(175b) Elucidating the Genetic Architecture of Isobutanol Tolerance in Escherichia Coli Through Targeted Genome Engineering and High Throughput Screening

Understanding the genetic architecture of complex phenotypes is of great fundamental interest and has important ramifications in biotechnology.  Metabolic engineering efforts have enabled microbial production of higher molecular weight alcohols as next-generation biofuels, but toxicity limits production.  Microbial stress tolerance is a complex multigenic trait intractable to traditional genetic study and rational engineering efforts. Most approaches to improving stress tolerance are therefore combinatorial, following a strategy of generating genotypic and phenotypic diversity in a population, then characterizing isolates with the desired properties. However, present methods explore relatively small genotype spaces and often fail to capture epistatic interactions between distal genetic loci. Guided by evolutionary-genomic studies, we are using Multiplex Automated Genome Engineering (MAGE) to generate combinatorial libraries of 38 mutations associated with isobutanol tolerance in E. coli.  Using a high-throughput microfluidic screening platform, we characterize phenotype distributions within the libraries and isolate variants with improved isobutanol tolerance.  Isolates are further characterized via detailed phenotyping and genotype analysis, allowing for systematic mapping of isobutanol tolerance phenotypes and genotypes.  Our work reveals prevalent epistasis between genetic loci and provides insights into biochemical mechanisms of tolerance, as well as generating improved strains of E. coli that may be immediately useful for isobutanol production.