Engineering an allosteric transcription factor to respond to new ligands

By N. Taylor, Alexander S. Garruss, Rocco Moretti, Sum Chan, M. Arbing, D. Cascio, Jameson K. Rogers, Farren J. Isaacs, Sriram Kosuri, David Baker and 3 more. 2015

310 citations recorded by the source corpus.

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.

Subfields: Genetics, Molecular Biology

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Citations in the corpus

Allostery in Its Many Disguises: From Theory to Applications (2019, background): “Since even in a highly structured protein ‘‘communication’’ between sites is likely mediated by multiple paths (Guo et al., 2015; Taylor et al., 2016), the questions of whether a given path can be rigorously validated against experimental data, or whether its specific role can be rationalized, need ”