Smart microbial cells couple catalysis and sensing to provide high throughput selection of an organophosphate hydrolase.

Abstract

Enzyme engineering for gain-of-function requires navigating a large combinatorial sequence space efficiently. Typically, many mutations are needed to get significant improvements while a single 'bad' mutation can inactivate the enzyme. To establish high throughput screening and achieve enhanced resolution between two variants, genetic libraries of organophos-phate hydrolase enzyme (PON1) were rapidly screened via an engineered positive feedback circuit: a PNP-specific transcrip-tion factor (TF) regulated expression of PON1, which catalyzed paraoxon breakdown and PNP production. Rare active mu-tant colonies, picked by simple visual fluorescence of a PON1-GFP (Green Fluorescent Protein) fusion, were characterized. In a single screening round, high (library-scale) throughput enabled discovery of enhanced paraoxon degradation activity in PON1 including structurally unexpected mutations.

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Identifiers

DOI

10.1021/acssynbio.0c00025

Journal

ACS Synthetic Biology

Year

2020