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Engineering an allosteric transcription factor to respond to new ligands

2015; Nature Portfolio; Volume: 13; Issue: 2 Linguagem: Inglês

10.1038/nmeth.3696

1548-7105

Noah D. Taylor, Alexander S. Garruss, Rocco Moretti, Sum Chan, Mark A. Arbing, Duilio Cascio, Jameson K. Rogers, Farren J. Isaacs, Sriram Kosuri, David Baker, Stanley Fields, George M. Church, Srivatsan Raman,

Microbial Metabolic Engineering and Bioproduction

The combination of computational protein design and single-site saturation mutagenesis enables engineering of allosteric transcription factors to respond to new small molecules. 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.