Over the past century, fossil fuels have been an abundant and cheap source for petroleum-derived chemicals and fuels. The production and burning of these carbon-derived fuels have led to adverse global environmental changes such as increase in air pollution, climate change and fluctuations in sea level. To replace dwindling petroleum resources and to curb emissions of CO2, it is critical to develop alternative and renewable resources for energy and fuels. Genetically engineered microorganisms that can directly produce medium and long chain hydrocarbons have been one of the most promising potential routes to renewable biofuel synthesis.Microbial fatty acids are an attractive source of precursors for a variety of renewable biofuels such as alkanes, alcohols, and biofuels. Enormous progress has been in engineering microbes to divert endogenous fatty acid synthesis and overproduce free fatty acids. However, there is an inherent problem of product toxicity that greatly reduces cell viability, increases cell lysis and product titers and there are previous reports suggesting membrane damage as the main mechanism of free fatty acid toxicity. In this work, we metabolically engineered Escherichia coli (E. coli) bacteria to overproduce medium chain free fatty acids and identified membrane stress as the leading factor in product toxicity. We found that membrane lipid composition can be altered by the direct incorporation of endogenously produced medium-chain fatty acids into lipids via the Aas pathway. The deletion of the aas gene and sequestering exported fatty acids reduces medium-chain fatty acid toxicity, partially restores normal lipid composition, and dramatically improves medium-chain fatty acid yields. In the second part of this thesis, we genetically engineered E. coli to produce fatty acid methyl esters (FAMEs) by direct in vivo methylation of free fatty acid in the strains discussed above. Insect Drosophila melanogaster Juvenile Hormone Acid O-Methyltransferase (DmJHAMT) was identified as a candidate to methylate a variety of endogenous medium chain fatty acids in E. coli. By introducing DmJHAMT in E. coli engineered to produce medium chain fatty, we obtain medium chain FAMEs at titers of 0.56 g/L, more than two orders of magnitude higher than titers previously achieved. This one-step conversion process was optimized by expression of rat Mat1A gene that increased the S-adenosyl-L-methionine cofactor pool and providing a physical sink to extract FAMEs from culture. The work presented here shows the viable method of producing microbial biodiesel by metabolical engineering of E. coli. Primary physiological stress associated with production of free fatty acid precursors was identified and higher titers of fatty acid production were reported. These free fatty acids were then converted to biodiesel in one step by expressing insect enzyme in E. coli. Although further work is needed for viable bacterial production of biodiesel, the simplicity of the pathway allows easier optimization and possibility to transport this pathway into photosynthetic microorganisms in the future
