title: 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.
authors.affiliation: Wyss Institute for Biologically-Inspired Engineering, Harvard University, Boston, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Noah D
authors.initials: ND
authors.lastname: Taylor
authors.affiliation: Wyss Institute for Biologically-Inspired Engineering, Harvard University, Boston, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Garruss
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, USA.|Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA.
authors.forename: Rocco
authors.initials: R
authors.lastname: Moretti
authors.affiliation: University of California Los Angeles-Department of Energy Institute for Genomics and Proteomics, University of California Los Angeles, Los Angeles, California, USA.
authors.forename: Sum
authors.initials: S
authors.lastname: Chan
authors.affiliation: University of California Los Angeles-Department of Energy Institute for Genomics and Proteomics, University of California Los Angeles, Los Angeles, California, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Arbing
authors.affiliation: University of California Los Angeles-Department of Energy Institute for Genomics and Proteomics, University of California Los Angeles, Los Angeles, California, USA.
authors.forename: Duilio
authors.initials: D
authors.lastname: Cascio
authors.affiliation: Wyss Institute for Biologically-Inspired Engineering, Harvard University, Boston, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Jameson K
authors.initials: JK
authors.lastname: Rogers
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.|Systems Biology Institute, Yale University, West Haven, Connecticut, USA.
authors.forename: Farren J
authors.initials: FJ
authors.lastname: Isaacs
authors.affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California, USA.
authors.forename: Sriram
authors.identifier: http://orcid.org/0000-0002-4661-0600
authors.initials: S
authors.lastname: Kosuri
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, USA.|Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA.
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.affiliation: Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA.|Department of Genome Sciences, University of Washington, Seattle, Washington, USA.|Department of Medicine, University of Washington, Seattle, Washington, USA.
authors.forename: Stanley
authors.initials: S
authors.lastname: Fields
authors.affiliation: Wyss Institute for Biologically-Inspired Engineering, Harvard University, Boston, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: George M
authors.initials: GM
authors.lastname: Church
authors.affiliation: Wyss Institute for Biologically-Inspired Engineering, Harvard University, Boston, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Srivatsan
authors.initials: S
authors.lastname: Raman
mesh_terms: D000494:Allosteric Regulation; D004269:DNA, Bacterial / Q000235:genetics; D004187:Disaccharides; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D005643:Fucose; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005818:Genetic Engineering; D056968:Lac Repressors / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D013395:Sucrose / Q000031:analogs & derivatives; D013402:Sugar Alcohols
chemical_list: D004269:DNA, Bacterial; D004187:Disaccharides; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D013402:Sugar Alcohols; D005643:Fucose; D013395:Sucrose; C026285:trichlorosucrose; C100052:gentiobiose; C014635:lactitol