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

BACKGROUND

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.

RESULTS

Thirty chaperones were overexpressed in JM109, and SecB recombinant strain was identified with the highest -butanol tolerance. The tolerance () of 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 , and mutant SecB was identified from 2800 variants rendering the highest butanol tolerance. Saturation mutagenesis on T10 site revealed that hydrophobic residues were required for high butanol tolerance of . Compared with wild-type (WT) SecB, the of SecB strain was further increased from 9.14 to 14.4% at 1.2% butanol, which was 5.3-fold of control strain. Remarkably, engineered with SecB could tolerate as high as 1.8% butanol (~ 14.58 g/L). The binding affinity of SecB 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.

CONCLUSIONS

SecB chaperone was identified by chaperone mining to be effective in enhancing butanol tolerance of . Maximum butanol tolerance of could reach 1.6% and 1.8% butanol by engineering single gene of SecB or SecB. Hydrophobic interaction is vital for enhanced binding affinity between SecB and cargo proteins, and therefore improved butanol tolerance.