Phase‐Separated Multienzyme Compartmentalization for Terpene Biosynthesis in a Prokaryote

Abstract Liquid–liquid phase separation (LLPS) forms biomolecular condensates or coacervates in cells. Metabolic enzymes can form phase‐separated subcellular compartments that enrich enzymes, cofactors, and substrates. Herein, we report the construction of synthetic multienzyme condensates that catalyze the biosynthesis of a terpene, α‐farnesene, in the prokaryote E. coli . RGGRGG derived from LAF‐1 was used as the scaffold protein to form the condensates by LLPS. Multienzyme condensates were then formed by assembling two enzymes Idi and IspA through an RIAD/RIDD interaction. Multienzyme condensates constructed inside E. coli cells compartmentalized the cytosolic space into regions of high and low enzyme density and led to a significant enhancement of α‐farnesene production. This work demonstrates LLPS‐driven compartmentalization of the cytosolic space of prokaryotic cells, condensation of a biosynthetic pathway, and enhancement of the biosynthesis of α‐farnesene.