Bacteriophage <scp>P22</scp> Virus‐Like Particles as Nanoscale Protein Scaffolds for Plant Synthetic Biology

Advancing the utility of plant synthetic biology requires the continued development of protein engineering tools. Self-assembling protein compartments, such as virus-like particles (VLPs), provide versatile scaffolds for synthetic biology. However, few plant-expressed VLPs have demonstrated broad amenability to protein engineering, restricting their applications to specific contexts. Here, the Enterobacteria phage P22 VLP is explored as a novel protein scaffold for plant synthetic biology, demonstrating its production in a eukaryote for the first time. Through transient expression in the biofactory plant Nicotiana benthamiana, the capacity for P22 VLPs to correctly assemble and direct encapsulation of recombinant protein cargo is demonstrated. The durability of this protein scaffold is explored through co-encapsulation of multiple cargo protein species and by encapsulation through direct fusion to the P22 coat protein. Finally, the ability to simultaneously program cargo encapsulation and external protein display on P22 VLPs in vivo is demonstrated through SpyTag/SpyCatcher-mediated protein conjugation. This work demonstrates the broad utility of P22 VLPs as nanoscale protein scaffolds for plant synthetic biology.