Bacterial Nanobioreactors--Directing Enzyme Packaging into Bacterial Outer Membrane Vesicles

Area of Science:

• Biotechnology
• Microbiology
• Enzyme Engineering

Background:

• Bacteria naturally release outer membrane vesicles (OMVs) containing various biomolecules.
• Phosphotriesterase (PTE) is an enzyme with potential applications but suffers from toxicity and instability.
• Existing methods for enzyme production and stabilization are often inefficient.

Purpose of the Study:

• To hijack bacterial OMV export pathways for simultaneous enzyme production, packaging, and release.
• To enhance the stability and production of active phosphotriesterase (PTE) using OMVs.
• To utilize the SpyCatcher/SpyTag (SC/ST) bioconjugation system for targeted OMV cargo loading.

Main Methods:

• Constructed a PTE-SpyCatcher (PTE-SC) fusion protein and a SpyTagged transmembrane porin (OmpA-ST).
• Coexpressed PTE-SC and OmpA-ST in bacteria to facilitate PTE packaging into OMVs via SC/ST interaction.
• Characterized the resulting PTE-loaded OMVs for size, quantity, enzyme kinetics, loading efficiency, and stability.

Main Results:

• Successfully produced and packaged active PTE into OMVs using the SC/ST system.
• PTE-loaded OMVs demonstrated native-like enzyme kinetics with paraoxon.
• Coexpression of OmpA-ST significantly improved PTE production by mitigating toxicity and enhanced enzyme stability against freeze-thaw cycles.

Conclusions:

• Bacterial OMV export pathways can be engineered to produce and stabilize active enzymes.
• The SC/ST system provides an effective strategy for loading specific proteins into OMVs.
• This approach offers a promising method for producing robust and stable enzyme preparations for various applications.