title: Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry.
abstract: We created a single cell sorting system to screen for enzyme activity in Escherichia coli producing 3,4 dihydroxy benzoate (34DHB). To do so, we engineered a transcription factor regulon controlling the expression of green fluorescent protein (GFP) for induction by 34DHB. An autoregulated transcription factor, pcaU, was borrowed from Acinetobacter sp ADP1 to E. coli and its promoter region adapted for activity in E. Coli. The engineered pcaU regulon was inducible at >5 μM exogenous 34DHB, making it a sensitive biosensor for this industrially significant nylon precursor. Addition of a second plasmid provided IPTG inducible expression of dehydroshikimate dehydratase enzyme (AsbF), which converts endogenous dehydroshikimate to 34DHB. This system produced GFP fluorescence in an IPTG dose-dependent manner, and was easily detected in single cell on flow cytometer despite a moderate catalytic efficiency of AsbF. Using fluorescence-activated cell sorting (FACS), individual cells carrying the active AsbF could be isolated even when diluted into a decoy population of cells carrying a mutant (inactivated) AsbF variant at one part in a million. The same biosensor was also effective for further optimization of itself. FACS on E. coli carrying randomized loci in the promoter showed several variants with enhanced response to 34DHB.
authors.affiliation: Bioscience Division, MS M888, Los Alamos National Laboratory, Los Alamos, NM 87545.
authors.forename: Ramesh K
authors.initials: RK
authors.lastname: Jha
authors.affiliation: Bioscience Division, MS M888, Los Alamos National Laboratory, Los Alamos, NM 87545.
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Kern
authors.affiliation: Bioscience Division, MS M888, Los Alamos National Laboratory, Los Alamos, NM 87545.
authors.forename: David T
authors.initials: DT
authors.lastname: Fox
authors.affiliation: Bioscience Division, MS M888, Los Alamos National Laboratory, Los Alamos, NM 87545 cems@lanl.gov.
authors.forename: Charlie E
authors.initials: CE
authors.lastname: M Strauss
mesh_terms: D000150:Acinetobacter / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D002469:Cell Separation; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D015723:Gene Library; D017930:Genes, Reporter; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism; D062385:Hydroxybenzoates / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D018085:Regulon; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
chemical_list: C520290:3,4-dihydroxybenzoate; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D062385:Hydroxybenzoates; C111304:PcaU protein, Acinetobacter; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; C044921:3-dehydroshikimate dehydratase; D006836:Hydro-Lyases