Using Metabolic Network Modeling to Design an Escherichia coli strain with Enhanced Outer Membrane Vesicle Production

Bacterial structures formed from the outer membrane and periplasmic components carry biomolecules to expel cellular material and interact with other cells. These outer membrane vesicles (OMVs) can encapsulate bioactive content, which gives OMVs a high potential as alternative drug delivery vehicles or novel vaccines. The biogenesis of OMVs has been associated with structural mechanisms such as interruptions in linkage between inner and outer membrane, pressure from accumulation of periplasmic material, and membrane curvatures induced by proteins.

Escherichia coli strains were engineered based on metabolic network modelling to find single gene deletion strains with enhanced OMVs production (ΔpoxB, ΔsgbE, ΔgmhA, and ΔallD) through in silico gene knockout strain design applied to an already hypervesiculating strain (JC8031). Two deletions (ΔsgbE, ΔallD) have been linked to decreased amino acid precursor synthesis and decreased assimilation of nitrogenated compounds. The two other deleted genes (ΔpoxB, ΔgmhA) are associated with lipopolysaccharide composition, lipid interaction, and intermembrane links. These deletions suggest that there is a relation between the metabolic activity and structural mechanisms that have been proposed as the cause of vesicle biogenesis.

These four mutants were obtained by genome editing using CRISPR-Cas9. Experiments on the recovery of OMVs from these strains showed enhanced OMV production in the four designed strains. Lipidomic analysis through LC-MS provided the lipid profile for E. coli OMVs. The lipid profile did not change significantly when comparing OMVs from the designed strains with the parent strain (JC8031).

The obtained results suggest that the vesicle production can be improved while the obtained vesicles are not altered in their composition, allowing further study for stability and integrity for use in therapeutic settings.