A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor

• Published in: Nucleic acids research 2016-08, Vol.44 (17)
• Main Authors: Jha, Ramesh K., Kern, Theresa L., Kim, Youngchang, Tesar, Christine, Jedrzejczak, Robert, Joachimiak, Andrzej, Strauss, Charlie E. Â M.
• Format: Article
• Language: English
• Subjects: BASIC BIOLOGICAL SCIENCES; Biological Science; whole-cell biosensor, transcription factor, protein design, high throughput screening, paraoxon, nerve agent, flow cytometry
• ISSN: 0305-1048; 1362-4962

A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires an access to an expanded repertoire of TFs. Using ligand docked homology models for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 107 variants of PobR, four were active when pNP was added as an inducer, with two mutants showing a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production and detect hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show that the fluorescence of the cells correlated with the catalytic efficiency of PTE variants, each cell expressed. High selectivity for similar molecules (4HB vs pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo- structures of PobR scaffold with computational models are notable successes presented in this work.