Biochemical Characterization of Extremophile Fatty Acid Metabolism Enzymes for Use in Algal-Based Biofuel Production

There is a renewed interest in renewable energy due to concerns over long-term fossil fuel supply, global warming, and global human population growth. Although promising alternative fuel sources have been derived from food crops (first-generation) and lignocellulose biomass (second generation), these feedstocks are not feasible for commercial use due to competition with food supplies and a requirement for technology development (pretreatment of biomass, enzymatic saccharification of the pretreated biomass, etc.) for the affordable conversion of lignocellulose biomass to fuel. For these reasons, there is a push to produce clean and renewable energy derived from algal biomass. The research reported here is focused on the biochemical characterization of fatty acid synthesis enzymes to augment microalgae-biofuel. Among microalgae, the highly productive, halophilic chlorophytes Dunaliella spp. are a rich source of lipids and have strong potential to be an economically viable source for renewable oil production.;To modify carbon flux through the fatty acid biosynthesis pathway and capture fatty acids incorporated into triacylglycerides, lipid biosynthesis genes from extremophiles were selected and biochemically characterized to establish their compatibility for functioning in Dunaliella to increase microalgal oil production. To this end, the acetyl-coenzyme A carboxylase (ACCase) from the bacterial halophile Chromohalobacter salexigens BAA-138 was recombinantly expressed in Escherichia coli to provide sufficient enzyme for biochemical characterization. The ACCase enzyme carries out the rate-limiting step during fatty acid synthesis (FAS), and it has been shown that increased ACCase expression in bacteria leads to elevated rates of FA production. Based on its rate-limiting role in the formation of fatty acids, it is proposed that by expressing a bacterial ACCase in microalgae the FAS limiting effects of transcriptional repression and feedback inhibition of the native microalgal ACCase could be mitigated.;In addition to increasing lipid production in microalgal strains, thermoactive thioesterases were also identified so that during high temperature conversion of lipids to fuel they could release the free fatty acids (FFAs) previously sequestered during algal cell growth as part of triacylglycerides. Therefore, heat stable thioesterases from Metallosphaera sedula DSM5348 and Sulfolobus solfataricus P2 were recombinantly expressed in E. coli to enable their biochemical characterization and evaluation for suitability for use in microalgae for improved biofuel production.