Fatty Acid Biosynthetic Manipulation in Microalgae Figure 6. Fatty acid analysis of C. reinhardtii strains expressing thioesterases. Fatty acid composition of Cr strains was determined by GC/MS analysis and comparison to authentic standards. Peak areas were integrated and compared to an external standard for quantification. Bar graphs denote abundances of (A) Myristic acid (14:0), (B) Palmitic acid (16:0), and (C) Oleic acid (18:1), and labels on the Y-axis correspond to the percentages of these fatty acids of the total fatty acid content. (D) Full GC/MS chromatograms of Cr strains expressing CrTE (red), UcTE (Blue) and wildtype CrTE (black). Three separate cultures of each strain were analyzed for fatty acid content and composition, and data were recorded and averaged with a mean deviation of 7% in each experiment. Statistical analyses were performed using SPSS (v13.0), and for all data analysis, a p-value,0.5 was considered statistically significant. doi:10.1371/journal.pone.0042949.g006 shows that CrTE is less selective for shorter chain acyl-ACPs, but it is still able to process them. In vivo characterization illustrates that CrTE can act upon 14:0-ACPs and 16:0-ACPs, as well as 18:1- ACPs, suggesting promiscuity in hydrolytic activity by CrTE. Docking fatty acid substrates of varied chain lengths and degrees of unsaturation provides further evidence that CrTE can hydrolyze both saturated and 18:1 fatty acids (Fig. S3). We propose that CrTE is a unique TE with both FatA and FatB character. Algal TEs remain largely uncharacterized, with the exception of a putative thioesterase (PtTE) from the diatom Phaeodactylum tricornutum [46]. PtTE is distinct from FatA and FatB TEs as well as prokaryotic TEs. However, overexpression of PtTE in E. coli increases the amount of monounsaturated fatty acids and total fatty acid content, similar to FatA TEs [46]. Upon sequencing of the C. reinhardtii genome [18], CrTE was initially annotated as a FatA TE based on homology to plant TEs. Yet, sequence alignment shows only dispersed regions of similarity with FatA TEs (Figs. 1, S1). Currently in the UniProt database, CrTE is designated a ‘Fat1’ TE [47]. Phylogenetic analysis reveals that CrTE exists in a separate clade from FatA and FatB TEs along with other algal TEs (Fig. S13). Perhaps the CrTE sits at the crossroads of thioesterase evolution, where it functioned as one of the first TEs to hydrolyze unsaturated fatty acids. This is the first evidence of a unique Fat1 TE with promiscuity and possibly high hydrolytic activity for saturated acyl-ACP substrates in algae. PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e42949