Enhanced Outer Membrane Vesicle Production in Escherichia coli: From Metabolic Network Model to Designed Strain...

Bacterial structures formed from the outer membrane and the periplasm components carry biomolecules to expel cellular material and interact with other cells. These outer membrane vesicles (OMVs) can encapsulate bioactive content, which confers OMVs with high potential as alternative drug delivery vehicles or as a platform for novel vaccine development. Single-gene mutants derived from Escherichia coli JC8031 were engineered to further enhance OMV production based on metabolic network modelling and in silico gene knockout design (ΔpoxB, ΔsgbE, ΔgmhA, and ΔallD). Mutants were experimentally obtained by genome editing using CRISPR-Cas9 and tested for OMVs recovery observing an enhanced OMV production in all of them. Lipidomic analysis through LC-ESI-QTOF-MS was performed for OMVs obtained from each engineered strain and compared to the wild-type E. coli JC8031 strain. The lipid profile of OMVs from the wild-type E. coli JC8031 did not change significantly confirmed by multivariate statistical analysis when compared to the mutant strains. The obtained results suggest that the vesicle production can be further improved while the obtained vesicles are not altered in their composition, allowing further study for stability and integrity for use in therapeutic settings.