing cells contained larger oil bodies at each time point. Neutral lipid content in all cell types increased considerably in N-starved cultures compared to N-replete conditions. DGAT2B and DGATElo cells contained larger and more numerous oil bodies relatively to WT and Pt_OtElo5 over the time. The increase and volume of oil bodies in P. tricornutum cells expressing PtDGAT2B and/or OtElo5 was consistent with the increase in neutral lipid content measured by mass spectrometry. To detect the impact of transgene expression on FA assembly in TAG further detailed analysis by ESI-MS/MS was undertaken. FA content in TAG showed that along with the different levels of TAG accumulation between strains, there were also differences in TAG FA profiles and molecular species composition under N-replete and N-deplete conditions. DGATElo contained a significantly higher proportion of DHA in TAG than other strains under both conditions (p=0.0003) (Fig 7 a, b). However, DHA levels were slightly decreased in all cell types under N starvation in agreement with previous observations that LC-PUFAs levels decrease in N-depleted medium .
Also, in agreement with previous reports [28, 31] the most abundant TAG species under standard conditions in P. tricornutum cells were C16 containing species, 48:1, 48:2 and 48:3. A similar pattern was observed in all transgenic cells. However, the levels of C16 FA-containing species and those consisting of a mix of C16-C18 FA (50:2 and 50:3) in DGAT2B cells increased 3-8 fold compared to WT, thus demonstrating enhanced channelling of C16 FA into TAG in the presence of PtDGAT2B. Elevated 54:6, 54:7 and 54:8 corresponding to DHA/DPA-containing TAG species, were detected in DGATElo cells (Additional file 8: Figure S3). The levels of TAG species containing C16 FA were dramatically changed with nitrogen treatment. Under N-deplete conditions levels of 48:1, 48:2 and 48:3 TAG were substantially increased in all cell types. DGAT2B and DGATElo cells contained elevated levels of 50:2 and 50:3 TAG. DHA-containing TAG species showed differing responses to N depletion. Notably, DHA TAG 54:7 increased up to 64% in DGATElo, whereas 54:8 and 54:6 increased up to 39% and 8.7% respectively. Minor new DHA-containing species, e.g. 56:8, 56:9, 56:10 and 56:11, were mainly observed in cells grown in N-deplete media (Additional file 8: Figure S3). The data collectively demonstrated that the broad substrate specificity associated with PtDGAT2B facilitated a pathway for DHA synthesis and incorporation into TAG as lipid droplets. Moreover, the combined expression of PtDGAT2B and OtElo5 increases lipid production without altering cellular growth.
Polar lipid accumulation in WT and transgenic cells under N-replete and N-deplete conditions
To better understand the impact of transgene expression on the lipidome of DGAT2B overexpressing cells and determine if the substantial changes in TAG composition under N stress are a result principally of de novo biosynthesis of TAG, as indicated by multivariate analysis, or by lipid remodelling, the polar lipid content of WT and transgenic cells was examined using ESI-MS/MS approaches. The structural reorganisation of chloroplast membranes in response to N depletion and recycling of membrane lipids into TAG has been reported in many microalgae strains. Nitrogen depletion is often accompanied by the movement of LC-PUFA from MGDG and DGDG to TAG. Assessment of the polar lipid classes illustrates a profile that was similar to that of the P. tricornutum Pt1 ecotype reported by Abida et al.,  and dominated by MGDG, PC, phosphatidylglycerol (PG) and DGDG under both N conditions (Additional file 9: Figure S4). The primary impact of N depletion on polar lipids in all strains was a reduction in all classes. Reviewing the individual polar lipid molecular species under N-replete conditions (Additional file 10; Figure S5), the most abundant species in MGDG of all cell types is 36:8, likely comprising of 20:5 and 16:3. Molecular species found in MGDG of DGAT2B cells show similar profiles to that of WT. Transgenic clones expressing the OtElo5 gene, have distinct MGDG profiles from that of WT and DGAT2B strains and are characterized by enhanced levels of 32:5 and 32:6 (a mix of 16:2 and 16:3) and lower levels of 36:8, correlating with a decrease of EPA due to OtElo5 activity in these transformants and consequently, reduced import of EPA into the chloroplast via a putative omega pathway  (Additional file 10: Figure S5). Under N-deplete conditions there was an overall reduction in EPA-containing 36:6, 36:8, 36:9 MGDG species in all cell types correlating with a general decrease in EPA content. MGDG 32:5 and 32:6 were also reduced considerably under N depletion. An assessment of other -glycolipid species showed differing responses in both DGDG and sulfoquinovosyldiacylglycerol (SQDG) (Additional file 10: Figure S5). Consistent with previous observations , 36:7 and 36:6 species were present at high levels in WT and are likely to contain 20:5 and 16:2/16:1. All transgenic clones contain molecular species similar to that of WT indicating that transgene expression did not have a significant impact on DGDG and SQDG. The profile of SQDG in all cells was dominated by 30:1 (14:0/16:1) and 32:1 (16:1/16:0) under N-replete conditions and showed minor decrease under N-deprived conditions. The main EPA-containing species of SQDG, 36.5 (20:5 and 16:0), were present only in WT and DGAT2B cells, correlating with reduced amounts of EPA in OtElo5-expressing clones. In contrast to Popko et al.,  a lower proportion of a betaine glycerolipid has been observed (Additional file 11: Figure S6). This may be due to the different culture growth conditions, e.g. media or light, or analytical methodology used in both experiments.
Under N-replete conditions the dominant phosphatidylcholine (PC) species in WT and DGAT2B were EPA-containing 36:5, 36:6, 38:7 and 40:10 (Additional file 12; Figure 7 a & b). The proportions of these species remained mostly unchanged under both N conditions. The PC pool of OtElo5 expressing strains in N-replete medium contained highly elevated levels of species incorporating newly-synthesized DPA and DHA. The major species in these transformants were those, consisting of the mix of LC-PUFAs and C16 FA: 38:5 (likely 22:5 and 16:0), 38:6 and 38:7 (a mix of 22:6 and C16:0/C16:1), and those with a mix of EPA, DPA and DHA, such as 40:7; 42:9, 42:10 and 42:11. Four new PC molecular species 42:8, 44:10, 44:11 and 44:12 likely containing DHA and DPA were detected in transgenic clones expressing OtElo5. An increase in DHA- and DPA-containing species suggests that the synthesis of these LC-PUFAs takes place in the PC pool. Species containing EPA (36:5 and 40:10) in OtElo5 expressers were markedly reduced, likely corresponding to changes in EPA levels. Interestingly, under N-limited conditions, levels of DHA containing species, 42:10 and 42:11, increased in DGATElo cells.
Analysis of PG showed a consistent predominance of C16-consisting 32:1 and 32:2 species, and EPA-containing 36:5 and 36:6 species in all the transgenic clones and WT under both N-conditions. Pt_OtElo5 cells contained higher 32:1 in PG compared to WT cells grown in replete and N-deprived medium, the significance of this is not clear. The measured amount of PG reduced in all cell types under N-deplete conditions (Additional file 12; Figure 7c & d). Notably, phosphatidylinositol (PI) of all cell types comprised mainly 16:1/16:0 and was stable with N treatment, indicating that this lipid pool was not remodelled in response to N deprivation (Additional file 12; Figure 7 e & f). Phosphatidylethanolamine (PE) profiles were very different between strains grown under N-replete and N-deplete conditions. In N-replete medium PE of WT and DGAT2B cells contained mainly 40:10 (20:5 and 20:5) and 42:11 (20:5 and 22:6) molecular species. New 42:10 (20:5 and 22:5) and enhanced levels of 42:11 species were detected in Pt_OtElo5 and DGATElo transgenic cells (Additional file 12; Figure 7 g & h). The presence of these new species can be attributed to expression of the OtElo5 gene as they are absent in WT and DGAT2B cells. Jointly the data for PI and PE indicates that they do not contribute significantly to the biosynthesis of TAG in response to N deprivation.
To complete our analysis, ESI-MS/MS profiling was used to determine the FA composition of lyso-PC in WT and transgenic cells under the different N treatments (Fig. 8). The major FAs in WT and DGAT2B cells grown under both conditions were 18:2, EPA and DHA, whereas in Pt_OtElo5 and DGATElo transgenic cells FA profiles were dominated by DPA and DHA, the products of Δ5-elongation of EPA and subsequent Δ4-desaturation. In addition, two new FAs, 22:4 (the product of 20:4n-6 Δ5-elongation) and 24:6 (probably the product of 22:6 elongation) were observed in Pt_OtElo5 and DGATElo transformants. There was a 2-fold increase in DHA-containing lyso-PC in Pt_OtElo5 compared to WT and DGAT2B and a 5-fold increase in DGATElo in N-deplete conditions (Fig. 8b). The rise in DHA levels correlated with a reduction of 16:0, 18:1, 18:2, 18:3 and EPA in the OtElo5 expressing strains. The levels of DHA-containing lyso-PC were 2-fold higher in DGATElo compared to that of OtElo5 cells. Overexpression of the DGAT2B gene on its own has only small impact on the DHA levels in N-replete cultures. These data indicate a crucial role lyso-PC in LC-PUFA synthesis and incorporation into complex lipid species.