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

Abstract

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.

Authors:

Jha, Ramesh K. ^[1]; Kern, Theresa L. ^[1]; Kim, Youngchang ^[2]; Tesar, Christine ^[2]; Jedrzejczak, Robert ^[2]; Joachimiak, Andrzej ^[3]; Strauss, Charlie E.  M. ^[1]

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+ Show Author Affiliations

1. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
2. Argonne National Lab. (ANL), Argonne, IL (United States)
3. Argonne National Lab. (ANL), Argonne, IL (United States); Univ. of Chicago, IL (United States)

Publication Date:

2016-08-30

Research Org.:

Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

Sponsoring Org.:

USDOD; USDOE

OSTI Identifier:

1396115

Report Number(s):

LA-UR-16-20060
Journal ID: ISSN 0305-1048

Grant/Contract Number:  

AC52-06NA25396

Resource Type:

Accepted Manuscript

Journal Name:

Nucleic Acids Research

Additional Journal Information:

Journal Volume: 44; Journal Issue: 17; Journal ID: ISSN 0305-1048

Publisher:

Oxford University Press

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; Biological Science; whole-cell biosensor, transcription factor, protein design, high throughput screening, paraoxon, nerve agent, flow cytometry