Self-Assembly of P22 Virus-like Particles in Liquid–Liquid Phase-Separated Condensates for Enhanced Enzyme Catalysis

Within cells, biomacromolecules self-assemble into ordered and hierarchical structures, facilitating the formation of efficient enzyme catalytic networks and cofactor recycling. However, generating similar structures outside their natural context to create an efficient catalytic system in vitro remains a challenge. This research describes an approach that confines nanosized enzyme-encapsulated virus-like particles (VLPs) within microsized condensates through intrinsically disordered region (IDR)-mediated liquid-liquid phase separation (LLPS), demonstrating enhanced reaction rates and cofactor recycling that surpass state-of-the-art free enzyme catalysis. Specifically, an engineered amine dehydrogenase (<i>Tthe</i>r<i>Am</i>DH_V10) and a formate dehydrogenase (<i>Cb</i>FDH) were coencapsulated within VLPs which were then assembled into condensates, creating a self-sustaining NADH regeneration system for producing chiral lactams. Compared to free enzyme systems, the phase-separated VLPs exhibited 1.2- to 28-fold improvements in NADH recycling efficiency, 2.1- to 6.1-fold enhancements in catalytic efficiency (<i>k</i>_cat/<i>K</i>_m), and a 1.3- to 19.9-fold increase in substrate conversion under the same conditions. Additionally, VLPs and their condensates demonstrated higher activity toward 15 out of 16 substrates compared to free enzyme systems. In large-scale synthesis, the dual-enzyme VLP condensates reduced NADH consumption to just 0.05% of the substrate concentration while still achieving a high substrate conversion at such low cofactor concentrations. Ultimately, these findings showed how condensed, catalytic VLPs are more effective than free enzymes for enzyme catalysis.