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.