Fabio: Fatty Acid Biosynthesis: Inducible, Orthogonal

Engineering heterologous fatty acid metabolic pathways (e.g., for the production of biofuels and other bioproducts) in E. coli (and in other organisms) is complicated and made much more difficult by concurrent and highly regulated endogenous fatty acid metabolism (and catabolism). Since fatty acid metabolism is essential for cellular growth, and the genes responsible for it are distributed across the genome, it is not possible to simply remove it wholesale; nor is it straightforward how to repress it in a controllable fashion. Our proposed approach is to remove from the genome (via CRISPR Cas9 approach) the genes responsible for endogenous fatty acid metabolism and place them (under native gene regulation) instead on a replicating artificial chromosome that can be controllably degraded (via CRISPR Cas9 and then subsequent endogenous nucleases).The heterologous fatty acid pathway (either plasmid borne or chromosomally-integrated) could then be induced in a manner orthogonal to and independent from the endogenous metabolism. Our main goal in this research is to decouple fatty acid biosynthesis in this system from, cell growth, self-regulation, consumption of fatty acids for other biomolecules such as phospholipids and lipopolysaccharides.