Fatty Acid Biosynthetic Manipulation in Microalgae Figure 1. Thioesterase sequence alignments. Structure-based sequence alignment of FatA TEs from Arabidopsis thaliana (AtTE_A) and Brassica napus (BnTE_A), Chlamydomonas reinhardtii (CrTE), and FatB TEs from Cuphea hookeriana (ChTE_B1 and ChTE_B2) and Umbellularia californica (UcTE_B). The Cys-His-Asn catalytic triad is shown by an asterisk (*). Conserved residues are highlighted in red and similar residues in blue boxes. doi:10.1371/journal.pone.0042949.g001 between ACP and partner FAS enzymes [32,33]. Examining electrostatic potential maps of Cr-cACP/CrTE models shows electrostatic complementarity, and docking predicts a 13.5 A˚ distance between Cr-cACP conserved serine and CrTE active site cysteine residue (Fig. S5). Virtually screening a panel of fourteen vascular plant TEs for interaction with Cr-cACP using a high-end computational cluster collecting data over several months [34] for each TE, U. californica TE (UcTE) and C. hookeriana TE (ChTE) emerged as promising candidates based on their ability to dock in proximity to the Cr- cACP Ser43 (Figs. 2B, S3). Careful examination of Cr-cACP with UcTE and ChTE revealed that these ACP-TE models closely resemble the Cr-cACP/CrTE model, indicating a possible productive interaction between Cr-cACP and plant TEs. In contrast, modeling C. reinhardtii mitochondrial ACP (Cr-mACP) with these same plant TEs shows poor docking, suggesting that not all algal ACPs will successfully bind, and thus not catalytically interact, with plant TEs (Figs. S4, S5). Although the electrostatic potential map of Cr-mACP is similar to Cr-cACP, docking Cr- mACP to CrTE illustrates a different binding motif than that of Cr-cACP/CrTE, and as a result, the distance between Cr-mACP serine and CrTE cysteine is predicted to be 18.8 A˚ (Fig. S5). Characterization of fatty acid acyl carrier proteins of C. reinhardtii To study ACP-TE interactions in vitro, we obtained both fatty acid ACPs identified in the C. reinhardtii genome, ACP1 and ACP2. Based upon alignment with plant mitochondrial and chloroplastic ACPs, homology, and BLAST analysis, ACP1 and ACP2 correspond to the mitochondrial and chloroplastic ACPs, respec- tively (Fig. S6, Text S1). ACPs were synthesized in E. coli codon bias and cloned into pET28b expression vectors bearing an N- terminal-His6 tag (Table S1). Whereas Cr-mACP (ACP1) formed insoluble aggregates when expressed in E. coli and required refolding [35], Cr-cACP (ACP2) was expressed in E. coli in soluble form. Because we anticipated a stronger interaction between the ACP of the chloroplast and the CrTE, Cr-cACP was used as a positive control in crosslinking experiments (for details about Cr- mACP, we refer to Text S1). After Ni-NTA purification, a mixture of apo-Cr-cACP and holo-Cr-cACP (Fig. 3A) was obtained. The conversion of holo-Cr-cACP to apo-Cr-cACP was catalyzed by ACP-hydrolase (ACPH) from Pseudomonas aeruginosa [36], which removed the 49-phosphopantetheine moiety from Ser43 of Cr- cACP (Figs. 3A, S7). The formation of apo-Cr-cACP was Figure 2. Thioesterase modeling, docking of ACP-TE protein-protein interactions, and blind substrate docking of fatty acid substrate to C. reinhardtii TE. (A) Docking of CrTE (grey) with Cr-cACP (blue) showing a ,10 A˚ distance between Cr-cACP Ser43 (orange) and the active site Cys306His270Asn268 triad (magenta) of CrTE. (B) Docked complex of CrTE (grey) and ChTE (yellow) showing similar binding modes of Cr-cACP to both plant and algal thioesterases. (C) Surface representation of blind docking of stearyl-49-phosphopantetheine to CrTE showing the thioester bond of the substrate in close proximity to the TE active site and stearate in the binding tunnel of CrTE. doi:10.1371/journal.pone.0042949.g002 PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e42949