Advances in microbial engineering have led to the development of metabolic pathways for producing higher molecular weight alcohols as next-generation biofuels. However these molecules are highly toxic to microbes, which may limit product titer and reduce volumetric productivity . Due to broad mechanisms of toxicity, tolerance to alcohols and other solvents is a complex trait that involves a diversity of cellular adaptations and responses that probably contribute synergistically to the overall phenotype . Guided by evolutionary-genomic studies , we are using Multiplex Automated Genome Engineering (MAGE)  to generate combinatorial libraries of 35 mutations associated with isobutanol tolerance in E. coli. Variants with improved isobutanol tolerance are isolated using high-throughput phenotype screening with a microfluidic platform and then further characterized by precisely determining isobutanol tolerance, isobutanol productivity, and genotype. This approach allows systematic correlation of isobutanol tolerance phenotypes and genotypes, yielding insights into mechanisms of tolerance, as well as generating improved strains of E. coli that may be immediately useful in the production of isobutanol.
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