Journal
ACS SYNTHETIC BIOLOGY
Volume 7, Issue 9, Pages 1990-1999Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.8b00052
Keywords
biosensors; ligand-dependent stabilization; directed evolution
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Funding
- NIH [P41GM103533, T32HG000035, F32GM122202]
- NATIONAL HUMAN GENOME RESEARCH INSTITUTE [T32HG000035] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [F32GM122202, P41GM103533, T34GM008563] Funding Source: NIH RePORTER
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The engineering of microorganisms to monitor environmental chemicals or to produce desirable bioproducts is often reliant on the availability of a suitable biosensor. However, the conversion of a ligand-binding protein into a biosensor has been difficult. Here, we report a general strategy for generating biosensors in Escherichia coli that act by ligand-dependent stabilization of a transcriptional activator and mediate ligand concentration-dependent expression of a reporter gene. We constructed such a biosensor by using the lac repressor, Lad, as the ligand-binding domain and fusing it to the Zif268 DNA-binding domain and RNA polymerase omega subunit transcription-activating domain. Using error prone PCR mutagenesis of lad and selection, we identified a biosensor with multiple mutations, only one of which was essential for biosensor behavior. By tuning parameters of the assay, we obtained a response dependent on the ligand isopropyl beta-D-1-thiogalactopyranoside (IPTG) of up to a 7-fold increase in the growth rate of E. coli. The single destabilizing mutation combined with a lad mutation that expands ligand specificity to D-fucose generated a biosensor with improved response both to D-fucose and to IPTG. However, a mutation equivalent to the one that destabilized Lad in either of two structurally similar periplasmic binding proteins did not confer ligand-dependent stabilization. Finally, we demonstrated the generality of this method by using mutagenesis and selection to engineer another ligand-binding domain, MphR, to function as a biosensor. This strategy may allow many natural proteins that recognize and bind to ligands to be converted into biosensors.
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