An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes

Abstract

In vivo selection systems are powerful tools for directed evolution of enzymes. The selection pressure of the systems can be tuned by regulating the expression levels of the catalysts. In this work, we engineered a selection system for laboratory evolution of highly active enzymes by incorporating a translationally suppressing cis repressor as well as an inducible promoter to impart stringent and tunable selection pressure. We demonstrated the utility of our selection system by performing directed evolution experiments using TEM β-lactamase as the model enzyme. Five evolutionary rounds afforded a highly active variant exhibiting 440-fold improvement in catalytic efficiency. We also showed that, without the cis repressor, the selection system cannot provide sufficient selection pressure required for evolving highly efficient TEM β-lactamase. The selection system should be applicable for the exploration of catalytic perfection of a wide range of enzymes.

Figure 1

Overview of in vivo selection systems with tunable selection pressure. (a) Inducible promoter-equipped selection system. (b) Inducible promoter and cis repressor-equipped selection system. P_inducible, inducible promoter; RBS, ribosome binding site; GOI, Gene of interest. In both selection systems, GOI is placed downstream of the inducible promoter. (c) General schematic of the selection-based directed evolution experiment conducted in this study. First, GOI is subjected to random mutagenesis to create a mutant library. Bacterial cells expressing EOI mutants are then cultivated under selective conditions, in which only improved mutants can grow. If the (a) selection system cannot provide higher selection pressure for the next evolutionary round, the (b) selection system is used instead. (d) The regulation of selection pressure of the (a) and (b) selection systems. The survival of cells is coupled to the catalytic activity of EOI. Selection pressure of the selection systems can be modulated by adjusting the concentration of the expression inducer. The EOI starting point requires a high level of expression to catalyze the growth-limiting reaction above a certain rate threshold. Improved EOI variants can be selected by reducing the EOI expression level. The (b) selection system can be used when the (a) selection system can no longer provide sufficient selection pressure.

Figure 2

Tuning the dynamic range of the in vivo selection system. (a) Heat map representing the average colony diameter of E. coli XL1-Blue expressing the wild-type TEM under the control of different expression vectors. Each TEM-expressing E. coli strain was grown on LB agar plates containing 50 µg/ml ampicillin (LB Amp50) and varying concentrations of aTc as well as on LB agar plates containing 30 µg/ml chloramphenicol (LB Cm30) at 37 °C for 24 h. E. coli XL1-Blue pAT containing no TEM gene and E. coli XL1-Blue pAT-TEM_c, which constitutively expresses TEM, were used as the negative and positive controls, respectively. Colony diameters were determined by ImageJ. The raw data are shown in Table S1. (b) The sequence alignment of the original cis repressor and its mutants. (c) Cis repressor secondary structures predicted by Mfold. Ribosome binding sites and start codons are highlighted in blue and green, respectively. The graphical representation of cis repressor structures was generated by VARNA.

Figure 3

Distribution of the YR5-2 mutations in a three-dimensional structure of the wild-type TEM β-lactamase (PDB ID: 1ERM). The Glu166, Ser70, and the Ω loop are shown in purple. Mutated residues in YR5-2 are shown in pink. The boronic acid transition state analog is shown in yellow. The S4T mutation, which is located in the signal sequence, is not shown. The figure was prepared using PyMOL.

Figure 4

Heat map representing the average colony diameter of E. coli XL1-Blue expressing evolved TEM variants under the control of the pAT and pAT-cr3 vectors. All selected gene variants were subcloned into pAT and pAT-cr3. Each TEM variant-expressing E. coli strain was grown on LB Amp50 containing varying concentrations of aTc and on LB Cm30 at 37 °C for 24 h. E. coli XL1-Blue containing the pAT plasmid was used as the negative control. Colony diameters were determined by ImageJ. The raw data are shown in Tables S5 and S6.

Figure 5

Heat map representing the average colony diameter of E. coli XL1-Blue expressing E166Y TEM variants selected from the fourth (a) and fifth round (b) of directed evolution. All selected gene variants were subcloned into pAT-cr3. Each TEM variant-expressing E. coli strain was grown on LB Amp50 containing varying concentrations of aTc and on LB Cm30 at 37 °C for 24 h. E. coli XL1-Blue containing the pAT-cr3 plasmid was used as the negative control. Colony diameters were determined by ImageJ. The raw data are shown in Tables S7 and S8.

Figure 6

Susceptibility of TEM variants to trypsin digestion. Purified TEM variants (0.5 mg/ml) were incubated with 0.01 mg/ml trypsin in 20 mM Tris–HCl pH 7.5 and 50 mM NaCl at 25 °C. Aliquots from different time points were analyzed by SDS-PAGE. The intensity of full-length protein bands was analyzed by ImageJ.