Engineering an Allosteric Transcription Factor to Respond to New Ligands

Nat Methods. 2016 Feb;13(2):177-83. doi: 10.1038/nmeth.3696. Epub 2015 Dec 21.

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.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Allosteric Regulation
  • DNA, Bacterial / genetics
  • Disaccharides
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / genetics*
  • Escherichia coli Proteins / metabolism*
  • Fucose
  • Gene Expression Regulation, Bacterial / genetics*
  • Genetic Engineering*
  • Lac Repressors / genetics*
  • Lac Repressors / metabolism*
  • Models, Molecular
  • Mutation
  • Protein Binding
  • Protein Conformation
  • Sucrose / analogs & derivatives
  • Sugar Alcohols

Substances

  • DNA, Bacterial
  • Disaccharides
  • Escherichia coli Proteins
  • Lac Repressors
  • LacI protein, E coli
  • Sugar Alcohols
  • Fucose
  • Sucrose
  • trichlorosucrose
  • gentiobiose
  • lactitol