Overproduction of β-barrel outer membrane proteins in Escherichia coli BL21(DE3) induces hypervesiculation

Aim: Gram-negative bacteria release outer membrane vesicles (OMVs) that fulfill many functions including survival during stress conditions, delivery of virulence factors, and nutrient acquisition. Additionally, they are increasingly used as an alternative for live bacteria in vaccine development and as a platform for bioengineering. Recently, OMVs have also been applied to express recombinant outer membrane proteins (OMPs) in their natural context as an alternative to the cumbersome reconstitution in liposomes. Here, we use an Escherichia coli strain that lacks four major OMPs for selective expression of the β-barrel assembly machinery (BAM) complex and PhoE in OMVs. Methods: OMV production of Escherichia coli BL21(DE3) and its omp8 derivative upon overexpression of BAM and PhoE is compared and characterized. Results: We find that overexpression of the BAM complex and PhoE causes a strong hypervesiculation phenotype, and the OMVs produced are intact and appear to recruit the BamA subunit of BAM and PhoE in their correctly folded and assembled conformations. Conclusion: While the exact mechanism of hypervesiculation remains to be elucidated, it contributes to the suitability of the BL21(DE3)omp8 host strain to produce recombinant OMP-enriched OMVs that can be used for various purposes, including structural analysis.