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 E. 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 minutes. Furthermore, we managed to enrich small molecular substrates simultaneously with enzymes in the compartments and achieved the acceleration of luciferin and catechol oxidation by 2.3 folds and 1.6 folds, respectively. We also developed a quantitative model to guide the further optimization of this de-mixed regulatory system. Our tool can thus be used to study the rapid redistribution of proteins, and reversibly regulate enzymatic reactions in E. coli .
Introduction
Up to now, there have been many endeavors contributed to expanding the toolbox of enzymatic reaction regulation in bacterial chassis likeE. coli [1]. Tuning the expression level of enzymes is straightforward but has many limitations including more metabolic burden of protein synthesis[2]. Recently, regulations on the spatial arrangement of enzymes have been proposed as a solution for adjusting reaction rate at a fixed enzyme concentration[3-5]. With the enzymes in the same pathway assembled on a DNA- or protein-based scaffold, the reaction productivity can be enhanced via the adjustment of spatial correlation of enzymes in the same pathway, as the result of minimizing the diffusion loss of intermediates[3, 6, 7]. In addition to those de novo designed proximity-based strategies, naturally existed bioreactors, known as bacterial microcompartments (BMCs), were hijacked to pack target enzymes of the same pathway together in a protein shell to mitigate intermediate losses[8]. However, the complexed properties of BMCs, made it challenging to be reused [9-11]. Furthermore, for all those strategies mentioned, it is hard to switch among different regulatory states in case it is needed to balance different metabolic pathways regularly[12].
Recently, LLPS-based technologies have been utilized as a convenient method to regulate the spatial distribution of target proteins, since a single protein segment can readily form compartments in cells[13], [14, 15]. There has been an integration of LLPS and proximity-based strategy to regulate pathway production[16, 17]. In vitrocondensates were also constructed to regulate enzymatic reactions via protein redistribution, and some of them proved it possible to regulate reactions via tuning the spatial correlation between enzymes and substates [15, 18, 19]. However, the attempt of regulating in vivo reactions based on the same strategy, especially those catalyzing the conversion of small organic molecules, was still at its infancy, [18, 20, 21], though there are over 30 important metabolic pathways involving small molecular reactions found to be regulated by condensates, including acetyl-CoA carboxylation and glutamine synthesis[22-24].
The main strategy of enriching molecules into protein-based condensates was specific molecular recognition. For example, substrate RNAs could be directly enriched by interior RNA binding proteins, while SUMOylation enzymes could be recruited to synthetic condensates via protein-protein recognitions[18, 25, 26]. Beyond macro-biomolecules, recent studies also observed the direct incorporation of organic dyes and other small molecules like ATP and GTP into the condensates formed in vitro , providing the potential of enriching substrates in enzymatic reactions via non-specific protein-substrate interactions (Figure 1A)[19, 27]. With the enrichment of substrate inside the condensates, the overall productivity of an enzymatic reaction can be regulated by switching on the redistribution of enzymes (Figure 1B).
Here, we developed a photo-activated switch in E. coli (PhASE) to dynamically regulate enzymatic reactions by tuning the spatial correlation of enzymes and substrates (Figures 1C and 1D). To start with, a scaffold protein was harnessed to form artificial compartments based on LLPS in E. coli , followed by fusing it and protein of interest (POI) with either member of an optogenetic protein pair, termed phase module and light-responsive module (Figures 1C). POI was confirmed to be reversibly recruited into LLPS-based compartments within seconds via light-activated interaction between two modules (Figure 1C). Taking pi-pi interaction as the scaffold-client interaction, we tested the enrichment of small molecules and found that their reaction rates could be changed by about 2 folds (Figure 1D)[28]. The PhASE strategy can thus be used for sensitively and reversibly regulating wide range of enzymatic reactions in E. coli via light-induced protein rearrangement.
Results