Algal Lipid Bodies: Stress Induction,Purification, and Biochemical Characterization in Wild-Type and Starchless Chlamydomonas reinhardtii

When the unicellular green soil alga Chlamydomonas reinhardtii is deprived of nitrogen after entering stationary phase in liquid culture, the cells produce abundant cytoplasmic lipid bodies (LBs), as well as abundant starch, via a pathway that accompanies a regulated autophagy program. After 48 h of N starvation in the presence of acetate, the wild-type LB content has increased 15-fold. When starch biosynthesis is blocked in the sta6 mutant, the LB content increases 30-fold, demonstrating that genetic manipulation can enhance LB production. The use of cell wall-less strains permitted development of a rapid “popped-cell” microscopic assay to quantitate the LB content per cell and permitted gentle cell breakage and LB isolation. The highly purified LBs contain 90% triacylglycerol (TAG) and 10% free fatty acids (FFA). The fatty acids associated with the TAGs are ∼50% saturated (C₁₆ and C₁₈) fatty acids and ∼50% unsaturated fatty acids, half of which are in the form of oleic acid (C_18:1). The FFA are ∼50% C₁₆ and ∼50% C₁₈. The LB-derived TAG yield from a liter of sta6 cells at 10⁷ cells/ml after starvation for 48 h is calculated to approach 400 mg. The LB fraction also contains low levels of charged glycerolipids, with the same profile as whole-cell charged glycerolipids, that presumably form LB membranes; chloroplast-specific neutral glycerolipids (galactolipids) are absent. Very low levels of protein are also present, but all matrix-assisted laser desorption ionization-identified species are apparent contaminants. Nitrogen stress-induced LB production in C. reinhardtii has the hallmarks of a discrete pathway that should be amenable to additional genetic and culture condition manipulation.There is keen interest in the development of technologies that harvest lipids from microalgae and convert them into diesel fuel (7, 11, 24, 43). A widely adopted approach is to identify algal species that are ascertained to be oleaginous (e.g., Neochloris, Nannochloropsis, and Dunaliella species) and focus on extracting and processing their oils (43). We have adopted a complementary approach, which is to start with the unicellular green soil alga Chlamydomonas reinhardtii, widely regarded as nonoleaginous (48), and utilize the highly developed molecular and genetic tools available for this organism (22) to investigate how its oil production might be enhanced. Such information could then, in the future, be applied to any algae that may prove promising for industrial application.Algal oil production is commonly quantitated by extracting cells with organic solvents, characterizing the lipid fraction, and expressing the lipid yield as the percent dry weight of the starting material (24, 19, 43). Many cellular components (e.g., pigments, cell walls, and starch) contribute to dry weight, and these may change in quantity and/or composition during the cell cycle and/or during fluctuations in growth conditions for a given alga; they are also likely to differ among different algal lineages. Therefore, this convention provides an ambiguous metric for assessing how much oil a given cell produces when genetic background and/or culture conditions are manipulated and may yield misleading between-species comparisons.A related difficulty with this convention is that the extract is expected to contain lipids from numerous sources, e.g., plasma membranes, endomembranes, chloroplasts, and lipid bodies (LBs) dedicated to the storage of triacylglycerides (TAG) and fatty acids (FA). Although all of these sources are of potential value for fuel production, it is problematic to evaluate the output of a particular pathway when the outputs from multiple pathways are being pooled.We have therefore elected to focus on the biosynthesis of LBs (also referred to as lipid droplets or oil bodies), since this process has the hallmark of a discrete and inducible pathway amenable to genetic and culture condition manipulation (37), and we have developed a simple and direct microscopic assay for quantitating LB production per cell that is independent of dry weight.The assay was used to study two parameters. (i) Increases in extractable TAG in numerous algal species, including C. reinhardtii (65), when cells are starved for nitrogen (N) have been reported previously (3, 4, 25, 26, 33, 43, 44, 49, 53, 57, 59). We demonstrate that, in C. reinhardtii, this increase correlates with a robust increase in LB production. (ii) N starvation has also been shown to stimulate a pronounced increase in the biosynthesis of starch granules in C. reinhardtii (31, 35, 45), prompting us to ask whether LB production might be enhanced if starch production was eliminated. We report that the cw15 sta6 starch null strain (66) deleted in ADP glucose phosphorylase (an essential enzyme in the starch biosynthetic pathway) produces twice as much LB-associated TAG as its parental cw15 strain under nitrogen stress, suggesting that carbon skeletons normally converted into starch are instead able to flow into the starvation-induced TAG biosynthetic pathway when the starch option is unavailable.We have also developed a simple LB purification procedure that has permitted analysis of LB composition. The bodies contain ∼90% TAG, ∼10% free FA (FFA), and minor levels of charged glycerolipids (CGLs). Protein levels are also minor, and no proteins with the expected features of “dedicated” LB proteins, like the oleosins or caleosins of plant seeds, were identified.