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.

Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity

“…Multiple in vivo mutagenesis strategies facilitate adaptive laboratory evolution for rapid screening of strongly tolerant expression hosts, including DNA replication proteins, RNAP and T7 RNAP fused with base deaminases [134][135][136][137]. Construction of artificial organelles allows for E. coli compartmentalization, which has the potential to accomplish precise PTMs [138,139]. T7 RNAP translational regulation Rapid screening of suitable expression hosts using the base editor and CRISPR/Cas9 to construct an T7 RNAP RBS library…”

Advances in biosynthesis of scopoletin

“…Synthetic pathways targeting to peroxisomes increased the production of fatty-acid-derived fatty alcohols, alkanes and olefins [ 58 ]. Condensation of Multienzymes in E. coli compartmentalized the cytosolic space into regions of high and low enzyme density and led to a significant enhancement of α-farnesene production [ 59 ]. The localization of different lipases into the organelles of lipolytic yeast was proposed to improve triglyceride derivative production by compartmentalization [ 60 ].…”

Interaction between the Anchoring Domain of A-Kinase Anchoring Proteins and the Dimerization and Docking Domain of Protein Kinase A: A Potent Tool for Synthetic Biology

“…Then the interacting peptides RIAD and RIDD were fused to RGG and pathway rate-limiting enzymes, respectively, to mediate the formation of multienzyme condensates in E. coli . That study provides a novel strategy for spatially organizing enzymes in prokaryotic hosts to improve the biosynthesis of valuable chemicals.…”

“…52 Recently, this research group also constructed synthetic multienzyme condensates to improve terpene biosynthesis in E. coli. 54 Specifically, RGG, an intrinsically disordered protein domain of LAF-1, was used as the scaffold protein to create membraneless condensates via liquid−liquid phase separation. Then the interacting peptides RIAD and RIDD were fused to RGG and pathway rate-limiting enzymes, respectively, to mediate the formation of multienzyme condensates in E. coli.…”

“…Recently, this research group also constructed synthetic multienzyme condensates to improve terpene biosynthesis in E. coli . Specifically, RGG, an intrinsically disordered protein domain of LAF-1, was used as the scaffold protein to create membraneless condensates via liquid–liquid phase separation.…”