While under nitrogen deprivations, there has been considerable uncertain­ty expressed whether the increase in TAG content is due to a reduction in the mass of the cell, rather than increase in TAG production [2]. Both the measured increase in TAG content per cell dry weight reported here (which accounted for the loss of cell mass during nitrogen limitation), and the observed changes in the FAME profile unequivocally demonstrate the overproduction and accumulation of TAG in N. oleoabundans under nitro­gen stress. Quantitative gene expression results also support these TAG production observations. In our study, most of the genes involved in the fatty acid biosynthetic pathway were up-regulated under — N conditions. The gene encoding for ACCase, the first enzyme in the pathway, was re­ported as down-regulated under — N. However, the biotin-containing sub­unit of ACCase, biotin carboxylase (BC), was significantly overexpressed. In photosynthetic organisms, two different forms of ACCase have been identified, one located in the plastid and the other located in the cytosol. The plastidal ACCase is a heteromeric multi-subunit enzyme that contains BC, whereas the cytosolic ACCase is a homomeric multifunctional protein that does not contain BC [27]. In our transcriptome analysis, we identified genes encoding for both forms of ACCase. In the plastid—the primary cite of lipid biosynthesis in microalgae—we have observed a significant increase in expression of the BC subunit of heteromeric isoform that cata­lyzes the very first step of carboxylation. On the other hand, the expression of homomeric ACCase, predominantly located in the cytosol where lipid biosynthesis does not typically occur, was repressed.

Although the overexpression of BC points to a key step in the pathway as a potential target to genetically engineer an improved oleaginous strain, mixed results for improving fatty acid synthesis in microalgae have been observed when ACCase is overexpressed [2]. Recent research has sug­gested that fatty acid synthesis may also be regulated by inhibition from the buildup of long chain fatty acyl ACPs [9]. Overexpressing genes that cleave ACP residues from the long chain fatty acyl ACPs is a condition observed in bacteria and recently in the microalga P. tricornutum to result in increased production of fatty acids [9]. In our study, genes encoding for these enzymes were highly overexpressed under the — N conditions. Therefore, a potential target for metabolic engineering in N. oleoabundans is the overexpression of thioesterases FatA and OAH that cleave off ACP residues.

Genes encoding enzymes involved in the steps downstream of fatty acid biosynthesis, including elongation and desaturation, have also displayed significant changes in transcription levels in response to nitrogen star­vation. In particular, the genes encoding AAD and delta-15 desaturase, which catalyze the formation of double bond between the 9th, 10th, 14th, and 15th carbon, respectively, were up-regulated under — N conditions. A similar observation has been reported by Morin et al. [37], where the gene encoding delta-9 fatty acid desaturase is up-regulated in the oleagi­nous yeast Y. lipolytica cultured under nitrogen limitation. As observed here, and supported by gene expression levels, nitrogen limitation alter the lipid profile towards higher saturation (increase in C18:1, and decrease in C18:2 and C18:3). The increased proportion of saturated fatty acids in TAG has been demonstrated to improve cetane number and stability of resulting biodiesel [38].

Based on the lipid metabolism genes discovered from our transcriptome assembly, the acyl-CoA dependent mechanism is the major contributor to TAG biosynthesis in N. oleoabundans. In our study, two genes associated with biosynthesis of TAG show significant changes in their expression under — N condition: one encoding GPAT and the other one encoding AGPAT. These enzymes catalyze the acyl-CoA-dependent acylation of positions 1 and 2 of glycerol-3-phosphate, respectively. The acylation of glycerol-3-phos­phate represents the first and committed step in glycerolipid biosynthesis, and likely the rate limiting step in the pathway as GPAT exhibits the lowest specific activity among all enzymes involved in the glycerol-3-phosphate pathway [39]. A recent proteomics study also reported significant up-regu­lation of TAG-related acyltransferases in parallel with accumulation of large quantities of lipid in C. vulgaris cultured under nitrogen limitation [13]. The overexpression of GPAT and AGPAT has been reported to increase seed oil accumulation in Arabidopsis and Brassica napus[40-42]. The up-regulation of these two genes also indicates an increase in the flow of acyl-CoA to­ward TAG biosynthesis. The final step of the TAG biosynthesis pathway is catalyzed by DGAT, the third acyltransferase. In our study, the gene encod­ing DGAT displays relatively no change in its expression under nitrogen limitation. This observation coupled with the significant increase in TAG production in the — N case, and previous proteomics studies that showed overexpression of DGAT in the C. vulgaris due to nitrogen limitation [13] provides evidence that DGAT expression in N. oleoabundans may be regulated post-transcriptionally. The post-transcriptional regulation of DGAT has pre­viously been documented in the oilseed rape Brassica napus [43].

Finally, the enrichment of intracellular starch increased during the — N case. Although starch synthase and AGPase encoding genes were repressed in — N, the gene encoding for a-amylase, responsible for the hydrolysis of starch to glucose monomers, was also repressed. The con­comitant accumulation of starch and lipids under nitrogen limitation has been reported in the nonoleaginous C. reinhardtii[44,45] and recently reported for N. oleoabundans[46]. This contrasts with recent reports in Micractinium pusillum where carbohydrate content was reduced and TAG production was increased under nitrogen limitation [19]. Genetic manipulations (sta6 mutant) that block starch synthesis in C. reinhardtii have resulted in a significant increase in TAG accumulation [47]. Under nitrogen limitation, the increased TAG content in N. oleoabundans and concomitant repression of starch synthase are analogous to the C. rein­hardtii sta6 mutant. These results extend the idea of blocking starch syn­thesis for improvement of TAG production to the oleagenous microalga N. oleoabundans.