Fatty Acid Biosynthetic Manipulation in Microalgae Figure 5. Thioesterase activity assay. Activity of plant and algal thioesterases and porcine pancreas type II lipase were determined by monitoring the hydrolysis of para-nitrophenylhexanoate for 16 hours at 30uC. (A) pH 7, TEs expressed in E. coli; (B) pH 8, TEs expressed in E. coli; (C) pH 7, TEs expressed in C. reinhardtii; (D) pH 8, TEs expressed in C. reinhardtii. doi:10.1371/journal.pone.0042949.g005 protein-protein interactions, the acyl chain attached to the ACP is important in guiding the thioester moiety towards the TE active site cysteine residue. Our modeling studies reveal a shallow hydrophobic binding cleft close to the CrTE active site (Fig. 2C). Perhaps there is a synergistic effect at work, whereby the acyl chain helps orient the fatty acid substrate in the correct conformation for hydrolysis to occur. To test whether the length of the acyl chain attached to the Cr- cACP factors into enzymatic hydrolysis by the CrTE, we synthesized a shorter chain derivative of 3 composed of only six carbons for activity-based crosslinking. Whereas C16 a-bromo- palmitic pantetheine probe 3 resulted in full conversion to an ACP-TE species, its six carbon analogue 6 yielded significantly less Cr-cACP/CrTE complex formation (Fig. 4C). These results indicate that in addition to ACP-TE interactions, chain length plays a role in the hydrolytic process. To further characterize the CrTE in vivo and test its specificity, transgenic C. reinhardtii overexpressing native CrTE was analyzed for fatty acid content and composition. Wildtype C. reinhardtii produces mostly 16:0 fatty acids and only negligible amounts of 14:0 and 18:1 [44]. The shift in fatty acid profile to increased levels of 14:0 by overexpressing CrTE is notable in that CrTE does not display high homology to plant FatB TEs, which have varied affinities for saturated fatty acyl-ACPs [10]. In fact, CrTE is more homologous to FatA TEs, which explains the slight increase in 18:1 (Fig. 6). The availability of acyl-ACP substrates for TE hydrolysis determines the fatty acid content observed. For example, when Arabidopsis thaliana FatB TE was expressed in E. coli, an organism which produces mostly 16:0, there was a significant increase in 14:0 [45]. Therefore, if CrTE has specificity for saturated acyl-ACPs, its overexpression most likely causes a stoichiometric imbalance of CrTE to Cr-cACP in the plastid, which results in premature hydrolysis of available acyl-ACPs and accumulation of shorter chain fatty acids. The observation that the pool of 16:0-ACPs was diminished in this strain compared to wildtype supports this hypothesis (Fig. 6). In plants, it is postulated that medium chain FatB TEs and FatA TEs are descendents of an ancient, ubiquitous FatB TE specific for 16:0-ACP [10]. A dedicated 16:0-acyl-ACP TE has not been detected in C. reinhardtii, but activity-based crosslinking studies with the 16:0-acyl-ACP probe indicate that CrTE can hydrolyze 16:0- ACP substrates. Crosslinking with the short-chain derivative (6) PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e42949