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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Jha RK,Kern TL,Fox DT,M Strauss CE

doi

10.1093/nar/gku444

subject

Has Abstract

pub_date

2014-07-01 00:00:00

pages

8150-60

issue

12

eissn

0305-1048

issn

1362-4962

pii

gku444

journal_volume

42

pub_type

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