Tunable In Vivo Co-localisation of Enzymes Within P22 Capsid-Based Nanoreactors

Abstract

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.

Protein‐basierte Nanopartikel: Von Wirkstofftransport zu Bildgebung, Nanokatalyse und Proteintherapie

“…[18] Viren bestehen aus einer Hülle von sich wiederholenden, symmetrischen Anordnungen von viralen Hüllproteinen (VCPs), die Verkapselung von Nukleinsäuren als Nutzlast ermöglichen. Die meisten Viren haben eine ikosaedrische oder helikale Virus-Kapside [18] Proteine und Nukleinsäure, weniger häufig kleine Moleküle Meist Ikosaeder, hohl, 20-120 Nanometer Wirkstofftransport [19] Bildgebung [20] Nanoreaktoren [21] Proteintherapie [22] Ferritin [4b, 23] Meist kleine Moleküle Ikosaeder, hohl, 12 nm…”

Design of a symmetry‐broken tetrahedral protein cage by a method of internal steric occlusion

“…For example, the outer surface of a symmetric protein cage will present many structurally and chemically equivalent motifs (e.g., many equivalent chain termini) for attachment or fusion—12 for symmetry T, 24 for symmetry O, and 60 for symmetry I. That feature is beneficial for some applications (e.g., vaccine‐like particles (Antanasijevic et al, 2020; Brouwer et al, 2021; Marcandalli et al, 2019; Walls et al, 2020), polyvalent binding (Divine et al, 2021; Miller et al, 2023), enzymatic materials (McConnell et al, 2020; McNeale et al, 2023), and imaging scaffolds (Castells‐Graells et al, 2023; Liu et al, 2018; Liu et al, 2019)). However, for other applications it may be desirable to functionalize (or present fusions) on a singular location.…”

Pore engineering as a general strategy to improve protein-based enzyme nanoreactor performance

“…This includes both in vivo applications related to metabolic engineering 2 and bioremediation 3 , in vitro applications as chemoenzymatic production systems 4 , and applications as delivery devices for biomedically relevant enzymes 5 . Encapsulating enzymes inside nanoreactors can result in a number of distinct advantages, including the controllable co-localization of enzymes 6 , the selective enrichment of substrates 7 , intermediate sequestration and channeling 8 , prevention of unwanted side reactions and toxicity 9 , increased enzyme stability and protease resistance [10][11][12][13] , and the ability to sequester and store molecules of interest in a soluble and non-toxic form 14 . Depending on the system, nanoreactors are often assembled in vitro using relatively harsh disassembly, mixing, and reassembly protocols [15][16][17][18] .…”