Evolution of enzymes with new specificity by high-throughput screening using DmpR-based genetic circuits and multiple flow cytometry rounds

Abstract

Genetic circuit-based biosensors are useful in detecting target metabolites or in vivo enzymes using transcription factors (Tx) as a molecular switch to express reporter signals, such as cellular fluorescence and antibiotic resistance. Herein, a phenol-detecting Tx (DmpR) was employed as a critical tool for enzyme engineering, specifically for the rapid analysis of numerous mutants with multiple mutations at the active site of tryptophan-indole lyase (TIL, EC 4.1.99.1). Cellular fluorescence was monitored cell-by-cell using flow cytometry to detect the creation of phenolic compounds by a new tyrosine-phenol-lyase (TPL, EC 4.1.99.2). In the TIL scaffold, target amino acids near the indole ring (Asp¹³⁷, Phe³⁰⁴, Val³⁹⁴, Ile³⁹⁶ and His⁴⁶³) were mutated randomly to construct a large diversity of specificity variations. Collection of candidate positives by cell sorting using flow cytometry and subsequent shuffling of beneficial mutations identified a critical hit with four mutations (D137P, F304D, V394L, and I396R) in the TIL sequence. The variant displayed one-thirteenth the level of TPL activity, compared with native TPLs, and completely lost the original TIL activity. The findings demonstrate that hypersensitive, Tx-based biosensors could be useful critically to generate new activity from a related template, which would alleviate the current burden to high-throughput screening.

Figure 1

Genetic enzyme-screening principle for evolutionary enzyme engineering. (a) Genetic libraries were constructed by site-saturation mutagenesis and staggered extension process. A genetic enzyme-screening system (GESS) was used to screen the constructed libraries for extracting active cells that triggered the genetic circuit to express a fluorescent reporter protein in the presence of phenol-lyase. Active variants with fluorescence were verified by HPLC or colorimetric assay. (b) Comparison of relative sensitivity for phenol detection by GESS (⦁), HPLC (◼) or colorimetric assay (▴). Inside panel shows the enlarged version ranging from 0 to 20 μM phenol. Each experiment was performed in triplicate.

Figure 2

Target residues for the development of phenol-lyase activity from an indole-lyase scaffold. (a) Overlapping schemes of β-elimination of l-tyrosine and l-tryptophan catalysed by phenol-lyase (thick line and black letters) and indole-lyase (thin line and grey letters), respectively. (b) Conserved residues in the active sites of indole-lyase (upper) and phenol-lyase (lower) within a distance of 6 Å from the substrate. The y-axis represents the conservation of residues in each of 50 enzymes from the NCBI database. Asterisks indicate the characteristic residues conserved differently between indole-lyase and phenol-lyase. (c) Structural representation of five characteristic residues (Asp¹³⁷, Phe³⁰⁴, Val³⁹⁴, Ile³⁹⁶, and His⁴⁶³) in tryptophan indole-lyase from Escherichia coli (PDB ID: 2C44).

Figure 3

High-throughput screening and development of new phenol-lyases from indole-lyase mutant libraries. (a) Fluorescence histograms of the 1^st and 2^nd mutant libraries obtained using flow cytometry. Cells were grown in M9 broth with 1 mM L-tyrosine. (b) Fluorescence image of the 3^rd mutant library on M9 agar plate containing l-tyrosine. White arrow indicates the 3R3 variant exhibiting substantial phenol-lyase activity. (c) HPLC analysis of products formed by the most beneficial indole-lyase variant sorted from each library. Citrobacter freundii phenol-lyase served as the positive control enzyme. (d) DNA shuffling of 2R11 phenol-lyase and wild-type indole-lyase. Seven hits from DNA shuffling were analysed using WebLogo to determine the frequency of each mutation. Wild-type indole-lyase residues are shown using black letters, while mutated residues are shown using red letters.

Figure 4

Characterization of synthesised phenol-lyase. (a) SDS-PAGE. M: marker; Lane 1: E. coli cell extracts expressing the 3R3 variant; Lane 2: purified 3R3 enzyme. (b) Effects of temperature on 3R3 phenol-lyase. (c) Effect of pH on 3R3 phenol-lyase. All experiments were performed in duplicate.

Figure 5

Effect of each mutation on 3R3 phenol-lyase activity. Relative activity of site-directed mutants of 3R3 phenol-lyase. Phenol-lyase activity was monitored for 1 h in 50 mM potassium phosphate buffer containing 50 µM pyridoxal 5-phosphate and 1 mM L-tyrosine. The concentration of generated phenol was examined using a colorimetric method. All experiments were repeated twice.

Figure 6

Homology modelling of wild-type indole-lyase and 3R3 phenol-lyase. Both modelled structures were generated using molecular dynamics and docking simulations. The docked ligand and Tyr⁷⁴ are represented as stick and line, whereas the 4 mutated residues are represented as balls and sticks, respectively. The distance between Tyr⁷⁴ and Cγ in the substrate is marked by a green line. The new indole-lyase has a more suitable orientation of the docked ligand regarding atomic distances between the substrate and active site residues.