Encapsulin Nanocontainers as Versatile Scaffolds for the Development of Artificial Metabolons

Area of Science:

• Synthetic biology
• Biotechnology
• Biocatalysis

Background:

• Constructing non-native biosynthetic pathways is key for producing valuable chemicals.
• Enzyme immobilization on scaffolds creates efficient metabolons.
• Encapsulins are robust, engineerable protein containers with biotechnological applications.

Purpose of the Study:

• To engineer encapsulins for concurrent surface functionalization and internal packaging.
• To create a catalytically competent two-enzyme metabolon using encapsulins.
• To assess the biocatalytic efficiency of the engineered encapsulin-based system.

Main Methods:

• Engineered encapsulins from *Thermotoga maritima* for dual enzyme localization.
• Utilized SpyCatcher bioconjugation for surface attachment of dihydrofolate reductase (DHFR).
• Packaged demethylase enzymes within the encapsulin lumen using peptide affinity tags.
• Introduced a pore-enlarging deletion to enhance metabolite exchange.

Main Results:

• Successfully created a two-enzyme metabolon within *T. maritima* encapsulins.
• Demonstrated cross-talk between surface-bound DHFR and lumen-packaged demethylase.
• Pore-enlarged encapsulins achieved biocatalytic speeds comparable to free enzymes in solution.

Conclusions:

• Encapsulins are highly engineerable scaffolds for creating functional biocatalytic systems.
• This work highlights the potential of encapsulin-based metabolons for diverse biocatalytic applications.
• Optimized metabolite exchange is crucial for enhancing the efficiency of encapsulated enzyme systems.