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Investigating fatty acid biosynthesis within the algal chloroplast using Chlamydomonas reinhardtii as a model


As finite petroleum reserves run their course and combustion-related CO₂ emissions rise concerns about global warming, humanity is faced with the challenge of finding new sources of energy that are carbon-neutral, renewable and sustainable to meet the growing demand. Photosynthetic organisms convert solar energy and CO₂ directly into metabolic products that can serve as fungible biofuels. Microalgae are particularly attractive as a biodiesel feedstock, as they produce oil in high yields, grow at fast rates in habitats not suitable for conventional agriculture, and do not compete with the food supply. However, oil accumulation occurs under environmental stress, which compromises biomass productivity, and algal fatty acids are not ideal for biodiesel quality. The ability to manipulate algal fatty acid biosynthesis would thus be a significant stride towards developing algae as a biodiesel feedstock. In fatty acid biosynthesis within an algal chloroplast, an acyl carrier protein (ACP) tethers the growing fatty acid as it undergoes iterative cycles of elongation, and a thioesterase (TE) domain catalyzes the release of a mature fatty acid from the ACP. As plant TEs specific for certain chain length fatty acids have altered the fatty acid profile of transgenic plants and bacteria, this has emerged as a promising strategy to modify algal fatty acid content to fashion an optimized biodiesel feedstock. The work outlined in this thesis aims to investigate intermolecular interactions in algal fatty acid biosynthesis to facilitate engineering. A novel strategy was employed in which a chemical probe inspired by the enzymatic activity of the algal TE was synthesized, attached to the algal ACP chemoenzymatically, and used to trap algal ACP-TE interactions in vitro. No protein- protein interactions were detected between plant TEs and the algal ACP in vitro, and thus plant TEs did not elicit the desired phenotype when engineered into the algal chloroplast. Using protein-protein interactions as a means to control product identity may shift the paradigm towards rationally designed engineering approaches to optimize algae as a bioenergy source. Renewable energy outreach and education has been an indispensable facet of this work to generate awareness and instill passion for sustainable energy in our future scientists

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