title: Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli.
abstract: BACKGROUND
  Isobutanol is a promising next-generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titer. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases of adaptation to exogenous isobutanol stress.
  
  
  RESULTS
  The adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in marC, hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced RpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodeling the cell envelope and surprisingly, stress response attenuation.
  
  
  CONCLUSIONS
  We have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to further efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis targets for future global phenotype engineering.
authors: Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
authors: Jeremy J
authors: JJ
authors: Minty
authors: Ann A
authors: AA
authors: Lesnefsky
authors: Fengming
authors: F
authors: Lin
authors: Yu
authors: Y
authors: Chen
authors: Ted A
authors: TA
authors: Zaroff
authors: Artur B
authors: AB
authors: Veloso
authors: Bin
authors: B
authors: Xie
authors: Catie A
authors: CA
authors: McConnell
authors: Rebecca J
authors: RJ
authors: Ward
authors: Donald R
authors: DR
authors: Schwartz
authors: Jean-Marie
authors: JM
authors: Rouillard
authors: Yuan
authors: Y
authors: Gao
authors: Erdogan
authors: E
authors: Gulari
authors: Xiaoxia Nina
authors: XN
authors: Lin
mesh_terms: D000440:Butanols / Q000633:toxicity*; D004361:Drug Tolerance; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019143:Evolution, Molecular; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial; D005838:Genotype; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D017422:Sequence Analysis, DNA
chemical_list: D000440:Butanols; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083497:AcrA protein, E coli; C083496:AcrB protein, E coli; C040507:isobutyl alcohol