Allosteric transcription factors (aTF) act as switches that transduce small molecule binding into transcriptional actuation to regulate various cellular processes. This switch-like behavior makes aTFs a cornerstone in synthetic biology applications. In metabolic engineering, they act as in vivo sensors to report on the level of target metabolites, allowing directed evolution of production pathways by revealing rare, high-producing cells. aTFs also play a pivotal role as switches to control information flow and feedback regulation in synthetic gene networks. Expanding aTFs beyond naturally-occurring aTF-small molecule pairs would greatly increase their utility. For example, new chemical recognition capabilities would facilitate the evolution of new biosynthetic routes for natural products and synthetic chemicals useful as therapeutics, novel materials and fuels. Additional switches with orthogonal chemical specificity would allow the engineering of higher-order synthetic circuits that function more robustly for applications outside the laboratory. However, redesigning an aTF to bind to a new molecule is challenging. Mutations in the ligand-binding site often disrupt allosteric communication with the DNA-binding domain, destroying the switch-like behaviour. Here, we present a general strategy to engineer an aTF to respond to new inducer molecules using the E. coli LacI protein as a test case. We evaluate thousands of candidate designs, derived from computational design to identify those that are both active as a switch and responsive to a target molecule. We enhance the activity of the initial hits toward greater specificity and stronger induction. We demonstrate the utility of this approach by engineering LacI variants to respond to gentiobiose, fucose, lactitol or sucralose with response comparable or superior to the wild-type LacI response to its synthetic inducer, IPTG. This technology enables us to build highly specific intracellular biosensors for small molecules toward many applications in synthetic biology and cellular engineering.

Designing highly specific protein-based small molecule biosensors

254th National Meeting (2017)

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