Abstract
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 Pseudomonas putida KT2440. Deletion of oprI 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 oprF, 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 gpsA, which initiates glycerophospholipid biosynthesis, in the ΔoprF 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 P. putida.
| Original language | English |
|---|---|
| Journal | ACS Synthetic Biology |
| DOIs | |
| State | Accepted/In press - 2025 |
| Externally published | Yes |
Funding
The authors thank Stefan Haugen and Kelsey Ramirez for analytical support. This material is based upon work supported in part by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science Program under Award Number DE-SC0022181-0003. This work was authored in part by the National Renewable Energy Laboratory (NREL) for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. This material is based upon work supported in part by the Center for Bioenergy Innovation (CBI), U.S. Department of Energy, Office of Science, Biological and Environmental Research Program under Award Number ERKP886. Additional support was provided in part by the Director’s Fellowship within the Laboratory Directed Research and Development (LDRD) Program at NREL. B.N.D. was supported in part by the National Science Foundation Graduate Research Fellowship under grant number (B.N.D. #DGE-2234667). The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.
Keywords
- hypervesiculation
- membrane vesicles
- outer membrane vesicles
- protein secretion
- proteomics