Improving n-butanol tolerance of Escherichia coli based on membrane stress response pathways

Abstract:

[Objective] To improve n-butanol tolerance of Escherichia coli by screening and engineering the signal transduction pathways thinvolved in solvent stress response.[Methods] Under butanol stress, expression of response regulator in membrane signal transduction pathways of E. coli was determined and analyzed using RT-PCR. Key regulating components of stress response pathway were deleted and over-expressed in E. coli through red-homologous recombination and one-step clone. Solvent tolerance and membrane hydrophobicity analysis of the deleted and over-expressed strains were conducted against six different organic solvents.[Results] Expression level of cpxR and baeR in two-component stress response pathways Cpx and Bae was increased by 8.3 and 3.3 folds, respectively, after n-butanol (0.8%, V/V) treatment for 10 h. Under the solvent stress of tetrahydrofuran (0.6%, V/V), toluene (0.1%, V/V) and cyclohexane (0.6%, V/V) for 10 h, the OD₆₀₀ of recombinant E. coli JM109/pQE80L-spy and E. coli JM109/pQE80L-nlpE were increased by 0.13-0.17 and 0.05-0.13, respectively, compared with the control (ΔOD₆₀₀ of 0.02-0.04). Organic solvent tolerance of E. coli was improved.[Conclusion] Two component stress response pathways, Bae and Cpx, participate in the response of butanol stress. Overexpression of Spy could effectively improve organic solvent tolerance of E. coli. This study provides theoretical guidance for elucidating the mechanisms of microbial organic solvents tolerance.

Key words:butanol;stress response pathway;Escherichia coli;organic solvent tolerance