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Global warming is a major issue of modern climate change, and caused by the greenhouse effect mainly due to carbon dioxide emitted by burning fossil fuels. To reduce the emission of carbon dioxide, scientists have focused on replacing fossil fuels with renewable energy. Biofuel, one form of renewable energy derived from the carbon sources of natural biomass, can contribute to reduced carbon dioxide emission. In this study, Escherichia coli was engineered for the production of fatty acid ethyl ester (FAEE), biodiesel derived from fatty acids. For production of FAEE, pyruvate decarboxylase (pdc), alcohol dehydrogenase II (adhB), acylCoenzyme A : diacylglycerol acyltransferase (atfA), and fatty acid synthesis (FAS) pathway genes were selected and cloned on to various plasmids. We designed and introduced various combinations of expression constructs in E. coli to enhance FAEE production. As a result, a 3.88-fold increase in FAEE production was achieved by modifying the expression levels of the genes involved in the initiation step and the β-oxidation step. The production of FAEE at different growth phases as well as the effect of the culture conditions such as temperature and carbon sources on FAEE productivity were studied. As a future study, scale-up studies through fermenter cultivation would contribute to mass production of biodiesel.