Tunable In Vivo Colocalization of Enzymes within P22 Capsid-Based Nanoreactors

The spatial organisation of enzymatic pathways through compartmentalisation is a mechanism used in nature for the regulation of multi-step biocatalytic processes. Virus-like particles (VLPs) derived from Bacteriophage P22 have been explored as biomimetic catalytic compartments. The in vivo co-encapsulation of enzymes is typically achieved via sequential fusion to the scaffold protein (SP), which results in an equimolar ratio of enzyme monomers. However, control over enzyme stoichiometry, which has been shown to influence pathway flux, is key to realising the full potential of P22 VLPs as artificial metabolons. Here we present a strategy for the stoichiometrically controlled in vivo co-encapsulation of cargo proteins within P22-based VLPs. Co-encapsulation was achieved via co-expression of cargo proteins with individual SP fusions using a dual plasmid system and verified for fluorescent protein cargo by Förster resonance energy transfer. This strategy was subsequently applied to a two-enzyme reaction cascade. L-homoalanine, an unnatural amino acid and chiral precursor to several drugs, can be synthesised from the readily available L-threonine by the sequential activity of threonine dehydratase and glutamate dehydrogenase. We find that scaffolding by this system has a profound impact on the activity of each enzyme and, using a purification strategy designed to isolate the range of particle forms that exist in vivo, that scaffolding of multimeric enzymes can be at unexpectedly high densities. This work demonstrates the controlled co-localisation of multiple heterologous cargo proteins in a P22-based nanoreactor and shows that careful consideration of loading densities of individual enzymes in an enzymatic cascade is required for the optimal design of synthetic metabolons.