Dual film-like organelles enable spatial separation of orthogonal eukaryotic translation

Engineering new functionality into living eukaryotic systems by enzyme evolution or de novo protein design is a formidable challenge. Cells do not rely exclusively on DNA-based evolution to generate new functionality but often utilize membrane encapsulation or formation of membraneless organelles to separate distinct molecular processes that execute complex operations. Applying this principle and the concept of two-dimensional phase separation, we develop film-like synthetic organelles that support protein translation on the surfaces of various cellular membranes. These sub-resolution synthetic films provide a path to make functionally distinct enzymes within the same cell. We use these film-like organelles to equip eukaryotic cells with dual orthogonal expanded genetic codes that enable the specific reprogramming of distinct translational machineries with single-residue precision. The ability to spatially tune the output of translation within tens of nanometers is not only important for synthetic biology but has implications for understanding the function of membrane-associated protein condensation in cells. [Display omitted] •2D phase separation was utilized to design orthogonal enzymes•Film-like organelles maintained distinct suppressor tRNA microenvironments•Dual film-like synthetic organelles enabled orthogonal translation in eukaryotes•Cells were equipped with two expanded genetic codes in addition to the canonical one Use of 2D phase separation and two instances of codon expansion in a synthetic biology approach enables control of selective protein synthesis at the tens of nanometers scale in live cells.