title: SoxR as a single-cell biosensor for NADPH-consuming enzymes in Escherichia coli.
abstract: An ultra-high-throughput screening system for NADPH-dependent enzymes, such as stereospecific alcohol dehydrogenases, was established. It is based on the [2Fe-2S] cluster-containing transcriptional regulator SoxR of Escherichia coli that activates expression of soxS in the oxidized but not in the reduced state of the cluster. As SoxR is kept in its reduced state by NADPH-dependent reductases, an increased NADPH demand of the cell counteracts SoxR reduction and increases soxS expression. We have taken advantage of these properties by placing the eyfp gene under the control of the soxS promoter and analyzed the response of E. coli cells expressing an NADPH-dependent alcohol dehydrogenase from Lactobacillus brevis (LbAdh), which reduces methyl acetoacetate to (R)-methyl 3-hydroxybutyrate. Under suitable conditions, the specific fluorescence of the cells correlated with the substrate concentration added and with LbAdh enzyme activity, supporting the NADPH responsiveness of the sensor. These properties enabled sorting of single cells harboring wild-type LbAdh from those with lowered or without LbAdh activity by fluorescence-activated cell sorting (FACS). In a proof-of-principle application, the system was used successfully to screen a mutant LbAdh library for variants showing improved activity with the substrate 4-methyl-2-pentanone.
authors.affiliation: IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich , D-52425 Jülich, Germany.
authors.forename: Solvej
authors.initials: S
authors.lastname: Siedler
authors.forename: Georg
authors.initials: G
authors.lastname: Schendzielorz
authors.forename: Stephan
authors.initials: S
authors.lastname: Binder
authors.forename: Lothar
authors.initials: L
authors.lastname: Eggeling
authors.forename: Stephanie
authors.initials: S
authors.lastname: Bringer
authors.forename: Michael
authors.initials: M
authors.lastname: Bott
mesh_terms: D000090:Acetoacetates / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D052196:Levilactobacillus brevis / Q000378:metabolism; D009249:NADP / Q000032:analysis / Q000378:metabolism*; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D059010:Single-Cell Analysis; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000090:Acetoacetates; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D014157:Transcription Factors; C070724:SoxR protein, Bacteria; D009249:NADP; C005217:methyl acetoacetate