Evolution, genomic analysis, and reconstruction of isobutanol tolerance in Escherichia coli

Abstract

Escherichia coli has been engineered to produce isobutanol, with titers reaching greater than the toxicity level. However, the specific effects of isobutanol on the cell have never been fully understood. Here, we aim to identify genotype–phenotype relationships in isobutanol response. An isobutanol-tolerant mutant was isolated with serial transfers. Using whole-genome sequencing followed by gene repair and knockout, we identified five mutations (acrA, gatY, tnaA, yhbJ, and marCRAB) that were primarily responsible for the increased isobutanol tolerance. We successfully reconstructed the tolerance phenotype by combining deletions of these five loci, and identified glucosamine-6-phosphate as an important metabolite for isobutanol tolerance, which presumably enhanced membrane synthesis. The isobutanol-tolerant mutants also show increased tolerance to n-butanol and 2-methyl-1-butanol, but showed no improvement in ethanol tolerance and higher sensitivity to hexane and chloramphenicol than the parental strain. These results suggest that C4, C5 alcohol stress impacts the cell differently compared with the general solvent or antibiotic stresses. Interestingly, improved isobutanol tolerance did not increase the final titer of isobutanol production.

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

“…In this study, increased ethanol tolerance was not linked to higher ethanol yields (SI Appendix, Fig. S6), which is in keeping with a recent study that examined Escherichia coli isobutanol tolerance and productivity (13).…”

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

“…However, it still faces some challenges, such as ameliorating the toxic effects of intermediates and products of interest (Keasling, 2010). Numerous studies have focused on increasing the tolerance of different production strains toward various biochemicals using different strategies (Alper, Moxley, Nevoigt, Fink, & Stephanopoulos, 2006; Atsumi et al, 2010; Dunlop et al, 2011; Goodarzi et al, 2010). An alternative to this approach is to identify alternative production hosts that are naturally tolerant to higher concentrations of the chemicals of interest.…”

Current knowledge on isobutanol production withEscherichia coli,Bacillus subtilisandCorynebacterium glutamicum

“…12 Thus, the isobutanol-induced growth arrest limits the overall productivity for an industrial scale application. To overcome isobutanol toxicity, Atsumi et al 13 recently isolated an isobutanol-tolerant E. coli strain by a sequential transfer method. However, the final strain was more tolerant to isobutanol, but showed much lower isobutanol formation, when compared with the parental strain E. coli JCL260/pSA55/pSA69.…”

“…However, the final strain was more tolerant to isobutanol, but showed much lower isobutanol formation, when compared with the parental strain E. coli JCL260/pSA55/pSA69. 13 More recently, Minty et al used experimental evolution combined with genome resequencing to identify the genotypic adaptations of E. coli lineages with increased isobutanol tolerance. 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.…”