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.