title: Genomic library screens for genes involved in n-butanol tolerance in Escherichia coli.
abstract: BACKGROUND
  n-Butanol is a promising emerging biofuel, and recent metabolic engineering efforts have demonstrated the use of several microbial hosts for its production. However, most organisms have very low tolerance to n-butanol (up to 2% (v/v)), limiting the economic viability of this biofuel. The rational engineering of more robust n-butanol production hosts relies upon understanding the mechanisms involved in tolerance. However, the existing knowledge of genes involved in n-butanol tolerance is limited. The goal of this study is therefore to identify E. coli genes that are involved in n-butanol tolerance.
  
  
  RESULTS
  Using a genomic library enrichment strategy, we identified approximately 270 genes that were enriched or depleted in n-butanol challenge. The effects of these candidate genes on n-butanol tolerance were experimentally determined using overexpression or deletion libraries. Among the 55 enriched genes tested, 11 were experimentally shown to confer enhanced tolerance to n-butanol when overexpressed compared to the wild-type. Among the 84 depleted genes tested, three conferred increased n-butanol resistance when deleted. The overexpressed genes that conferred the largest increase in n-butanol tolerance were related to iron transport and metabolism, entC and feoA, which increased the n-butanol tolerance by 32.8±4.0% and 49.1±3.3%, respectively. The deleted gene that resulted in the largest increase in resistance to n-butanol was astE, which enhanced n-butanol tolerance by 48.7±6.3%.
  
  
  CONCLUSIONS
  We identified and experimentally verified 14 genes that decreased the inhibitory effect of n-butanol tolerance on E. coli. From the data, we were able to expand the current knowledge on the genes involved in n-butanol tolerance; the results suggest that an increased iron transport and metabolism and decreased acid resistance may enhance n-butanol tolerance. The genes and mechanisms identified in this study will be helpful in the rational engineering of more robust biofuel producers.
authors.affiliation: Department of Chemical Engineering, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Luis H
authors.initials: LH
authors.lastname: Reyes
authors.forename: Maria P
authors.initials: MP
authors.lastname: Almario
authors.forename: Katy C
authors.initials: KC
authors.lastname: Kao
mesh_terms: D020001:1-Butanol / Q000494:pharmacology*; D000222:Adaptation, Physiological / Q000187:drug effects / Q000235:genetics*; D002462:Cell Membrane / Q000187:drug effects / Q000235:genetics; D055028:Comparative Genomic Hybridization; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D020869:Gene Expression Profiling; D055786:Gene Knockout Techniques; D005798:Genes, Bacterial / Q000235:genetics*; D005820:Genetic Testing; D015698:Genomic Library; D007700:Kinetics; D058977:Molecular Sequence Annotation
chemical_list: D020001:1-Butanol