Rapid regulations of metabolic reactions in Escherichia coli via light‐responsive enzyme redistribution

Abstract

Protein-based condensates have been proposed to accelerate biochemical reactions by enriching reactants and enzymes simultaneously. Here, we engineered those condensates into a photo-activated switch in Escherichia coli (PhASE) to regulate enzymatic reactions via tuning the spatial correlation of enzymes and substrates. In this system, scaffold proteins undergo liquid-liquid phase separation (LLPS) to form light-responsive compartments. Tethered with a light-responsive protein, enzymes of interest (EOIs) can be recruited by those compartments from cytosol within only a few seconds after a pulse of light induction and fully released in 15 min. Furthermore, we managed to enrich small molecular substrates simultaneously with enzymes in the compartments and achieved the acceleration of luciferin and catechol oxidation by 2.3and 1.6-folds, respectively. We also developed a quantitative model to guide the further optimization of this demixed regulatory system. Our tool can thus be used to study the rapid redistribution of proteins, and reversibly regulate enzymatic reactions in E. coli.

Phase-Separated Synthetic Organelles Based on Intrinsically Disordered Protein Domain for Metabolic Pathway Assembly in Escherichia coli

“…These physical properties were similar to those of several other motifs that have been reported to be able to spontaneously form MLOs in E. coli cells. ,,, A recent study proved that a tandem repeat of the RGG domain (RGG-RGG-GFP) formed fluid-like condensates in E. coli, while a single RGG domain (RGG-GFP) was not sufficient for phase separation into condensates .…”

“…These physical properties were similar to those of several other motifs that have been reported to be able to spontaneously form MLOs in E. coli cells. 10,13,15,21 A recent study proved that a tandem repeat of the RGG domain (RGG-RGG-GFP) formed fluid-like condensates in E. coli, while a single RGG domain (RGG-GFP) was not sufficient for phase separation into condensates. 18 In this work, however, RGG-sfGFP successfully triggered the formation of a liquid−liquid phase separation (LLPS) based compartment by induction with 0.5 mmol IPTG at 25 °C for 8 h (Figure 1C) Thus, we speculated that the level of the intracellular RGG protein is one of the key factors for the occurrence of phase separation and the formation of protein condensates.…”

Spatiotemporal Organization of Functional Cargoes by Light-Switchable Condensation in Escherichia coli Cells

“…coli . Despite these successes in metabolic and cellular engineering, reversible and repeatable recruitment and release of clients remain to be addressed for native-like dynamic regulation of cellular functions, , which is crucial for enhancing cell fitness and robustness.…”

“…Light-controlled optogenetic tools are intriguing for dynamic regulation , due to their precise control over protein–protein interactions in a spatiotemporal and reversible manner. As optogenetic molecules are relatively large in molecular weights and prone to aggregation upon overexpression in prokaryotic cells, fusion expression of optogenetic molecules with scaffolding and client proteins often leads to the formation of insoluble and inactive aggregates, ,,, which adversely affects the functioning of the resulting condensates and the realization of reversible and repeatable regulation. To address this challenge, here, we propose a modular framework for the organization of synthetic condensates by decoupling condensate formation and client recruitment/release (Scheme ).…”

“… 15 , 16 Intriguingly, polypeptide domains that bind specific DNA sequences have also been utilized to recruit plasmid DNA into synthetic condensates for transcriptional regulation in E. coli . 17 Despite these successes in metabolic and cellular engineering, reversible and repeatable recruitment and release of clients remain to be addressed for native-like dynamic regulation of cellular functions, 17 , 18 which is crucial for enhancing cell fitness and robustness.…”