Fatty Acid Biosynthetic Manipulation in Microalgae Figure 3. Schematic of activity-based crosslinking between CrACP and TEs. (A) Apo-CrACP is formed by treating holo-CrACP with ACP hydrolase from P. aeruginosa [36], removing the pantetheine moiety from the conserved serine of CrACP. Presence of apo-CrACP is confirmed using a one-pot fluorescent labeling method [37], detected by visualization of a resulting SDS-PAGE gel at 365 nm. (+) formation of fluorescent crypto-CrACP; (2) control reaction in which fluorescent pantetheine analogue 1 was omitted. (B) Activity-based crosslinking scheme. Apo-CrACP is incubated with 2 or 3, Sfp, ATP, CoA-A, CoA-D, and CoA-E to generate the corresponding crypto-CrACPs 4 and 5. Upon incubation of crypto-CrACP with TE, protein- protein interactions trigger a site-specific covalent crosslinking reaction with the chloroacrylamide in 4 or the a-bromoamide in 5, forming an ACP-TE crosslinked complex. (C) Predicted enzymatic mechanism of the hydrolytic release of a fatty acid by CrTE using a Cys-Asn-His catalytic triad. (D) Mechanism of irreversible crosslink between TE and crypto-CrACP containing a reactive bromide on the carbon a to the site of nucleophilic attack by the TE. doi:10.1371/journal.pone.0042949.g003 not functionally interact with Cr-cACP in vivo and thus were unable to hydrolyze fatty acids attached to Cr-cACP. Interestingly, the strain overexpressing native CrTE resulted in a short circuiting of fatty acid chain elongation to increase myristic acid (14:0) content by 2.5 fold (p-value,0.05) (Fig. 6). These results signify the first time protein-protein interactions have been correlated with product control in algae. Discussion Fatty acid biosynthesis is orchestrated by a modular enzymatic complex within the algal plastid, similar to machinery present in PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e42949