Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions Jillian L. Blatti1, Joris Beld1, Craig A. Behnke2, Michael Mendez2, Stephen P. Mayfield3, Michael D. Burkart1* 1 Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, United States of America, 2 Sapphire Energy Inc., San Diego, California, United States of America, 3 Division of Biological Sciences, University of California San Diego, La Jolla, California, United States of America Abstract Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes. Citation: Blatti JL, Beld J, Behnke CA, Mendez M, Mayfield SP, et al. (2012) Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions. PLoS ONE 7(9): e42949. doi:10.1371/journal.pone.0042949 Editor: Elena A. Rozhkova, Argonne National Laboratory, United States Received January 26, 2012; Accepted July 16, 2012; Published September 13, 2012 Copyright: 2012 Blatti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was provided by the United States Department of Energy (DOE) DE-EE0003373; United States National Science Foundation (NSF) 0742551; Rubicon Postdoctoral Fellowship; and California Energy Commission CILMSF 500-10-039. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: MM and CB are employees of Sapphire Energy, which has competing interest in the biofuel arena. SPM was a founder of Sapphire and thus owns significant shares in the company. The research in this manuscript is performed through a US Department of Energy-funded collaboration between Sapphire Energy and UCSD. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. Therefore, the rest of the authors declare no competing interest. * E-mail: mburkart@ucsd.edu Introduction In our quest to replenish diminishing reserves of fossil fuels with high energy alternatives while mitigating CO2 emissions, micro- algae have emerged as an attractive option to convert solar energy directly into fungible fuels [1]. However, for microalgal biofuels to be used at industrial scale, productivity must be enhanced and consistency made tunable [2]. For example, to develop biodiesels to function in existing petroleum-based infrastructure, fatty acids from microalgae must be altered to more closely mimic conventional diesel [3]. Fusion of the powerful tools of systems biology and metabolic engineering could enable us to develop microalgal strains capable of producing commercially viable quantities fatty acids with desired chain lengths [4]. New advances in algal genetic engineering will allow us to fashion viable fuels and commodities from microalgal metabolic pathways [5,6], yet our knowledge of algal fatty acid biosynthesis remains incomplete. Without a detailed understanding of enzyme activity, timing, and regulation, the engineering of biofuel products from these pathways will struggle to meet our growing energy demands. Fatty acid biosynthesis has been successfully manipulated in oilseed crops to produce fatty acids with novel compositions [7]. In pioneering work, Voelker and coworkers achieved short circuiting of fatty acyl chain elongation by expressing a laurate (12:0)-specific thioesterase from the California bay plant (Umbellularia californica) in the seeds of Arabidopsis and rapeseed (Brassica napus) to increase laurate by 24 and 58%, respectively [8]. Since the discovery that heterologous expression of thioesterases can influence the lipid profile of an organism [8], plant TEs have been engineered into a variety of plant species effectively altering their oil content [7]. By terminating fatty acid biosynthesis, the TE functionally determines the length and identity of the fatty acid end product [9]. Plant FatA TEs select for oleoyl (18:1)-ACP substrates and FatB TEs preferentially hydrolyze ACPs loaded with saturated fatty acids [10]. Some plants have evolved FatB TEs capable of prematurely siphoning short chain fatty acids for incorporation into seed storage oil [11]. Of the range of fatty acids found in Nature, saturated medium chain fatty acids (C8–C14) are ideal for biodiesel because they have properties that mimic current diesel fuels [4]. Recently, plant FatB TEs were genetically engineered into diatoms (Phaeodactylum tricornutum) [12] and cyanobacteria (Synechocystis sp. PCC6803) [13] with the goal of creating a superior biodiesel feedstock, but these efforts were met with limited success. De novo fatty acid biosynthesis occurs within an algal plastid by action of a type II fatty acid synthase (FAS), a modular multi- domain enzymatic complex where each activity is encoded onto a PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e42949