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 or 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.

Structural hierarchy controlling dimerization and target DNA recognition in the AHR transcriptional complex

“…Changes in the dimer interfaces of glucocorticoid receptor (GR), one of the hormone nuclear receptors, was found to allosterically affect DNA sequencespecific signaling (44); allosteric changes are crucial for communications between domains and alters the GR structure in response to target DNA binding (45,46). Structural allostery is virtually broadly recognized as an important mechanism for signaling switches of transcription factors and has been used to engineer transcription factors for versatile response to diverse signaling cues (47,48), suggesting that structural allostery might be a general mechanism for nuclear receptors to respond to their ligands. It is plausible that broad AHR ligands might induce diverse structural allostery that might modify target DNA recognition and the C-terminal TAD and lead to distinctly different biological consequences.…”

Design and application of a lactulose biosensor

“…Apart from allolactose and IPTG, an IPTG analog, n-propyl 1-thio-β- d -galactoside, was reported to exhibit a comparative inducing effect on LacI33. Attempts have been made to engineer it to response to compounds other than its natural inducers, but high induction specificity is still a challenge5. The LacI-L5 mutant obtained here exhibited responsiveness towards lactulose but was not induced by other six disaccharides tested, among which the structures of lactose, melibiose and epilactose only differ from lactulose in the sugar residue at the reducing end.…”

“…Engineering the effector specificities of regulatory proteins is an efficient way to develop customized small-molecule biosensors as well as novel gene switches. Some regulatory proteins have been engineered to response to compounds other than their natural inducers345, however, only a few have been confirmed as functional in vivo biosensors in practice678. The specificity, sensitivity and robustness are important features for high-quality in vivo biosensors.…”

Biofuel metabolic engineering with biosensors

“…Of 17 designs that were experimentally characterized, two were functional for DIG-binding, and optimization of one of these constructs using site-saturation mutagenesis coupled with selections resulted in a 75-fold improvement in binding affinity [54]. Similarly, Taylor and coworkers have reported the redesign of the lac repressor transcription factor for a number of novel inducers using a combination of computational protein design, mutagenesis and gene shuffling [55]. As computational design of binding improves, a potential application of such work would be in tuning the affinity of a biosensor, and thus the operational range, for various applications.…”