Figure 1 Working principle of SDFA. Target ADH is translocated
to the extracellular environment by a MsfGFP-guided secretion system.
Subsequently, the enzyme activity of ADH is determined by a cascade
reaction in which NADH produced by the ADH-catalyzed reaction was used
by diaphorase to reduce resazurin into resorufin, which is highly
fluorescent. When excessive amount of resazurin and diaphorase were
provided, the rate of NADH formation was proportional to the rate at
which the red fluorescence increased.
Figure 2. Catalytic activity of PfODH toward various substrates
determined by the coupled enzyme assay. The increase of resorufin
fluorescence (Ex/Em=535/588 nm) was monitored for the PfODH-catalyzed
reactions with (A) (R )-2-octanol, (B)1-(3-Methylphenyl)ethanol, (C) 4-Fluoro-α-methylbenzyl alcohol,
and (D) 1-Phenylethanol respectively. All assays were performed
in a final reaction volume of 100 μl with the following conditions: 10
µg/ml purified PfODH, 2 U/ml diaphorase, 0.5 mM resazurin, 0.5 mM
alcoholic substrate, 0.5 mM NAD+, 0.1 M Tris solution
(pH 8.0). Assay reactions without adding purified PfODH served as the
controls for each substrate reaction. RFU: relative fluorescence units.
Figure 3 . Fluorescence image of the supernatants of the cells
expressing the MsfGFP/eGFP fusion proteins or eGFP (upper part of the
figure). The supernatant samples were excited by blue light (Safe
Imager™ 2.0 Blue Light Transilluminator, Thermo) and the image was taken
through an amber filter unit. SDS-PAGE analysis of the concentrated
supernatant samples of the cells expressing the MsfGFP/eGFP fusion
proteins or eGFP (lower part of the figure).
Figure 4. Catalytic activity of the secreted MsfGFP fusion
proteins determined by the coupled enzyme assay. The increase of
resorufin fluorescence (Ex/Em=535/588 nm) was monitored for the
reactions catalyzed by the secreted MsfGFP fusion proteins. (A)MsfGFP-GlyDH and eGFP-GlyDH were characterized by using 1,3-butanediol
as the substrate. (B) MsfGFP-CpSADH and eGFP-CpSADH were
characterized by using (S )-2-octanol as the substrate.(C) MsfGFP-PfODH and eGFP-PfODH were characterized by using
(R )-2-octanol as the substrate. (D) MsfGFP-BDH_KP and
eGFP-BDH_KP were characterized by using Meso-2,3-butanediol as the
substrate. The assay was carried out by mixing the supernatant sample
and the assay reagent mixture at volumetric ratio of 1:1. All assays
were performed in a final reaction volume of 100 μl with the following
conditions: 50 μl supernatant sample, 2 U/ml diaphorase, 0.5 mM
resazurin, 0.5 mM alcoholic substrate, 0.5 mM NAD+,
0.05 M Tris solution (pH 8.0). RFU: relative fluorescence units.
Figure 5. Catalytic
activity of ADHs normalized by the fluorescence of MsfGFP. Reactions
catalyzed by various concentrations of MsfGFP-PfODH with substrate
(R )-2-octanol. Red fluorescence represents the fluorescence
intensity of resorufin (Ex/Em=535/588 nm) recorded at a time point
within the linear part of the progress curve of MsfGFP-PfODH catalyzed
reactions. The same time point measurement was taken for all the
reactions with various enzyme concentrations. Green fluorescence
represents the fluorescence intensity of MsfGFP-PfODH. The normalized
signal is calculated by dividing the red fluorescence signal by the
green fluorescence signal. The assay was carried out by mixing the
MsfGFP-PfODH sample and the assay reagent mixture at volumetric ratio of
1:1. All assays were performed in a final reaction volume of 100 μl with
the following conditions: 50 μl enzyme sample, 2 U/ml diaphorase, 0.5 mM
resazurin, 0.5 mM alcoholic substrate, 0.5 mM NAD+,
0.05 M Tris solution (pH 8.0). Each bar indicates the mean value of
three biological replicates and the corresponding error bar indicates
the standard deviation. RFU: relative fluorescence units.
Figure 6. Schematic overview of the SDFA workflow for the
directed evolution of ADHs. 1-2: Mutated ADHs’ genes were fused to the
C-terminus of MsfGFP to create a plasmid library. 3-4: The host cells
were transformed with the constructed plasmids and were spread on
selection agar plates. 5: Grown mutant colonies were inoculated in
liquid medium and induced for enzyme expression. 6: The cell cultures
were centrifuged down to obtain the supernatants that contained the
secreted enzymes. The green fluorescence of the supernatants from the
enzyme mutants was determined. 7: Assay reagents were added to the
supernatant samples to monitor the red fluorescence. 8: The catalytic
activity of each mutant was normalized to identify positive hits.
Figure 7. The predicted conformations of (R)-2-octanol (cyan
ball-stick) and NAD
+ (yellow stick) in the active site
of PfODH. The catalytic residues (S148 and Y161) are shown in blue
sticks. The critical residues (G99, H150, and Y193), which affect the
substrate specificity and enantioselectivity, are shown in green sticks.
The substrate, (
R )-2-octanol, was built by the builder tool of
PyMol (DeLano Scientific) in productive conformation. Model structure of
PfODH–NAD
+ complex was constructed based on homology
modeling method using Swiss-Model server
(
https://swissmodel.expasy.org). The structure of glucose
dehydrogenase from
Bacillus megaterium IWG3 (K. Yamamoto et al.,
2001), BmGDH (PDB: 1GCO), was selected as the template. They share 40%
sequence identity.