Protein-protein interactions between the TE and ACP control the identity of the fatty acid end product, as illuminated by activity-based crosslinking. Here, we demonstrate for the first time that activity-based chemical probes can be used to trap FAS enzymes and study their interactions with endogenous biosynthetic machinery in algal chloroplasts. We have verified that in vitro activity-based crosslinking translates to expected phenotypes in vivo, as shown by engineering TEs into the Cr chloroplast and analyzing fatty acid content. Characterization of algal fatty acid biosynthetic enzymes has unveiled distinct features compared to established bacterial and plant type II FAS systems, exemplified by a unique TE detected in C. reinhardtii. Our results demonstrate that synergy between protein-protein interactions and substrate recog- nition mediates the hydrolysis of fatty acids by CrTE. This work offers a new fundamental understanding of algal fatty acid synthase to guide future metabolic engineering. It is the first demonstration of protein-protein interactions used as a mecha- nism to control product identity. We successfully designed and implemented activity-based probes as a means to understand the molecular recognition between the algal ACP and algal/plant TEs. In addition, we interrogated the specificity of the TE using three different activity-based probes. This novel approach may be used to facilitate the engineering of fatty acid identity in algae and other organisms of interest for energy production. Materials and Methods Protein modeling and docking For detailed methods we refer to the supporting information (Text S1). Proteins were modeled using Swissmodel [48] and docked using the Cluspro server [34]. The top 10 balanced models were manually examined using PyMol [49] and the distances between TE active site cysteine and ACP serine residues measured. Autodock was used to blindly dock fatty acid substrates onto the model of CrTE [50] (Figs. S2, S3, S4). Sequence alignment Sequence-based alignments of ACPs and TEs were produced using TCoffee [51], and figures were created using ESPript [52]. Transit peptides were predicted using ChloroP1.1 [53] and TargetP1.1 [54]. Full TE and ACP sequence alignments are provided in Figs. S1 and S6. Protein expression and purification Cr-mACP, Cr-cACP, CrTE, UcTE, and ChTE proteins were expressed in E. coli and purified according to standard protocols and TEs reduced prior to use. ACP hydrolase (ACPH) was used to form apo-Cr-cACP (Figs. 3A and S7), confirmed by MS analysis (Fig. S8). Thioesterase activity was confirmed kinetically by monitoring hydrolysis of para-nitrophenylhexanoate (Fig. 5) [40]. Activity-based crosslinking as a determinant of functional binding Crosslinking reactions contained phosphate buffer, pantetheine analogue (2, 3 or 6 in DMSO), MgCl2, ATP, ACP, CoA-A, CoA- D, CoA-E and Sfp [26]. After 1 h at 37uC, TE was added to the reaction mixture and further incubated at 37uC overnight. Anti- FLAG resin or Ni-NTA resin was added directly to the reaction mixtures at room temperature, centrifuged, washed, and proteins eluted with 1 M arginine (pH 3.5) or 100 mM imidazole. As a negative control, enzymes were incubated without pantetheine analogue. Fatty Acid Biosynthetic Manipulation in Microalgae Engineering TEs into C. reinhardtii chloroplast UcTE, ChTE, and CrTE genes were codon-optimized for the Cr chloroplast and cloned into plastid expression vectors (16). Microprojectile bombardment was used to transform C. reinhardtii with TE constructs [55]. PCR was used to screen for integration of TE genes into the chloroplast genome (Fig. S11) and expression of TEs was validated by Western blot analysis (Fig. S12) [42]. Plant and algal TE activity was confirmed kinetically by monitoring hydrolysis of para-nitrophenylhexanoate (Fig. 5) [40]. Growth of transgenic C. reinhardtii strains and GC/MS analysis of fatty acid content Chlamydomonas reinhardtii strains were grown on TAP media [56] agar plates supplemented with carbendazim, ampicillin and cefotaxime [57] under constant illumination. Transgenic strains were kept under kanamycin selection. Flasks containing TAP media were inoculated with single colonies and grown under constant shaking in a greenhouse. After 3 days, cultures were centrifuged and the cell pellet resuspended in methanolic acid. The suspension was incubated for 30 min at 65uC and fatty acid methyl esters extracted using hexanes. Fatty acid composition was determined by GC/MS analysis (Fig. 6). Statistical analyses were performed using SPSS (v13.0), and for all data analysis, a p- value,0.5 was considered statistically significant. Supporting Information Figure S1 Sequence alignment of FatA and FatB TEs with CrTE. Structure-based sequence alignment of FatA TEs from Arabidopsis thaliana (AtTE_A), Brassica napus (BnTE_A), Chlamydomonas reinhardtii TE (CrTE) and FatB TEs from Cuphea hookeriana (ChTE_B1) and (ChTE_B2) and Umbellularia californica (UcTE_B). Conserved residues are highlighted in red and similar residues appear in blue boxes. The end of transit peptide sequences are indicated by a black arrow. Cys-Asn-His catalytic triad is indicated by an asterix under each residue. Symbols are denoted as: a, a-helices; b, b-strands; g, 310 helices; TTT, strict a- turn; TT, strict b-turn. Sequence-based alignments were produced using TCoffee [S1]. The figure was created using ESPript [S2]. (TIF) Figure S2 Docking of fatty acid PPTs to CrTE. Autodock4 was used to blindly dock various fatty acid-PPTs to CrTE. (A) C16:0-PPT, (B) C16:4-PPT, (C) C18:0-PPT, (D) C18:1-PPT and (E) C18:3-PPT. Left: cartoon representation of CrTE (grey) and top 10 models of substrate as sticks. Right: surface representation of CrTE (grey) and top 10 models of substrate as sticks. [PPT: phosphopantetheine]. (TIFF) Figure S3 Ensemble representation of protein-protein docking of Cr-cACP with (A) UcTE, (B) ChTE and (C) CrTE. (TIF) Figure S4 Structural docking simulation of Cr-mACP to CrTE, UcTE, and ChTE. Docking of C. reinhardtii mitochon- drial ACP (Cr-mACP) to thioesterases showing less favorable docking characteristics (,50% of the ACP models docks to the tentative active site of the TEs). (A) UcTE (B) ChTE (C) CrTE. (TIF) Figure S5 Electrostatic potential maps of modeled Cr- cACP/CrTE and Cr-mACP/CrTE complexes. (A) Cr-cACP (green) docked to CrTE (grey) showing a 13.5 A˚ distance between conserved serine on Cr-cACP and active site cysteine of CrTE; (B) PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e42949