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.
Highlights
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
Experimental evolution and phenotypic characterization of end populations E. coli EcNR1, a derivative of E. coli K12 MG1655 harboring a l Red prophage integrated at the bio locus, was evolved by serial passaging of six independent populations for approximately 500 generations on isobutanol spiked M9 minimal medium supplemented with 50 g/L carbon source and 0.25 mg/L biotin
Populations were evolved on two different carbon sources, with three populations evolved with 50 g/L glucose as the sole carbon source and another three populations evolved with 50 g/L xylose as the sole carbon source
Summary
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. Various cellular responses to alcohol stress have been observed, including induction of general stress response (such as upregulation of chaperonins), active efflux of alcohols, synthesis of protective metabolites, alteration of membrane and cell surface properties, adaptations in energy metabolism, changes in cellular morphology, and metabolic degradation of alcohols; some or all of these responses may be present in a given organism [6]. Systems biology studies of E. coli response to isobutanol and the closely related compound n-butanol have revealed that multiple stress response systems are induced by these alcohols, leading to global changes in gene transcription and proteome composition. The transcriptomic and proteomic response of E. coli to n-butanol stress has been characterized, with especially strong induction of oxidative and cell envelope stress responses observed; it was subsequently demonstrated that n-butanol exposure results in increased intracellular generation of reactive oxygen species, and oxidative stress gene knockouts led to decreased tolerance [8]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
