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\begin{document}

\title{Rapid regulations of metabolic reactions in Escherichia coli via
light-responsive enzyme redistribution}



\author[1]{Zikang Huang}%
\author[2]{Lize Sun}%
\author[2]{Genzhe Lu}%
\author[2]{Hongrui Liu}%
\author[2]{Zihan Zhai}%
\author[2]{Site Feng}%
\author[2]{Ji Gao}%
\author[2]{Chunyu Chen}%
\author[2]{Chuheng Qing}%
\author[2]{Meng Fang}%
\author[2]{Bowen Chen}%
\author[2]{Jiale Fu}%
\author[2]{Xuan Wang}%
\author[3]{George Chen}%
\affil[1]{Tsinghua University}%
\affil[2]{Affiliation not available}%
\affil[3]{School of Life Sciences, Tsinghua University }%


\vspace{-1em}



  
  \date{May 23, 2024}


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\selectlanguage{english}
\begin{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.%
\end{abstract}%



\sloppy


\textbf{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 \emph{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 \emph{E. coli}
.

\textbf{Introduction}

Up to now, there have been many endeavors contributed to expanding the
toolbox of enzymatic reaction regulation in bacterial chassis
like\emph{E. coli} \textsuperscript{{[}1{]}}. Tuning the expression
level of enzymes is straightforward but has many limitations including
more metabolic burden of protein synthesis\textsuperscript{{[}2{]}}.
Recently, regulations on the spatial arrangement of enzymes have been
proposed as a solution for adjusting reaction rate at a fixed enzyme
concentration\textsuperscript{{[}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\textsuperscript{{[}3, 6, 7{]}}. In
addition to those \emph{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\textsuperscript{{[}8{]}}. However, the complexed properties of
BMCs, made it challenging to be reused \textsuperscript{{[}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\textsuperscript{{[}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\textsuperscript{{[}13{]},} \textsuperscript{{[}14, 15{]}}. There
has been an integration of LLPS and proximity-based strategy to regulate
pathway production\textsuperscript{{[}16, 17{]}}. \emph{In
vitro}condensates 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 \textsuperscript{{[}15, 18, 19{]}}. However, the attempt
of regulating \emph{in vivo} reactions based on the same strategy,
especially those catalyzing the conversion of small organic molecules,
was still at its infancy, \textsuperscript{{[}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\textsuperscript{{[}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\textsuperscript{{[}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 \emph{in vitro} , providing the potential of
enriching substrates in enzymatic reactions via non-specific
protein-substrate interactions (Figure 1A)\textsuperscript{{[}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 \emph{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 \emph{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)\textsuperscript{{[}28{]}}. The
PhASE strategy can thus be used for sensitively and reversibly
regulating wide range of enzymatic reactions in \emph{E. coli} via
light-induced protein rearrangement.

\textbf{Results}

\emph{2.1 Introduction of LLPS-based compartments into E. coli}

Since the establishment of LLPS-based compartment in prokaryotes has not
been recorded in detail in previous studies\textsuperscript{4}, we
introduced several phase modules and recorded condensate nucleation,
growth, and maintenance in \emph{E. coli} . The major phase module we
tested included FUSLCD (fused in sarcoma low complexity domain) fused
with truncated GCN4. FUSLCD was known for its ability to phase separate
via pi-pi interaction\textsuperscript{{[}28, 29{]}}, while GCN4 could
further facilitate phase separation via
oligomerization\textsuperscript{{[}30{]}} (Figures 2A and 2B). This
fusion protein formed membraneless compartments in the following
experiments. During the dynamic formation process, some condensates were
observed to nucleate around cell poles and move around them (Figure 2C,
Video S1), while others emerged elsewhere in the cytosol, then moved
around along the cellular membrane, and fused with each other,
especially with the ones near cell poles (Figure 2D, Video S2). These
phenomena are much like the behavior of liquid
droplets\textsuperscript{{[}31{]}}. Interestingly, nearly all
condensates finally took up the intracellular space in a regular
pattern, much like polar bodies\textsuperscript{{[}32{]}}, and the
pattern was homogeneous across different cells and different expression
levels of this phase module (Figure 2E, Supplementary Figure 1). Other
phase separating proteins, such as cryptochrome 2 (CRY2), known for its
homo-oligomerization after a period of blue light induction, formed
compartments at only one pole of the cell (Supplementary Figure
2)\textsuperscript{{[}33{]}}.

To further investigate the mechanism underlying the pole-localized
pattern of LLPS-based compartment, it was attempted to dissect genome
exclusion and pole attraction of protein aggregates by expressing phase
module in elongated \emph{E. coli} (\selectlanguage{greek}Δ\selectlanguage{english}\emph{ftsZ} ), which had duplicated
genomes but no septum between them. It turned out that the condensates
formed regularly among genomes rather than only targeting the poles
(Supplementary Figure 3). This phenomenon was similar to the results of
Winkler \emph{et. al} , indicating that the curvature of a cell wall did
not play a role in the pole-localization\textsuperscript{{[}34, 35{]}}.
Additionally, it was found that the compartments formed only along one
side of the cellular major axis, opposite to the side nucleoids occupied
(Supplementary Figure 3). Combining those results together, it was
reasoned that the unfavorable contact between nucleoid and phase module
and the stochastic movements of the droplet-like condensates could
determine its polar localization.

Then, the fluidity of the compartment formed by the phase module was
investigated, which showed similar recovering capability as that in
eukaryotes (Figure 2F)\textsuperscript{{[}36{]}}. Remarkably, the
compartments formed in our experiments were smaller compared to those
formed inside eukaryotic cells or \emph{in vitro} due to the limited
space inside a bacterial cell\textsuperscript{{[}19, 21, 37{]}}, which
could be the reason why they showed whole condensate bleaching. The
recovery of fluorescence could be the result of protein exchange from
the other compartments in the cells mediated by diffusing scaffold
proteins in cytosol, instead of the periphery of the bleached site
(Figure 2F). This property indicated that protein (and other molecules)
were allowed to diffuse across the condensate boundary, providing the
basis for POI recruitment\textsuperscript{{[}19{]}}.

\emph{2.2 Light-induced recruitment of POI into LLPS-based compartments}

To recruit POI into compartments rapidly and dynamically, the
light-responsive module was designed to sense the dynamic light signal.
Together with the phase module, the system was termed the
Photo-Activated Switch in \emph{E. coli} (PhASE) (Figure 3A). The phase
module acted as a scaffold to form isolated compartments from the
cytosol of \emph{E. coli} , while the light-responsive module remained
evenly distributed in the cell until triggered by induction signals.
After the light induction, the light-responsive module would be
recruited into the compartments formed by the phase module (Figure 1C).
Light-responsive protein pair CIB1 and CRY2 were chosen to respond to
the blue light signal, between which the binding affinity would increase
with the light intensity\textsuperscript{{[}38{]}}. CIB1 was included as
a part of the phase module, localizing permanently in compartments
isolated from the cytosol, while CRY2 was included as a part of the
light-responsive module, distributing evenly inside the cell in
darkness. POI was represented by mCherry (Figure 3A). During the light
induction process, the light-responsive module was captured and fixed
when diffused into the LLPS-based compartment. Since protein diffusion
is the major time-consuming step in this process, the regulation can be
completed in the time scale of seconds theoretically, estimated by the
protein diffusion rate in \emph{E. coli} \textsuperscript{{[}39, 40{]}}.
Indeed, it was found that right after 8 seconds of 488 nm laser intense
stimulation, the reorganization of mCherry into the compartments was
completed. The concentration of mCherry inside could reach more than 15
folds compared to the cytosol (Figure 3B) and maintained almost
unchanged over 30 minutes (data not shown). The LLPS-based compartments
labeled with GFP were captured after the stimulation process, showing
perfect overlap with mCherry signal (Figure 3B). When stimulated with
the laser of lower intensity, the recruited amount of mCherry reduced,
with the enrichment reaching only about 2 folds (Figure 3C).
Furthermore, this state was highly reversible, with the fastest recovery
time less than 10 minutes, and a fully reversed state was attained
within less than 15 minutes (Figure 3C, Video S3). We next tried to
stimulate the system several times after reversion and found that the
induction effect was consistent and robust at least in the first three
rounds (Figure 3C, Video S3). The overall reduction of the mCherry
signal could be the effect of laser bleaching, yet the fold enrichment
of POI in the compartment remained roughly the same under the same
intensity of light during different induction rounds. Remarkably, like
other condensates formed by proteins\textsuperscript{{[}41{]}}, their
aging and solidification were observed. Their fluidity decreased along
time and was almost undetectable after 10-hour IPTG induction at
16\textsuperscript{o}C. However, under such states, POIs could still be
recruited in seconds, suggesting a different mechanism for protein
entrance other than the dynamic component exchange between interior and
exterior layer (Figure 3, Supplementary Figure 4). In summary, PhASE\#1
was verified reorganizing POI within seconds in a reversible manner.

PhASE\#2 system was constructed using similar light-responsive
components as PhASE\#1 but substituting FUSLCD with tandem SIM and SUMO
repeats (Figure 3D), known for phase separating via multi-valent
interactions\textsuperscript{{[}42{]}}. The fold enrichment of POI
inside the compartment after light induction could be over 10-folds
(Figure 3E). Additionally, the interaction between phase module and
light-responsive module in this system can be conveniently redesigned by
adding different number (valency) of SIM or SUMO to light-responsive
module genetically\textsuperscript{{[}42{]}}, so that the fold
enrichment of POI can still be tuned even light source is fixed.

\emph{2.3 Regulation of metabolic reactions via mass action}

Besides the enzyme concentration, another critical component influencing
a biochemical reaction rate is the substrate concentration. If both
enzymes and substrates are enriched in the same compartment, the
reactions can be accelerated. If they are separated from each other, the
enzymatic reactions can be shut down (Figure 1B). Considering that the
main driving force of FUSLCD LLPS is pi-pi interaction, compounds
enriched with pi-electrons could be included in the compartment as
clients theoretically\textsuperscript{{[}13, 28{]}}. To explore the
regulatory effect of PhASE\#1 on enzymatic reactions, the enzyme of
interest (EOI) was tethered to the light-responsive module as the
experimental group (EXP), so that it could be recruited to the
membraneless compartments according to light-induced CRY2-CIB1
interaction. In the control groups (CTRL), EOIs were fused with mCherry
to perform catalysis in a fully dispersed state, excluding the influence
of protein fusion on enzymatic activity (Figure 4A). Then, both groups
were treated with light and the reaction rate difference between them
was derived.

As a proof of concept, the coelenterazine (CLZ) oxidation reaction
catalyzed by Renilla luciferase (Rluc) was tested, which was a reporter
system frequently harnessed for \emph{in vivo}
studies\textsuperscript{{[}43{]}} (Figure 4B), and the luminescence
generated during the process was used as a direct measurement of
reaction rate\textsuperscript{{[}44{]}} (Figure 4C). After the light
induction, EOIs were successfully recruited into the membraneless
compartments (Supplementary Figure 4A). In the first 20 minutes after
CLZ addition, the EXP group displayed a significantly higher oxidation
rate compared to that of the CTRL group, with 2.6 times of
coelenteramide (CLM, the oxidative product of CLZ) accumulation
difference. Since the overall substrate and enzyme concentration
remained the same between these two groups (Figure 4B), we deduced that
CLZ should have been enriched by the compartment from the cytosol,
otherwise the limitation of enzyme movement in cells would not benefit
the reaction efficiency. Remarkably, the concentration of bacteria did
not have a major impact on this reaction at a proper range as long as
the overall enzyme concentration remained at the same level
(Supplementary Figure 5). With four biological replicates, it was
concluded that PhASE\#1 could indeed regulate this enzymatic reaction.

Subsequently, more biochemical reactions were conducted for possible
practical applications. Catechol 2,3-dioxygenase (XylE) catalyzes the
oxidation of catechol, which is a key metabolite in many organic
degradation pathways\textsuperscript{{[}45, 46{]}} (Figure 4D). Like the
design above, a modified light-responsive module tethered with XylE was
constructed and was demonstrated to be successfully enriched into the
LLPS-based compartments (Supplementary Figure 4B). Both incubated under
daylight, the EXP group showed a higher reaction rate compared to that
of the CTRL group at the same enzyme and substrate levels, with 1.6
times 2-hydroxymuconate semialdehyde (2-HMS, the oxidative product of
catechol) accumulation difference 4.5 minutes after the substrate
addition (Figure 4E). From these results, the regulatory ability of
PhASE on enzymatic reactions was demonstrated. Additionally, they also
provided evidence for the principle of regulating metabolic reactions
via changing the distribution correlation of enzymes and substrates.

Furthermore, we have built a quantitative model based on mass action and
Michaelis-Menten equation to explore the parameters affecting the
regulatory capacity of a de-mixed system on biochemical reactions, in
which the acceleration of the reaction via the colocalization of the
substrate and enzyme has been verified. Interestingly, we also found
that the size of the compartments had significant effect on the
regulatory capacity.

\textbf{Discussion}

Tuning metabolic reactions via regulations on the enzyme expression
level has been studied comprehensively\textsuperscript{{[}1, 47{]}}. Yet
in many cases, changing the concentration of protein can cause
additional metabolic burden and a waste of time\textsuperscript{{[}2,
40{]}}. Here, we engineered protein-based condensates into a reaction
regulator named PhASE, changing reaction rate via the mechanism of mass
action. Without the need for protein synthesis during regulation, our
tool enabled the control of enzymatic reactions at a higher temporal
resolution, completing in a few seconds. Also, the reversibility of
PhASE allows the dynamic regulation of reaction velocity, while the
traditional regulations via chemical inducers and non-inducible
scaffolds can hardly be reversed\textsuperscript{{[}3, 6{]}}.
Additionally, our exploration of chemical interactions between small
molecular client and protein scaffold provided the potential of
regulating vast metabolic reactions via condensate-based
approach\textsuperscript{{[}24{]}} (Figures 1D, 4B and 4E).

However, there are still some considerations that should be taken into
using PhASE. It can be difficult to find such protein scaffolds for
every specific chemical to enrich them at arbitrary folds. Remarkably,
too strong interactions between chemical client and protein scaffold may
increase reaction efficiency either since the scaffold will compete
substrate binding with enzymes. Besides, to design those compartments in
specific sizes can be difficult, except for tuning the expression level
of scaffold proteins (Figure 2). Inappropriate fractional volume of the
condensates can lead to limited regulatory capacity (Figure S7).

Nevertheless, our system has many potential applications. With a very
high regulatory temporal resolution and reversibility, the PhASE system
allows the switch of enzymatic reactions in a higher frequency, while
the pulsatile activation of different pathways has been proved to
enhance biosynthetic production\textsuperscript{{[}12{]}}. Besides
metabolic regulations, PhASE system also has the potential to regulate
and study various biological activities. By quickly changing the spatial
correlation of substrates and enzymes, it is suitable for studying
transient biochemical reactions and the switch of signaling pathways
(Figure 2). Some of these processes last only for a few seconds, which
can hardly be rebuilt and studied via transcriptional and translational
control, which achieves the maximum regulatory effect in minutes or even
hours\textsuperscript{{[}42, 48{]}}. Additionally, the fold change of
reaction rate between two regulatory states may be increased even more
if our approach can be integrated with the proximity-based regulation of
metabolic pathways\textsuperscript{{[}16{]}}.

\textbf{Material and Methods}

\emph{4.1 DNA manipulation}

All constructs were assembled using available restriction enzymes and T4
ligase, or Hi-Fi assembly from New England BioLabs (NEB) and other basic
molecular cloning approaches. To construct the light-responsive LLPS
system PhASE\#1, we acquired gene sequences of FUSLCD (FUS residues
1-212, NP\_004951.1), CRY2 (NP\_171935.1), CIB1 (NP\_195179.2), and GCN4
(residues 2-34, 1W5I\_A) from Pilong Li's lab at Tsinghua University.
The sequences of the other system, PhASE\#2, contained SIM (E3
SUMO-protein ligase PIAS2 isoform X1 residues 505-527, XP\_006722634.1)
and SUMO (XP\_012635485.1), were synthesized by Ruibiotech directly.
Since we only used CRY2 as a light-responsive element, we added an MBP
tag to avoid its aggregation\textsuperscript{{[}49{]}}. Plasmids
(Supplementary Plasmid list) contain genes encoding Rluc (Renilla
luciferase, AGU01696.1), XylE (catechol 2,3-dioxygenase,
WP\_011005909.1), fluorescence proteins were acquired from iGEM parts
distribution. All sequence ID could be found on NCBI.

\emph{4.2 Plasmid transformation}

For plasmid amplification, 1 \selectlanguage{greek}μ\selectlanguage{english}L plasmid (approximately 100 ng
\selectlanguage{greek}μ\selectlanguage{english}L\textsuperscript{-1}) was transformed to 50 \selectlanguage{greek}μ\selectlanguage{english}L \emph{E. coli} DH5a
\selectlanguage{english}competent strain (TIANGEN). It was then incubated on ice for 25 minutes
followed by 90 seconds heat shock at 42. Subsequently, it was mixed
with 450 \selectlanguage{greek}μ\selectlanguage{english}L LB medium and incubated for an hour using a rotary shaker
(ZQZY-BG, Shanghai Zhichu). For protein expression, \emph{E.
coli}BL21(DE3) strain was employed (TIANGEN and TSINGKE). 100 \selectlanguage{greek}μ\selectlanguage{english}L
Competent cell culture was used for co-transformation of two plasmids,
the cultures were added with 900 \selectlanguage{greek}μ\selectlanguage{english}L LB medium to recover the cells. For
single plasmid transformation, the protocol was the same as for \emph{E.
coli} DH5a \selectlanguage{english}amplification. For plasmid construction using HiFi assembly
(NEB), 5 \selectlanguage{greek}μ\selectlanguage{english}L product were transformed to 100 \selectlanguage{greek}μ\selectlanguage{english}L bacterial culture,
followed the same protocol as that for amplification.

\emph{4.3 Strains, media, and culture conditions}

LB medium and plates were used for \emph{E. coli} cultivation. Kanamycin
and ampicillin were added at 100 mg L\textsuperscript{-1} when used
alone, 50 mg L\textsuperscript{-1} when combined. For plasmid
amplification and molecular cloning, we used \emph{E. coli} DH5a,
\selectlanguage{english}cultivated at 37, 220 r.p.m. For protein expression, \emph{E.
coli}BL21(DE3) strains (TIANGEN and TSINGKE) were used with regulations
on protein expression by \emph{lacO} in the presence of different
concentrations of IPTG, respectively. Seed cultures were grown at 37,
220 r.p.m, and adjusted to OD\textsubscript{600} of 0.6-1.0 for IPTG
induction. Since intrinsically disordered proteins were known to form
inclusion bodies at high expression levels and may cause the misfolding
of fused proteins, all of them were expressed at 16 and 220
r.p.m\textsuperscript{{[}49{]}}, while the light responses and enzymatic
reactivities were studied at room temperature or appropriate
temperatures.

\emph{4.4 Confocal microscopy}

All images shown in this paper were captured by Nikon A1 LFOVT. To assay
compartment formation (Figures 2C and 2D) and light responses (Figures
3B, 3C and 3E) for a long period of time, we used an LB agar pad to fix
1-5 \selectlanguage{greek}μ\selectlanguage{english}L overnight \emph{E. coli} under a microscope. The solid pad layer
was made from 400 \selectlanguage{greek}μ\selectlanguage{english}L 1\% LB agarose, on a glass coverslip-bottomed (20
mm in diameter) 35 mm Petri dish (D35-20-1-N). To observe bacterial
morphology and compartment localization pattern (Supplementary Figure
2-4), recruitment of enzymes into compartments (not shown), and other
short period observation, we used glass slides to fix them. All images
captured were under a 100X oil immersed lens. 488 nm laser was used both
for the observation of phase module distribution and the inducing light
signal to trigger POI recruitment. 561 nm laser was used for the
observation of POI recruitment. ProLong Live Antifade Reagent (P36975,
Invitrogen) was used to avoid photo-bleaching of mCherry (1:50
dilution). Intense light induction was performed using region of
interest (ROI) to guide 488 nm lasers, 3\% lasted for 8 seconds (Figures
3B and 3E), while weak light induction was conducted under 2\% laser
intensity for 6 seconds. FRAP assays were carried out using slightly
higher laser power with ROI as a single pixel.

\emph{4.5 Image analysis}

All confocal data analyses were performed on NIS Element Analysis. All
parameters in the same time series were maintained the same. All
quantified fluorescence intensity was calculated before images were
smoothed for display usage. The contrast of images was enhanced by
adjusting LUT.

\emph{4.6 Live staining of bacterial DNA}

NucBlue Live Cell Stain ReadyProbes reagent (R37605, Invitrogen) was
used to label \emph{E. coli} nucleoid. 2 drops of the solution were
added to 1 mL bacterial culture and incubated in darkness for 15
minutes. The observation should be completed in 45 minutes.

\emph{4.7 Coelenterazine oxidation assay}

CTRL and EXP bacteria were collected by centrifugation of 5,000-8,000
r.p.m for 2 minutes and washed twice using PBS. Then, 100 \selectlanguage{greek}μ\selectlanguage{english}L PBS culture
was added to each well of the 96-well plate, and the mCherry
fluorescence level of EXP groups and CTRL groups were adjusted to match
each other. Prior to each assay, 10 mM CLZ ethanol solution was diluted
in PBS to a final concentration of 250 \selectlanguage{greek}μ\selectlanguage{english}M and incubated for 45 minutes
to an hour. During the study, 3 \selectlanguage{greek}μ\selectlanguage{english}L CLZ diluted solution was added to
each well\textsuperscript{{[}43{]}}. The luminescence of 490 nm was
measured at 21-27. CLZ was purchased from Invitrogen (C2944). Thermo
Scientific Varioskan LUX was used for quantitating reactions. SkanIt
software was used for data management.

\emph{4.8 Catechol oxidation assay}

CTRL and EXP group bacteria were collected by centrifugation of
5,000-8,000 r.p.m for 2 minutes and washed twice using PBS. Then, 100 \selectlanguage{greek}μ\selectlanguage{english}L
PBS culture were added to each well of 96-well plate, and the mCherry
fluorescence level of EXP groups and CTRL groups were matched to each
other. Prior to each assay, 100 mM catechol water solution was diluted
to 10 mM working solution, 2.5 \selectlanguage{greek}μ\selectlanguage{english}L of which was then added to each well.
The absorbance of oxidation product 2-HMS at 377 nm can be measured at
19-22. Catechol was purchased from Damas-beta (120-80-9). Thermo
Scientific Varioskan LUX was used for quantitating reactions. SkanIt
software was used for data management.

\textbf{Reference}

{[}1{]} Wang, X., Han, J. N., Zhang, X., Ma, Y. Y.\emph{, et al.} ,
Reversible thermal regulation for bifunctional dynamic control of gene
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\textbf{Figure legend}

\textbf{Figure 1. Photo-activated switch for controlling enzymatic
reactions.}

\textbf{A.} \textbf{Illustration of a model enzymatic
reaction.}Substrates (S) can be converted to product (P) by enzymes (E),
during which light emission (yellow) by non-stable intermediates
directly reflects reaction rate. \textbf{B.} \textbf{Enzymatic reaction
organized by subcellular compartments.} Under the same overall
concentration of enzymes and substrates, a reaction will be accelerated
if enzymes and substrates are co-localized in a limited space, resulting
in increasing local substrates concentration, thus increasing effective
collision between substrates and enzymes (right). On the other hand, if
enzymes are isolated from substrates, the reaction rate will decrease
(left).\textbf{C.} \textbf{General design of rearranging enzymes
distribution by light.} Each member in a light-responsive protein pair
is fused with either phase separating protein or an enzyme of interest
(EOI) to form phase module or light-responsive module. The phase module
can phase separate into a membraneless compartment. Upon light
induction, these two modules will bind to each other, and thus EOI can
entry the compartment. \textbf{D.} \textbf{General principle of
concentrating chemicals into the compartment.} Substrates will be
enriched in the compartment if the substrate-protein interactions are
similar to protein-protein interactions promoting phase separation
(e.g., pi-pi interaction).

\textbf{Figure 2. Construction of LLPS-based compartment in \emph{E.
coli}.}

\textbf{A.} \textbf{Illustration of PhASE\#1 phase module design.}FUSLCD
can phase separate \emph{in vivo} , while GCN4 can further facilitate
it. CIB1 is one of the two members in the light-responsive protein pair.
See \textbf{Figure 3A} for further explanation.\textbf{B.}
\textbf{Illustration of the mechanism behind compartment construction.}
As its concentration rises beyond a threshold, the phase module will
self-aggregate into membraneless compartments with a unique chemical
environment. \textbf{C, D.} \textbf{The process of compartment formation
in \emph{E. coli}.} Most compartments nucleated around cell poles and
could move around (C). Some aggregates emerged elsewhere, moved towards
an end of the bacteria cell, and finally fused with other condensates
(D). Scale bar, 1 \selectlanguage{greek}μ\selectlanguage{english}m. \textbf{E. Homogeneous compartment formation in
\emph{E. coli}.} Almost every bacterium contains compartments localized
at their poles, when expressing PhASE\#1 phase module. Phase module was
under T7 promoter controlled by \emph{lacO} . Either 0.25 mM or 1 mM
IPTG was added to induce its expression. For more IPTG concentration
gradients, please refer to Supplementary Figure 1. Scale bar, 2.5 \selectlanguage{greek}μ\selectlanguage{english}m.
\textbf{F.} \textbf{Fluidity of PhASE\#1 phase module\emph{in vivo}.}
Fluorescence Recovery After Photo-bleaching (FRAP) was used to
characterize the fluidity of the fusion protein. The recovery of
fluorescence could be due to rapid protein exchange with the surrounding
cytoplasm. The fluorescence signal of the highlighted condensate was
normalized to that on the other side of the bacterium. Scale bar, 1 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure 3. The recruitment of POI into protein compartment.}

\textbf{A. Illustration of PhASE\#1 system design.} CIB1 and CRY2 could
bind to each other under 488 nm light. \textbf{B, C. Induction of
PhASE\#1 system by light of different intensity.} With intense 488 nm
laser irradiation for 8 seconds, the protein compartments could enrich
POI up to 15-fold compared to cytosol concentration. GFP signal was not
shown since its capture also need 488 nm laser but note that the
compartment formed by phase module had formed before the induction
process. The merged image was captured after the end of stimulation
process. More confirmations of the co-localization of two modules after
light were shown in Supplementary Figure 4 (B). With laser irradiation
of lower intensity for 3 seconds, the protein compartments enriched POI
at about 2-fold. After 10 to 15 minutes, the system fully reversed to
previous state and was ready for the second round of induction (data not
shown) and the third round of induction (1:08:19). With the same set of
parameters, this system could be induced to almost the same enrichment
fold as the first round. Since the bacteria were actively dividing, the
intensity profile of the last time point was not precisely overlapped
with that of former time points (C). \textbf{D.} \textbf{Illustration of
PhASE\#2 system design.} SIM and SUMO are utilized for compartment
formation. \textbf{E. Induction of PhASE\#2 system by light.} With
intense laser irradiation for 8 seconds, the protein compartments could
enrich POI for more than 10-fold. Scale bar, 1 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Fig 4. Regulation of single-step enzymatic reactions by PhASE\#1
system.}

\textbf{A. Module design of PhASE\#1 system.} In the experimental
strain, both phase module and light-responsive module tethered with
enzymes of interest (EOI) were expressed, resulting in cells containing
a LLPS-based compartment that could enrich both enzymes and substrates.
In the control (CTRL) strain, only a mCherry fused EOI was transformed,
mimicking traditional manufacturing process, with enzymes and substrates
dispersed in cells\textbf{. B, C. Coelenterazine (CLZ) oxidation
kinetics.} CLZ can be oxidized to CLM by Renilla Luciferase (Rluc).
During the reaction test, the overall concentration of Rluc in both
groups has no significant difference (B). Reaction rate from 4 to 20
time points from both groups, reflected by luminescence at 490 nm, were
compared to each other using paired t-test (1-3 time points were before
the addition of substrates). The experimental group has a significantly
higher reaction efficiency (C). This experiment has four biological
replicates. \textbf{D, E. Catechol oxidation kinetics.} 1,
2-Dihydroxybenzene (Catechol) can be oxidized to 2-hydroxymuconate
semialdehyde (2-HMS) by catechol 2,3-dioxygenase
(XylE)\textsuperscript{31}. During the reaction test, the overall
concentration of XylE in both groups has no significant difference (D).
Reaction rates from 4 to 20 time points were calculated in both groups
from the light absorbance of 2-HMS and compared using paired t-test (1-3
time points were before the addition of substrates). The experimental
group has a significantly higher reaction rate compared to control
group. (E). This experiment has four biological replicates.

\textbf{Acknowledgements}

We thank members of Pilong Li's lab for helpful discussions and kindly
provision of plasmids. We also thank Dr. Peng Li for his team and lab
training and members of iGEM Tsinghua team (2018) for their guidance in
brainstorming. This work was supported by Tsinghua University Initiative
Scientific Research Program, Tsinghua Xuetang Program, and Student
Research Training Program. This research was also financially supported
by grants from the Ministry of Science and Technology of China (Grant
No. 2018YFA0900200), National Natural Science Foundation of China (Grant
No. 31961133019; No. 21761132013; No. 31870859), and Center of Life
Sciences of Tsinghua-Peking University.

\textbf{Author contributions}

Z.H, G.L, H.L, G.Q.C contributed to the overall design of the PhASE
system. Z.H, L.S, G.L, Z.Z, S.F, J.G, C.C, C.Q contributed to molecular
cloning; Z.H, L.S, G.L, H.L collected and analyzed imaging data
together; Z.H, L.S, J.G and X.W tested the application of PhASE system
on metabolic reactions and analyzed the data. Z.H drafted the article
and figures, which were revised by X.W and G.Q.C. Under the approval of
X.W and G.Q.C, the article was submitted to scientific journals.

\textbf{Data availability}

All original data presented in this paper can be requested from the
corresponding authors.

\textbf{Competing interests}

The authors declare no competing interests.

\textbf{Disclosure}

The manuscript does not contain experiments using animals.

The manuscript does not contain human studies.

\textbf{Additional information}

Supplemental material is available.

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