Isobutanol tolerance in Ralstoniaeutropha

Background Ralstonia eutropha is bacterium known to naturally produce polyhydroxybutyrate (PHB) as carbon storage during nutrient starvation. Previously studies [1] showed that it is possible through the incorporation of an engineered biosynthetic pathway, to redirect carbon flux from PHB to the production of Isobutanol (IBT), a biofuel largely studied to replace the current fossil fuels in existing automobile engines. However R. eutropha, is unable to grow in the presence of IBT at concentrations above 0.2% (v v) which decreases its potential for industrial scale production. In order to minimize toxicity to the cells, we studied IBT tolerance to develop an IBT tolerant strain. We selected tolerant strains through experimental evolution and we confirmed the existence of mutations in 2 genes of R. eutropha evolved strains, the homologues of acrA and acrA6 in Escherichia coli , which were previously described as being related to IBT tolerance [2,3]. Those 2 genes were deleted from the wild type and engineered IBT-producing strains in order to evaluate improvement in IBT tolerance.