Compartmentalization of proteins into organelles is a promising strategy for enhancing the productivity of engineered eukaryotic organisms. However, approaches that co-opt endogenous organelles may be limited by the potential for unwanted crosstalk and disruption of native metabolic functions. Here, we present the construction of synthetic non-endogenous organelles in the eukaryotic yeast Saccharomyces cerevisiae, based on the prokaryotic family of self-assembling proteins known as encapsulins. We establish that encapsulins self-assemble to form nanoscale compartments in yeast, and that heterologous proteins can be selectively targeted for compartmentalization. Housing destabilized proteins within encapsulin compartments afford protection against proteolytic degradation in vivo, while the interaction between split protein components is enhanced upon co-localization within the compartment interior. Furthermore, encapsulin compartments can support enzymatic catalysis, with substrate turnover observed for an encapsulated yeast enzyme. Encapsulin compartments therefore represent a modular platform, orthogonal to existing organelles, for programming synthetic compartmentalization in eukaryotes.
