(29w) Engineering Self-Assembled Nanoreactors for Enhanced Biocatalysis

Living cells have evolved to use self-assembled protein structures to spatially organize sequential enzymes to entail facilitated intermediate transfer, enhanced reaction rate and controlled metabolites flux at branched metabolic nodes. Examples include multi-enzyme complexes, metabolons, and microcompartment. These self-assembled protein reactors inspire devise artificial ones in engineered biosynthetic systems to gear metabolic flux, thereby improving product yield. In this work, we developed an interactive protein cage that can be used as a scaffold for multi-enzyme spatial organization. In vitro, we show that consecutive enzymes in the menaquinone biosynthesis with different sizes and shapes can be targeted to the surface of the engineered protein cage in high density, yielding spherical, monodispersed and homogenous nano-reactors with superior catalytic properties. Modulated reaction rate was achieved by altering the distance between attached enzymes. Using a pair of fluorescent proteins as models, proteins assembled with the protein nanoparticles in complex intracellular environment spontaneously. In engineered Escherichia coli, three key enzymes in mevalonate pathway have been co-localized on the exterior of the protein cage, leading to an 8.5-fold increase of lycopene production by streamlining metabolic flux towards its biosynthesis. This work presents a versatile route to multienzyme spatial organization for applications in biosynthetic industry and studying the mechanisms of naturally occurring nano-reactors.