In our study, several genes encoding enzymes involved in the intracel­lular breakdown of fatty acids and lipids are significantly repressed under — N (Table 3). Repressing p-oxidation is a clear strategy for maintaining a higher concentration of fatty acids within a cell. In contrast, most of the identified lipases (with the exception of triacylglycerol lipases) are overexpressed during nitrogen limitation. Upon closer examination, the up-regulated lipases are mostly phospholipases associated with hydrolyz­ing cell wall glycerophospholipids and phospholipids into free fatty acids, potentially for incorporation into TAGs. A known result of nitrogen limi­tation induced autophagy in C. reinhardtii is the degradation of the chlo — roplast phospholipid membrane [47,48]. Moreover, the overexpression of lipases during nitrogen limitation in C. reinhardtii has previously been hypothesized to be associated with the reconstruction of cell membranes [10]. In addition to phospholipases, we have identified an enriched num­ber of transcripts for phospholipid metabolic processes and lipid transport in the — N case (Figure 4B). The up-regulation of genes encoding for enzymes that produce free fatty acids is also consistent with the fact that the PDAT enzyme associated with the acyl-CoA-independent mechanism of TAG synthesis (which utilizes phospholipids, rather than free fatty ac­ids, as acyl donors) was not recovered in our assembled transcriptome.

12.3 CONCLUSIONS

Assembling the transcriptome and quantifying gene expression responses of Neochloris oleoabundans under nitrogen replete and nitrogen limited conditions enabled the exploration of a broad diversity of genes and path­ways, many of which comprise the metabolic responses associated with lipid production and carbon partitioning. The high coverage of genes en­coding for full central metabolic pathways demonstrates the completeness of the transcriptome assembly and the repeatability of gene expression data. Furthermore, the concordance of metabolite measurements and ob­served physiological responses with gene expression results lends strength to the quality of the assembly and our quantitative assessment. Our find­ings point to several molecular mechanisms that potentially drive the over­production of TAG during nitrogen limitation. These include up-regula­tion of fatty acid and TAG biosynthesis associated genes, shuttling excess acetyl CoA to lipid production through the pyruvate dehydrogenase com­plex, the role of autophagy and lipases for supplying an additional pool of fatty acids for TAG synthesis, and up-regulation of the pentose phosphate pathway to produce NADPH to power lipid biosynthesis. These identified gene sequences and measured metabolic responses during excess TAG production can be leveraged in future metabolic engineering studies to im­prove TAG content and character in microalgae and ultimately contribute to the production of a sustainable liquid fuel.

12.4 METHODS