Engineering an allosteric transcription factor to respond to new ligands

Abstract

Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.

Figure 1

General workflow for designing new ligand binding in an aTF. Schematic diagram showing the steps in the design workflow.

Figure 2. Characterization of Rosetta-designed variants responding to new inducers

(a–f) Data for fucose (a,b), lactitol (c,d) and sucralose (e,f). Amino-acid substitution profiles with heat maps indicating fold induction are included (a,c,e). We computed these values by normalizing the induction value of each clone by the number of mutations it contained and reporting the highest such value per unique position and amino acid pair. Dose-response curves for variants and wild-type (WT) LacI induced with the target ligand are also shown (b,d,f). Error bars represent s.d. of fold induction from three biological replicates.

Figure 3. Characterization of gentiobiose-responsive variants from the protein-wide single-amino-acid substitution library

(a) Amino-acid substitution profile with heat map indicating fold induction. Substitutions are classified into four groups on the basis of their location in the protein structure: ligand-binding pocket (cyan), dimerization interface (purple), DNA-binding domain (green), and otherwise unclassified (brown). (b) LacI structure (PDB identifier 1LBG) that includes wild-type side chains with residue substitutions in gentiobiose-responsive variants showing greater than 4.0-fold induction highlighted and colored by classification. (c) Dose-response curves for four gentiobiose-responsive variants (colored by classification of substitutions) and WT LacI. Secondary mutations are due to synthesis errors. Error bars represent s.d. of fold induction from three biological replicates; error bars are not displayed where they overlap plot markers.

Figure 4. Ligand cross-reactivity of LacI variants

(a–d) For top variants displayed in Figures 2 and 3, a dose response was determined for indicated ligands. Values displayed represent the highest fold induction at any ligand concentration. Variants displayed were designed for binding to gentiobiose (a), fucose (b), lactitol (c) and sucralose (d). Error bars represent s.d. of fold induction from three biological replicates.

Figure 5. Activity maturation of LacI

(a) Induction response of WT LacI and three variants toward gentiobiose and IPTG. Q291H is a promiscuous variant found during the initial screen. Activity-matured variants Q291H,A266L,T276I and Q291H,T276L,S279G were found after shuffling with I^s variants. (b) Induction response of WT LacI and three LacI variants toward sucralose and IPTG. Quadruple mutant I160S,H163W,S191A,L196R was uncovered in the initial sucralose-response screen. Activity-matured variants N125S,I160S,H163W,S191A,L196R,R303L and N125S,I160S,H163W,S191A,L196R were found after shuffling of a library of sucralose-responsive variants. Error bars represent s.d. of fold induction from three biological replicates.

Figure 6. Crystal structure and GFP induction with ligand of sucralose-binding LacI design variant (D149T,S193D,V150A,I156L)

(a) Zoomed-in view of sucralose bound to LacI quadruple mutant. Designed residues D149T and S193D are shown in green; V150A and I156L are outside the field of view. Other key interactions of native residues are shown in yellow. (b) Backbone C-α structural superposition of WT LacI (pink) and sucralose-binding LacI variant (blue). Designed residues are shown in green, and loops undergoing substantial conformational change are marked. (c) Fold induction response of the sucralose-binding variant with sucralose and IPTG at 100 mM ligand concentration. PDB identifiers of the LacI variant in apo and sucralose-bound forms are 4RZS and 4RZT, respectively.