(623al) Engineering E. Coli for Improved Efflux of Fuel Molecules

Foo Jee Loon, Matthew Wook Chang, Chi Bun Ching, Susanna Su Jan Leong

School of Chemical and Biomedical Engineering, Nanyang Technological University

62 Nanyang Drive, Singapore 637459, Singapore

Escherichia coli is the production host used for the production of many biofuels. However, many organic solvents are toxic to bacteria and the tolerance of bacteria to these solvents has been found to involve exportation of the molecules out of the cells through efflux pump systems. In the context of biofuel production, these efflux pumps could be employed to confer greater solvent tolerance of the production host as well as promote secretion of the produced biofuel out into the external medium, thus increase the efficiency and reduce cost of biofuel production.

Of the few efflux pump systems identified in E. coli, the AcrAB-TolC is probably the best characterised. In order to facilitate the use of the AcrAB-TolC system for higher efflux efficiency, we have performed directed evolution on the AcrB inner membrane transporter for increased efflux of organic molecules, in this case, n-octane. Mutant libraries of AcrB were generated through random mutagenesis and variants with accelerated extrusion of intracellular n-octane were isolated after enrichment screening based on the n-octane tolerance of E. coli transformed with the mutant genes. The engineered AcrB mutants could be utilized in E. coli for more efficient biofuel production. Additionally, the information gathered from the mutants would enable us to further our understanding of the pump and aid rational design of the efflux system in the future.