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Biological production of hydrocarbons is an attractive strategy to produce drop-in replacement transportation fuels. Several methods for converting microbially produced fatty acids into reduced compounds compatible with petrodiesel have been reported. For these processes to become economically viable, microorganisms must be engineered to approach the theoretical yield of fatty acid products from renewable feedstocks such as glucose. Strains with increased titers can be obtained through both rational and random approaches. While powerful, random approaches require a genetic selection or facile screen that is amenable to high throughput platforms. Here, we present the use of a high throughput screen for fatty acids based on the hydrophobic dye Nile red. The method was applied to screening a transposon library of a free fatty acid overproducing strain of Escherichia coli in search of high producing mutants. Ten gene targets were identified via primary and secondary screening. A strain comprising a clean knockout of one of the identified genes led to a 20% increase in titer over the baseline strain. A selection strategy that combines these findings and can act in an iterative fashion has been developed and can be used for future strain optimization in hydrocarbon producing strains. © 2011 American Institute of Chemical Engineers Environ Prog, 2012