Fig 4. Regulation of single-step enzymatic reactions by PhASE#1
system.
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. 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. D, E. Catechol oxidation kinetics. 1,
2-Dihydroxybenzene (Catechol) can be oxidized to 2-hydroxymuconate
semialdehyde (2-HMS) by catechol 2,3-dioxygenase
(XylE)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.