HAMID RISMANI-YAZDI, BERAT Z. HAZNEDAROGLU, CAROL HSIN, and JORDAN PECCIA

12.1 BACKGROUND

Important advantages of microalgae-based biofuels over first generation biofuels include algae’s greater solar energy conversion efficiency com­pared to land plants [1], the ability of oleaginous microalgae to utilize non-arable land and saline or waste-water, and their high content of energy dense neutral lipids that can be readily transesterified to produce biodiesel [2,3]. Under stress conditions such as nutrient deprivation or high light intensity, several species of oleaginous microalgae can alter lipid biosyn­thetic pathways to produce intracellular total lipid contents between 30 to 60% of dry cell weight (DCW) [4]. Triacylglycerides (TAGs) are the dominant form of lipids produced under these conditions. The excess pro­duction of TAGs in microalgae is thought to play a role in carbon and energy storage and functions as part of the cell’s stress response [5].

Due to the limited understanding of microalgae genetics and physiol­ogy, lipid metabolism from higher plants and bacteria have been the basis from which the accumulation of TAGs in microalgae has been modeled [5]. TAGs and polar membrane lipids are synthesized from fatty acids, that are primarily produced in the chloroplast [6]. The committed step in

fatty acid biosynthesis starts with the conversion of acetyl CoA to malo — nyl CoA through the enzyme acetyl CoA carboxylase (ACCase). In some plants, there is evidence that both photosynthesis — and glycolysis-derived pyruvate could be endogenous sources of acetyl CoA pool for fatty acid biosynthesis [5]. Fatty acid production in E. coli is regulated through feedback-inhibition by long chain fatty acyl carrier proteins (ACP) [7,8], and a recent study in the microalgae Phaeodactylum tricornutum demon­strated that overexpression of genes that encode for the thioesterases that hydrolyze the thioester bond of long chain fatty acyl ACPs resulted in a significant increase in fatty acid production [9]. Recent nitrogen depriva­tion studies in the model, nonoleaginous microalga Chlamydomonas rein — hardtii have also suggested an important role for lipases in restructuring the cell membrane under nitrogen limitation in order to supply fatty acids for TAG biosynthesis [10].

The stress-induced production of TAGs provides an opportunity to observe differential gene expression between high and low TAG accu­mulating phenotypes. Because multiple pathways are associated with the enhanced production of neutral lipids in microalgae, transcriptomic studies are an appropriate tool to provide an initial, broad view of carbon partitioning [11] and regulation of TAG biosynthesis during microalgae stress responses. However, the most promising strains thus far identified by growth experiments and lipid content screening [4,12] do not have se­quenced, fully annotated genomes [13-15]. In microalgae, transcriptomic studies have instead focuses on model organisms that are not oleaginous but have sequenced genomes [10,16]. There is a growing number of ole­aginous microalgae from which de novo transcriptomes have been assem­bled and annotated but comprehensive quantitative gene expression analy­sis in these microalgae has not yet been performed [14,17-19]. Recently, a de novo assembled-transcriptome was used as a search model to enable a proteomic analysis of the oleaginous microalga Chlorella vulgaris that demonstrated up-regulation of fatty acid and TAG biosynthetic pathways in response to nitrogen limitations [13].

In the present study, we quantitatively analyzed the transcriptome of the oleaginous microalga Neochloris oleoabundans to elucidate the met­abolic pathway interactions and regulatory mechanisms involved in the accumulation of TAG. N. oleoabundans (a taxonomic synonym of Ettlia oleoabundans[20]) is a unicellular green microalga belonging to the Chlo — rophyta phylum (class Chlorophyceae). It is known to produce large quan­tities of lipids (35 to 55% dry cell weight total lipids and greater than 10% TAGs) [4,12,21] in response to physiological stresses caused by nitrogen deprivation. To produce differences in lipid enrichment, N. oleoabundans was cultured under nitrogen replete and nitrogen limited conditions and major biomolecules including total lipids, TAGs, starch, protein, and chlo­rophyll were measured. The transcriptome was sequenced and assembled de novo, gene expression was quantified, and comparative analysis of genes, pathways and broader gene ontology categories was conducted. The results provide new insight into the regulation of lipid metabolism in oleaginous microalgae at the transcriptomic level, and suggest several potential strategies to improve lipid production in microalgae based on a rational genetic engineering approach.

12.2 RESULTS