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. Here, 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 via 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 manifests LLPS-driven compartmentalization of the cytosolic space of prokaryotic cells, condensation of a biosynthetic pathway, and enhancement of of the biosynthesis of 𝛼-farnesene.

Results

Formation of RGG protein condensate in E. coli

    LLPS-mediated protein clustering in E. coli cells

Compartmentalizing biosynthetic enzymes to increase 𝛼-farnesene production in E. coli

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