S155: Engineering biofuel tolerant microbes using efflux pumps

Many compounds being considered as candidates for next generation biofuels are toxic to microorganisms. This introduces an undesirable trade-off when engineering metabolic pathways for biofuel production because strains must balance production against survival. Cellular export systems, such as efflux pumps, provide a direct mechanism for reducing biofuel toxicity. To identify novel biofuel pumps, we constructed a library of heterologously expressed efflux pumps in E. coli and tested it against a representative set of biofuel candidates. In order to efficiently screen for improved tolerance, we devised a competitive growth assay to identify pumps that improved survival. A computational model suggests that competitive exclusion will result in a dramatic overrepresentation of strains containing beneficial pumps. We tested these predictions experimentally, finding that in the presence of a stressor, beneficial pumps quickly distinguish themselves. We identify specific efflux pumps that confer resistance to biofuel candidates. Several pumps discovered using this strategy have never been characterized for biofuel tolerance. The end goal of this study is to develop host microbes that are not limited by toxicity for the production of higher levels of biofuel.