For microbial fuel production, the efficiency with which fuel can be exported from the cell is likely to have significant influence on production titer. Build-up of fuel molecules may directly reduce titer, and may also cause significant intracellular stress, leading to feedback inhibition of fuel production. Transport systems, such as efflux pumps and ABC-transport systems in bacteria and yeast, are documented to export a broad range of substrates, including solvents, and provide a valuable engineering route to relieve fuel accumulation-related stress and improve production titer.
We focus on investigating the role of native, as well as heterologously expressed, RND efflux pumps in E. coli. Targeted studies focus on the well-characterized E. coli AcrAB-TolC system, and efflux pumps from solvent resistant bacteria such as Pseudomonas putida S12. Because efflux pumps are likely to be specific to certain fuel molecules and stressors, a wider range of native and heterologous efflux pump systems must be tested against different fuel compound exposure, growth conditions, and in different engineered hosts. To address our broad goal of improving solvent resistance using efflux pumps, a high-throughput approach has been initiated to create a library of expression vectors representing all efflux pumps from E. coli as well from other organisms known to be naturally resistant to solvents.