Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules

Background: The depletion of fossil fuels and the rising need to meet global energy demands have led to a growing interest in microbial biofuel synthesis, particularly in Escherichia coli, due to its tractable characteristics. Besides engineering more efficient metabolic pathways for synthesizing biofuels, efforts to improve production yield by engineering efflux systems to overcome toxicity problems is also crucial. This study aims to enhance hydrocarbon efflux capability in E. coli by engineering a native inner membrane transporter, AcrB, using the directed evolution approach. Results: We developed a selection platform based on competitive growth using a toxic substrate surrogate, which allowed rapid selection of AcrB variants showing enhanced efflux of linear and cyclic fuel molecule candidates, n-octane and alpha-pinene. Two mutants exhibiting increased efflux efficiency for n-octane and alpha-pinene by up to 47% and 400%, respectively, were isolated. Single-site mutants based on the mutations found in the isolated variants were synthesized and the amino acid substitutions N189H, T678S, Q737L and M844L were identified to have conferred improvement in efflux efficiency. The locations of beneficial mutations in AcrB suggest their contributions in widening the substrate channel, altering the dynamics of substrate efflux and promoting the assembly of AcrB with the outer membrane channel protein TolC for more efficient substrate export. It is interesting to note that three of the four beneficial mutations were located relatively distant from the known substrate channels, thus exemplifying the advantage of directed evolution over rational design. Conclusions: Using directed evolution, we have isolated AcrB mutants with improved efflux efficiency for n-octane and alpha-pinene. The utilization of such optimized native efflux pumps will increase productivity of biofuels synthesis and alleviate toxicity and difficulties in production scale-up in current microbial platforms.