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[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 in vivo studies[43] (Figure 4B), and the luminescence generated
during the process was used as a direct measurement of reaction
rate[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[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.
Discussion
Tuning metabolic reactions via regulations on the enzyme expression
level has been studied comprehensively[1, 47]. Yet
in many cases, changing the concentration of protein can cause
additional metabolic burden and a waste of time[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[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[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[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[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[16].
Material and Methods