title: Manipulating fatty acid biosynthesis in microalgae for biofuel through protein-protein interactions.
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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Jillian L
authors.initials: JL
authors.lastname: Blatti
authors.forename: Joris
authors.initials: J
authors.lastname: Beld
authors.forename: Craig A
authors.initials: CA
authors.lastname: Behnke
authors.forename: Michael
authors.initials: M
authors.lastname: Mendez
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Mayfield
authors.forename: Michael D
authors.initials: MD
authors.lastname: Burkart
mesh_terms: D000213:Acyl Carrier Protein / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D056804:Biofuels; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism; D005227:Fatty Acids / Q000096:biosynthesis*; D015870:Gene Expression; D058086:Microalgae / Q000235:genetics / Q000378:metabolism*; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism; D030821:Plants, Genetically Modified; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry / Q000378:metabolism
chemical_list: D000213:Acyl Carrier Protein; D056804:Biofuels; D005227:Fatty Acids; D010940:Plant Proteins; D011506:Proteins; D013869:Thiolester Hydrolases