Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli

Abstract

BackgroundIsobutanol 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.ResultsThe 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.ConclusionsWe 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.

AcrB drug-binding pocket substitution confers clinically relevant resistance and altered substrate specificity

“…In MexD, a homolog of AcrB in Pseudomonas aeruginosa, SNPs in the large periplasmic loops were detected after selection on carbenicillin in the laboratory and the mutations broadened the resistance spectrum of these bacteria to include β-lactam antibiotics (33). Substitutions within AcrB have been detected following selection with various solvents but, in contrast to this study, the changes tended to be away from the drug-binding pocket and did not alter antimicrobial resistance (34)(35)(36)(37). Mutations at codon 288 of E. coli acrB that were selected in vitro with the efflux pump inhibitor NMP altered susceptibility to efflux pump inhibitors and to some antimicrobial substrates (38).…”

Genetic Determinants forn-Butanol Tolerance in Evolved Escherichia coli Mutants: Cross Adaptation and Antagonistic Pleiotropy betweenn-Butanol and Other Stressors

“…SetA is an efflux pump capable of transporting different sugars, and the expression of setA was found to be increased under glucose/phosphate stress (5,23), potentially alleviating the accumulation of nonmetabolized sugar phosphates. However, SetA also transports, albeit inefficiently, other substrates, such as the antibiotics kanamycin and neomycin, as well as glucosides and galactosides with alkyl or aryl substituents (6)(7)(8)(9)(10)(11)(12)24). Since there are no sugar analogs in the medium, it is currently unclear whether the overexpression of setA contributes to osmoprotection in MY2 and MY4 mutants.…”

“…Several nonnative microbial systems have been engineered for its production, including Escherichia coli (1), Lactobacillus brevis (2), Pseudomonas putida (3), Bacillus subtilis (3), and Saccharomyces cerevisiae (4). However, this solvent is highly toxic to microorganisms, imposing a limit on the productivity of bio-based production and leading to the development of simultaneous fermentation and separation techniques to mitigate the toxic effects of the biofuel (5) and efforts to identify the genetic determinants and molecular mechanisms associated with n-butanol tolerance for reverse engineering of more robust strains (6)(7)(8)(9)(10)(11)(12). Prior strain engineering efforts include overexpression of GroESL in Clostridium acetobutylicum (resulting in a 50% improvement in total growth in 0.75% [vol/vol] n-butanol) (12) and recently in E. coli (resulting in a 2.8-fold increase in total growth after 48 h in 0.75% [vol/vol] n-butanol) (11) and overexpression of gene CAC1869 in C. acetobutylicum (resulting in an 81% increase in total growth after 12 h) (13).…”

Current knowledge on isobutanol production withEscherichia coli,Bacillus subtilisandCorynebacterium glutamicum

“…Thereby, the authors identified a set of mutations (marC, hfq, mdh, acrAB, gatYZABCD, rph) common in several isobutanol tolerant lineages and they speculated that rpoS and post-transcriptional regulators such as hfq are promising targets to improve isobutanol production with E. coli. 14 Since we observed that the best C. glutamicum producer (strain Iso7) showed under aerobic conditions a more than 2-fold lower Y P/S (unpublished results), we tried to transfer the process conditions from the bottle to a bioreactor and thus developed a two phase fermentation. C. glutamicum Iso7 was cultivated in the first phase under aerobic conditions.…”