Enhancing butanol tolerance of Escherichia coli reveals hydrophobic interaction of multi-tasking chaperone SecB

BackgroundEscherichia coli has been proved to be one promising platform chassis for the production of various natural products, such as biofuels. Product toxicity is one of the main bottlenecks for achieving maximum production of biofuels. Host strain engineering is an effective approach to alleviate solvent toxicity issue in fermentation.ResultsThirty chaperones were overexpressed in E. coli JM109, and SecB recombinant strain was identified with the highest n-butanol tolerance. The tolerance (T) of E. coli overexpressing SecB, calculated by growth difference in the presence and absence of solvents, was determined to be 9.13% at 1.2% (v/v) butanol, which was 3.2-fold of the control strain. Random mutagenesis of SecB was implemented and homologously overexpressed in E. coli, and mutant SecB(T10A) was identified from 2800 variants rendering E. coli the highest butanol tolerance. Saturation mutagenesis on T10 site revealed that hydrophobic residues were required for high butanol tolerance of E. coli. Compared with wild-type (WT) SecB, the T of SecB(T10A) strain was further increased from 9.14 to 14.4% at 1.2% butanol, which was 5.3-fold of control strain. Remarkably, E. coli engineered with SecB(T10A) could tolerate as high as 1.8% butanol (similar to 14.58g/L). The binding affinity of SecB(T10A) toward model substrate unfolded maltose binding protein (preMBP) was 11.9-fold of that of WT SecB as determined by isothermal titration calorimetry. Residue T10 locates at the entrance of hydrophobic substrate binding groove of SecB, and might play an important role in recognition and binding of cargo proteins.ConclusionsSecB chaperone was identified by chaperone mining to be effective in enhancing butanol tolerance of E. coli. Maximum butanol tolerance of E. coli could reach 1.6% and 1.8% butanol by engineering single gene of SecB or SecB(T10A). Hydrophobic interaction is vital for enhanced binding affinity between SecB and cargo proteins, and therefore improved butanol tolerance.