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