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

Abstract

Background Escherichia 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. Results Thirty 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 (~ 14.58 g/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. Conclusions SecB 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. Electronic supplementary material The online version of this article (10.1186/s13068-019-1507-7) contains supplementary material, which is available to authorized users.

Microbial production of advanced biofuels

“…The importance of cell wall engineering for biofuel production has also been recently reviewed (Sandoval and Papoutsakis 2016) . Recent examples for other targeted studies are the systematic evaluation of chaperones to aid in butanol production (Xu et al 2019) . Transporters have specifically been an attractive target due to their ability to export the final product, and have been developed for hemiterpenes (e.g.…”

Trends in Synthetic Biology in the Bioeconomy of Non-food-Competing Biofuels

“…However, product toxicity is one of the main bottlenecks in achieving the maximum production of biofuels. With that in mind, Xu et al [132] used host deformation engineering to alleviate the solvent toxicity in fermentation. Likewise, new synthetic promoters in E. coli provide better biochemicals and biofuels production by fine-tuning gene expression levels [113].…”

Synthetic Biology and Metabolic Engineering Employing Escherichia coli for C2–C6 Bioalcohol Production

“…(3) disruption of succinylglutamate desuccinylase (AstE) (Guo et al, 2019); (4) overexpression of the chaperone protein SecB or its mutation, SecB T10A (Xu et al, 2019); and (5) overexpression of the membrane-targeted tilapia metallothionein, OmpC-TMT (Chin et al, 2017).…”

“…Low n-butanol tolerance is also limiting for the economic viability of n-butanol production. A number of studies have identified strategies that improve tolerance: (1) mutation of genes related to cis-regulatory elements ( yqjA , yabI , and rob ) or the efflux pump subunit, acr B ( Jeong et al, 2017 ; He et al, 2019 ); (2) disruption of the transmembrane protein, TqsA ( He et al, 2019 ); (3) disruption of succinylglutamate desuccinylase (AstE) ( Guo et al, 2019 ); (4) overexpression of the chaperone protein SecB or its mutation, SecB T10A ( Xu et al, 2019 ); and (5) overexpression of the membrane-targeted tilapia metallothionein, OmpC-TMT ( Chin et al, 2017 ).…”