Engineered Membrane Vesicle Production via or Deletion Has Distinct Phenotypic Effects in .

Membrane vesicle (MV) production is a natural phenomenon in Gram-negative bacteria and represents an emerging synthetic biology tool for the secretion of biomolecules or bioproducts. Manipulation of membrane components has proven successful in enhancing MV production. However, the impact of membrane disruptions on strain fitness and protein composition warrants further investigation for the use of MVs in industrial bioprocesses. Here, we identify and characterize two genetic engineering strategies for inducing hypervesiculation─deletion of genes for the outer membrane porin OprF or the lipoprotein OprI─in the commonly used platform KT2440. Deletion of generated up to a 1.5-fold increase in MVs, larger MVs with a greater proportion of outer membrane proteins, and no significant impact on strain fitness compared to wild type. In contrast, deletion of relative to wild type, generated up to a 4-fold increase in MVs but diminished growth, permeabilized membranes, and increased cytosolic protein packaging. Both hypervesiculation phenotypes increased nontargeted and MV-targeted mNeonGreen extracellular signal by up to 6-fold, demonstrating vesiculation as a mechanism for protein secretion. Despite increased blebbing of MVs from gene deletions, proteins involved in membrane biosynthesis were not elevated relative to wild type. Overexpression of which initiates glycerophospholipid biosynthesis, in the Δ background improved the membrane integrity by 37% and maintained MV formation, highlighting the importance of membrane biosynthesis in restoring the membrane in hypervesiculating strains. Together, this study provides genetic engineering strategies with corresponding phenotypic outcomes toward providing a synthetic biology toolset for MV deployment in .