Application of Synthetic Biology for Increasing Anaerobic Biodiesel Production in Escherichia coli

The ever-increasing demand for transportation biofuels requires new and novel approaches to solve the complexities associated with efficient biofuel production. Ethanol, the most common biofuel, has physical limitation associated with difficulty of separations and issues with water contamination and as such is not a long-term transportation fuel solution. (Lou & Singh, 2010; Wheals, Basso, Alves, & Amorim, 1999) Biodiesel is seen as a possible alternative to ethanol due to its hydrophobicity and also has comparable energy density and cetane number to its petroleum derived counterpart. (Kalscheuer, Stölting, & Steinbüchel, 2006) Because of feedstock limitations, biodiesel produced from vegetable oils is limited by the supply of vegetable oil crops which creates scaling issues and land usage concerns. (Kalscheuer et al., 2006) An alternate method for biodiesel (fatty acid ethyl esters, FAEEs) generation was proposed which would bypass the need for vegetable oils by utilizing the fatty acids and ethanol made in engineered Escherichia coli. (Kalscheuer et al., 2006) FAEE is not water soluble, so water contamination in fuel supplies seen with ethanol is not likely to cause damage to fuel infrastructure and has similar combustion properties to petroleum based diesel. (Lou & Singh, 2010)

The goal of this project is to apply metabolic engineering and synthetic biology principles to engineer E. coli for efficient anaerobic production of FAEE from fermentable sugars. The strain utilized for this project builds upon previous work in which elementary mode analysis was used to design an E. coli strain that minimizes fermentative by-products under anaerobic conditions. The engineered strain provided an optimized platform to employ plug-and-play modular principles for fully endogenous FAEE biosynthesis.

In order to produce the desired esters, two parallel pathways were introduced, the first produced fatty-acyl-CoA, and the second the alcohol of interest. These two molecules are then catalyzed by a wax-ester synthase to produce desired biodiesel. Pathway flux engineering principles were employed to balance the metabolic fluxes of the two pathways that complete for the common substrate, pyruvate. The results show the dynamic range of module fluxes that can be achieved by varying promoter strength, operon orientation, and plasmid copy number.