(642d) Functional Targeting of Membrane Transporters and Enzymes to Peroxisomes

Engineered peroxisomes hold promise as a highly versatile platform for compartmentalizing engineered metabolic pathways for the biosynthesis of high-value products, insulating the engineered pathway from other cellular factors. However, native peroxisomes will often lack the required substrates and cofactors in their lumen to support heterologous reactions; accordingly, non-native membrane proteins (MPs) must be recruited to the peroxisome membrane. We developed a robust, modular “chauffeur” strategy that enables MP protein folding in the ER followed by trafficking to the peroxisome membrane via an engineered interaction in the cytosol with a transmembrane domain natively trafficked from the ER to the peroxisome. We demonstrated the modularity of this strategy by successfully applying it on multiple MP cargoes, including heterologous plant MPs, and observed increased titers for a monoterpene biosynthetic pathway. This strategy overcomes the challenges posed by uncertain MP folding and sorting mechanisms and expands the repertoire of pathways that can be compartmentalized into peroxisomes.