Systems Biology Analysis of Chlorella vulgaris Figure 5. Multiple sequence alignment for acetyl-coA acyltransferase (ACAT) peptides identified via MS/MS analysis. Despite high sequence identity (Expect values,1e-124), ACAT was not identified using Chlorophyta sequence databases as search models in MS/MS analysis. Notably, underlined peptides differed by just a single amino acid substitution between C. vulgaris and C. variabilis, preventing positive identification. Using the C. vulgaris transcriptome as a sequence search database yielded 7 peptide identifications at a confidence interval .95%. doi:10.1371/journal.pone.0025851.g005 lated 1.6-fold. Similar to ACCase, MAT abundance was upregulated, approximately 1.4-fold, under nitrogen-deplete conditions. Enzymes catalyzing condensation reactions were also upregulated, with the exception of the approximately 2-fold down- regulation of KAR, the enzyme catalyzing the conversion of 3- ketoacyl-ACP to 3-hydroxyacyl-ACP. A protein identified via sequence alignment as a putative (non-specific) fatty acid thioesterase, proposed to terminate fatty acid elongation, displayed no change in abundance [1]. Although the absolute spectral counts for the components of the TAG biosynthetic pathway were generally lower than those of the fatty acid biosynthetic pathway (especially in the replete samples) the change in protein abundance was far more pronounced, one to two orders of magnitude greater than those observed in the fatty acid biosynthetic pathway. The corresponding p-values for observed changes for all components of the FA and TAG biosynthetic pathways were less than 0.05 (indicating ,5% chance that the difference is random). The largest increase in abundance was observed for DGAT, with greater than 100-fold spectral count increase. DAGK showed no statistically significant change in abundance in the nitrogen- deplete state, implying that acylation of LPA is the preferred path to PA synthesis under these conditions. Discussion Chlorella vulgaris UTEX395 as a commercially relevant algal strain Selection of suitable algal strains will be a critical step in realizing the full potential of commercial-scale photosynthetic algal cultivation for biofuel and bioproduct production. An ideal production strain will have the attributes of fast growth at acceptable cell density, cultivation robustness, and high lipid- accumulation capacity and productivity [12]. Genetic engineering, though perhaps problematic from a regulatory or community acceptance standpoint, can, at a minimum, help establish upper limits to lipid productivity and guide classical genetics or breeding programs. In addition, the glycerolipid profile of a strain will have a large impact upon the lipid’s potential as a biofuel feedstock, as chain length and degree of unsaturation are critical for conversion to both biodiesel and renewable diesel and jet fuel. For example, a C18:3 fatty acid requires 7 moles of hydrogen per mole of fatty acid ester for full saturation, while C18:0 requires only 4 moles of hydrogen. As such, the glycerolipid profile of a microalgal species can ultimately have a significant impact on production costs (Robert McCormick, NREL, personal communication). The green alga, C. reinhardtii, has been extensively examined as a model organism due to its ease of cultivation in a laboratory setting and its ability to be genetically manipulated. As such, investigation of C. reinhardtii has perhaps contributed more to the elucidation of the fundamental underpinnings of microalgal biology than any other microalga to date. However, this species lacks the intrinsic high-lipid productivity of many oleaginous microalgae. For example, under nitrogen-deplete conditions, wild-type C. reinhardtii starchless mutant only produces ,20% lipid on a dry cell weight basis [12]. Conversely, the fatty acid profile, production, and accumulation capacity of C. vulgaris under nitrogen-deplete conditions suggests it presents an ideal feedstock for biodiesel production. Nitrogen limitation dramat- ically increases the desirable C18:1 fatty acid content at the expense of less desirable C18:3, C18:2, C16:1 and C16:0 content (Figure 1c). As such, nutrient-limiting conditions result in the accumulation of an oil having improved properties as a feedstock PLoS ONE | www.plosone.org 9 October 2011 | Volume 6 | Issue 10 | e25851