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Tunable In Vivo Colocalization of Enzymes within P22 Capsid-Based Nanoreactors
Donna McNeale1,2, Lygie Esquirol1,3, Shoko Okada3
1Centre for Cell Factories and Biopolymers, Griffith Institute for Drug Discovery, Griffith University, QLD 4111, Australia.
ACS Applied Materials & Interfaces
|March 30, 2023
Summary
Researchers developed a method to control enzyme amounts within bacteriophage P22 virus-like particles (VLPs). This allows for precise tuning of biocatalytic nanoreactors for efficient synthesis of valuable compounds.
Area of Science:
- Biotechnology
- Synthetic Biology
- Biocatalysis
Background:
- Bacteriophage P22 virus-like particles (VLPs) serve as biomimetic catalytic compartments.
- Current methods achieve equimolar enzyme concentrations, limiting control over pathway flux.
- Precise enzyme stoichiometry is crucial for optimizing artificial metabolons.
Purpose of the Study:
- To develop a tunable strategy for controlling the stoichiometry of co-encapsulated proteins within P22 VLPs.
- To apply this strategy to an enzymatic cascade for the synthesis of l-homoalanine.
- To investigate the impact of enzyme loading density and stoichiometry on biocatalytic activity.
Main Methods:
- Utilized sequential fusion to scaffold proteins for in vivo co-encapsulation of cargo proteins.
- Employed Förster resonance energy transfer (FRET) to verify stoichiometric control of fluorescent proteins.
- Constructed and analyzed a two-enzyme cascade (threonine dehydratase and glutamate dehydrogenase) for l-homoalanine synthesis.
Main Results:
- Demonstrated tunable stoichiometric control over co-encapsulated proteins in P22 VLPs.
- Observed that enzyme loading density affects activity, with higher activity at lower densities suggesting molecular crowding effects.
- Showed that increasing the loading of a rate-limiting enzyme can enhance overall cascade activity.
Conclusions:
- Established a method for in vivo colocalization of multiple proteins in P22-based nanoreactors.
- Highlighted the necessity of controlled enzyme stoichiometry for optimizing nanoscale biocatalytic compartments.
- Provided insights into enzyme behavior and activity within crowded nanoreactor environments.
