confirmed by mass spectrometry (Fig. S8). A one-pot chemoenzy- matic method was used to validate in vitro post-translational modification of apo-Cr-cACP [37]. As shown in Fig. 3A, incuba- tion of apo-Cr-cACP with Sfp, a surfactin synthetase-activating PPTase from B. subtilis [38], coenzyme A biosynthetic enzymes (CoA-A, CoA-D, and CoA-E), ATP, and fluorescent pantetheine analogue 1 resulted in the formation of fluorescently-labeled crypto- Cr-cACP. This conversion validated proper folding and activity of Cr-cACP and confirmed our ability to successfully load the activity-based probe onto ACP for further studies of protein- protein interactions [39]. Expression, purification, and activity of Cr, Uc, and Ch thioesterases CrTE, UcTE, and ChTE genes were synthesized in E. coli codon bias and subcloned into pET21a expression vectors bearing a C-terminal FLAG tag (Table S1). Expression in E. coli and FLAG affinity chromatography yielded pure TE protein. After reduction, plant and algal thioesterase activity was confirmed kinetically by monitoring hydrolysis of para-nitrophenylhexanoate [40]. In vitro crosslinking assay to screen TEs for functional binding to Cr-cACP and design of thioesterase substrate mimic Recently, we developed a crosslinking assay based on enzymatic activity that interrogates specific protein complex formation in FAS, PKS, and NRPS modular synthases using pantetheine probes [26]. Work in our laboratory has demonstrated that activity-based crosslinking serves as a quantitative measure of the strength of functional binding interactions between ACP and modular FAS domains [41]. In this assay, selectivity arises from protein-protein interactions, rather than purely protein-substrate specificity, as shown by examining biochemically unrelated carrier proteins modified with the same alkylating functionality [26]. This method is far more selective than traditional techniques aimed at interpreting protein-protein interactions, thus preventing false positive readings from non-specific protein complex formation. To investigate ACP-TE interactions, we sought to design a chemical probe that would selectively react with the active site of the TE. Chloroacrylic pantetheine probe 2 was used in previous studies to generate a site-specific crosslink between E. coli ACP and E. coli KS, triggered by protein-protein recognition [26]. Based on sequence alignment (Fig. 1), the CrTE uses an active site cysteine as its catalytic residue during fatty acid biosynthesis, similar to the KS domain. However, the TE is fundamentally different from the KS, as it only acts upon the final acyl-ACP substrate, whereas the KS acts on intermediate chain lengths. When a mature fatty acid is assembled on the ACP, the TE first catalyzes acyl transfer to its active site cysteine residue (Fig. 3C). The product is then hydrolyzed as water attacks the pendant thioester to yield a free fatty acid. Modeling studies of CrTE illustrate a shallow hydrophobic tunnel at the surface of the enzyme, where the acyl chain presumably lies (Fig. 2C). Previously, a panel of pantetheine probes was used to examine the compatibility of ACP-KS partners, which established that substrate recognition also plays a critical role in catalysis [25]. Hypothesizing that the fatty acyl chain factors into enzymatic hydrolysis by the TE, we designed an activity-based substrate mimic containing a reactive bromide a to the site of nucleophilic attack by the TE and a long aliphatic tail (Fig. 3D). To test the substrate specificity and selectivity of the CrTE using activity-based probes, we designed a shorter a- bromohexyl pantetheine analogue composed of only six carbons Fatty Acid Biosynthetic Manipulation in Microalgae (6). Chemical synthesis of a-bromopalmitic pantetheine probe 3 and its shorter analogue 6 (Text S1) enabled us to study ACP-TE interactions using three different activity-based probes (2, 3, and 6). Manipulating the CoA biosynthetic pathway [39], functiona- lized CoA analogues were formed in vitro from chloroacrylamide 2 or a-bromopalmitamide 3 (Fig. 3B). Sfp [38] was used to attach substrate mimics to Ser43 of apo-Cr-cACP, generating reactive crypto-Cr-cACPs 4 and 5 (Fig. 3B) [37]. A crosslinked complex was observed between crypto-Cr-cACP and CrTE using both probes, indicating a positive interaction between the Cr-cACP and CrTE (Fig. 4B). Importantly, incubation of a-bromopalmitic crypto-Cr- cACP 5 with CrTE resulted in full conversion of CrTE to a Cr- cACP/CrTE complex, indicating a strong functional interaction between the two proteins (Fig. 4B). In contrast, reacting chloroacrylic crypto-Cr-cACP 4 with CrTE did not result in a high yield of ACP/TE protein complex formation (Fig. 4B). To study the effect of the length of the acyl chain attached to Cr-cACP on functional binding to CrTE, a shorter a-bromohexyl pantetheine probe 6 (Text S1) was used for activity-based crosslinking. In agreement with its predicted natural activity, a higher quantitative yield of crosslinked complex was detected between CrTE and longer chain a-bromopalmitic-ACP (Fig. 4C), indicating that chain length factors into enzymatic hydrolysis by the TE. Based on the results of our initial computational docking screen of plant TEs, UcTE and ChTE were tested for their ability to crosslink with crypto-Cr-cACP. Contrary to modeling studies, no crosslink was detected between heterologous plant TEs and Cr- cACP due to an absence of protein-protein recognition (Fig. 4D). Crypto-Cr-cACPs formed from either chloroacrylic pantetheine probe 2, a-bromopalmitic pantetheine probe 3, or a-bromohexyl pantetheine probe 6 did not result in a crosslinked complex, indicating vascular plant TEs did not functionally interact with Cr- cACP in vitro (Fig. S9). Interestingly, C. reinhardtii mitochondrial ACP (Cr-mACP) did not orient favorably when docked onto CrTE, UcTE or ChTE (Figs. S4, S5). Indeed, a crosslinked complex was not detected between Cr-mACP and the CrTE of the chloroplast, demonstrat- ing selective recognition between ACP and TE domains (Fig. S10). Engineering thioesterases into Cr chloroplast and fatty acid analysis of transgenic strains To determine whether plant TEs functionally interact with the Cr-cACP in vivo and to validate our in vitro activity-based crosslinking results, CrTE, UcTE, and ChTE were engineered into the C. reinhardtii chloroplast (Tables S2, S3). A high level of protein expression was achieved by codon-optimizing the plant TE sequences for the C. reinhardtii chloroplast and using endogenous promoters and RNA elements to drive expression [21]. Micro- projectile bombardment transformed C. reinhardtii strain 137c (mt+) with thioesterase-harboring plastid expression vectors [21]. PCR confirmed integration of the TE genes and homoplasmicity (Fig. S11) (Table S3) and Western blot analysis validated expression of the TEs in the C. reinhardtii chloroplast (Fig. S12) [42]. After purification from Cr lysate and reduction, plant and algal thioesterase activity was validated kinetically by monitoring hydrolysis of para-nitrophenylhexanoate (Fig. 5) [40]. Transgenic algal strains were grown in a greenhouse to late log phase, harvested, and fatty acids converted into their correspond- ing methyl esters for GC/MS analysis (Text S1). Compared to wildtype, no significant change in fatty acid content or compo- sition was detected in transgenic C. reinhardtii strains expressing the UcTE or ChTE (Fig. 6). This was an indication that plant TEs did PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e42949