(798a) Genome Evolution and Engineering for Elucidating the Genetic Architecture of Isobutanol Tolerance In Escherichia Coli

Understanding the genetic architecture underlying complex phenotypes is of great fundamental interest and also has important ramifications in biotechnology.  Metabolic engineering efforts have enabled microbial production of many fuels and commodity chemicals, but frequently toxicity limits production.  Microbial stress tolerance is a complex multigenic trait that is intractable to traditional genetic study and rational engineering efforts. We are developing an innovative evolutionary-genomics methodology for elucidating and improving complex phenotypes. Our approach entails experimental evolution of stress tolerance followed by genome re-sequencing to identify acquired mutations, genomic and functional dissection to reverse engineer mechanisms of tolerance, and targeted genome engineering for further phenotype improvement. As a proof-of-concept, we have been studying E. coli tolerance to isobutanol, a promising next-generation biofuel. Through our evolution and genome re-sequencing work (Minty et al. Microb Cell Fact 2011 10:18), we identified 247 genetic loci correlated with isobutanol tolerance.  We are currently performing targeted mutagenesis of selected genetic loci using Multiplex Automated Genome Engineering (MAGE), a recently developed technology that entails repeated cycles of high efficiency recombination using libraries of mutagenic DNA oligonucleotides (Wang et al. Nature 2009 460).  This strategy enables rapid exploration of vast genotype space without being constrained to adaptive walks. Improved strains isolated from mutant libraries 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 in the production of isobutanol.