Diesel fuel accounts for a large portion of the fuel used in the United States and may be difficult to replace with ethanol and other biofuels.  Escherichia coli makes straight-chain fatty acids that can be converted for use in diesel engines and is able to grow on multiple carbon sources, making E. coli an excellent platform for diesel fuel production.  This research focuses on developing E. coli as an industrial source of fatty acids for diesel production.  Initial steps based on metabolic modeling include knocking out genes involved in beta-oxidation of fatty acids as well as overproducing genes involved in precursors of fatty acid synthesis.  Further metabolic engineering includes heterologous expression of a plant thioesterase leading to the production of free fatty acids in E. coli.  These steps have led to a greater than 10-fold increase in free fatty acid production and to further enhance fatty acid synthesis, a high-throughput screen for fatty acid production was developed.  After optimization and validation of the screen, a random transposon library was generated in the fatty acid overproducing strain of E. coli and screened to identify genes outside of existing metabolic models that affect fatty acid synthesis.  Future work will focus on incorporating genes identified in the transposon library into the E. coli diesel platform as well as examining other libraries for novel genes that lead to the overproduction of fatty acids.