Engineering post-transcriptional regulation via synthetic membraneless organelles.

Artificial membraneless organelles (MLOs) are emerging as spatial organizers in synthetic biology, yet their applications remain largely confined to post-translational regulation. Here, we engineer synthetic condensates that act as modular hubs for targeted mRNA sequestration, enabling programmable post-transcriptional control in Escherichia coli. By incorporating orthogonal RNA-binding protein-aptamer interaction into the compartment, the designed MLOs can enhance translation through the integration of the translation machinery or repress specific pathways by isolating target transcripts. Tailoring condensate properties allows flexible functional optimization. We demonstrate rescue of bacterial cell division by sequestering a division inhibitor mRNA, and significantly improve the production of lacto-N-tetraose and lacto-N-neotetraose through compartmentalization or suppression of competing metabolic genes. Our work expands the functional repertoire of synthetic MLOs beyond enzyme scaffolding to encompass RNA-centric regulation, offering an adaptable strategy for reprogramming cellular functions in synthetic biology and biomanufacturing.