Designer biosensors that respond to new small molecules

Genetic regulatory systems inducible by small molecules are indispensable tools for building genetic circuits, assaying gene function, and high-throughput metabolic engineering. Microbial allosteric transcription factors (aTFs) constitute a major class of proteins that confer such small-molecule inducible gene regulation, but aTF–inducer pairs are currently limited by those that naturally occur. Altering molecular specificity in these proteins is difficult because mutations that affect inducer binding may also destroy the allosteric behavior.  Here we engineer an allosteric transcription factor, LacI from E. coli, to respond to four new inducer molecules, including two which are fully synthetic. We employ computational protein design and multiplex assembly of 13,600 specified gene variants to engineer the LacI ligand binding pocket for new inducer recognition. We use a two-stage enrichment screen that identifies LacI variants which allosterically regulate gene expression in response to fucose, gentiobiose, lactitol or sucralose. Following activity maturation, we identify LacI variants responsive to these new inducers that exhibit specificity and induction response values comparable to the wild-type LacI response to its synthetic inducer, IPTG. We expect this approach to be a generalized method for altering aTF inducer specificity because it relies only on sequence and structure information, which is available for dozens of additional aTFs. The ability to create designer sensors and switches will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.