Engineered peroxisomes hold promise as a highly versatile platform for compartmentalizing engineered metabolic pathways, insulating them from native cellular factors to prevent undesired crosstalk. However, native peroxisomes often lack the required substrates and cofactors in their lumen; accordingly, non-native membrane proteins (MPs) must be recruited to the peroxisomal membrane to support heterologous pathways requiring these molecules. We developed a robust, modular “chauffeur” strategy that enables MP folding in the ER followed by trafficking to the peroxisomal 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 redirecting multiple MP cargoes, including heterologous plant MPs, and observed increased titers for a monoterpene biosynthetic pathway. This strategy overcomes the challenges of misfolding and sorting of MPs to the peroxisome and, accordingly, expands the repertoire of pathways that can be compartmentalized into this organelle.
