Rapid design and prototyping of biocatalytic virus-like particle nanoreactors

Abstract Protein cages are attractive as molecular scaffolds for the fundamental study of enzymes and metabolons, and for the creation of biocatalytic nanoreactors for in vitro and in vivo use. Virus-like particles (VLPs) such as those derived from the P22 bacteriophage capsid protein make versatile self-assembling protein cages and can be used to encapsulate a broad range of protein cargos. In vivo encapsulation of enzymes within VLPs requires fusion to the coat protein or a scaffold protein. However, the expression level, stability and activity of cargo proteins can vary upon fusion. Moreover, it has been shown that molecular crowding of enzymes inside virus-like particles can affect their catalytic properties. Consequently, testing of numerous parameters is required for production of the most efficient nanoreactor for a given cargo enzyme. Here we present a set of acceptor vectors that provide a quick and efficient way to build, test and optimise cargo loading inside P22 virus-like particles. We prototyped the system using yellow fluorescent protein then applied it to mevalonate kinases, a key enzyme class in the industrially important terpene (isoprenoid) synthesis pathway. Different mevalonate kinases required considerably different approaches to deliver maximal encapsulation as well as optimal kinetic parameters, demonstrating the value of being able to rapidly access a variety of encapsulation strategies. The vector system described here provides an approach to optimise cargo enzyme behaviour in bespoke P22 nanoreactors. This will facilitate industrial applications as well as basic research on nanoreactor-cargo behaviour. biorxiv;2022.02.10.479872v1/UFIG1F1ufig1