Engineering Biofuel Tolerance Using Efflux Pumps

A major limitation for the efficient production of biofuels is that their intracellular accumulation leads to a disruption of membrane integrity, inhibition of growth, and cell death. Therefore, the engineering of efflux pumps to facilitate the cellular export of biofuels has proven a valuable strategy for improving production efficiency. We previously used directed evolution to develop pumps that secrete short-chain alcohols; the presence of these engineered pumps confer improved alcohol tolerance of Escherichia coli. Moreover, we have designed a stress-response system which dynamically optimizes the efficiency of efflux pumps by fine-tuning their expression levels.

Here, we describe our efforts to extend this approach to engineer the efflux pumps of Pseudomonas putida, which confer a high tolerance to several biofuels, including n-butanol. To exploit the use of these pumps for biofuels and achieve further enhanced tolerance in this promising production host, we apply targeted modifications to a native pump of P. putida that is homologous to our successful variant from E. coli. We further integrate the dynamic stress-response system for optimizing the expression levels and investigate the effect of the system on biofuel tolerance of E. coli and P. putida. The engineered strains represent highly promising host systems for enabling the large-scale production of biofuels.