Smart Microbial Cells Couple Catalysis and Sensing to Provide High-Throughput Selection of an Organophosphate Hydrolase.
Ramesh K JhaCharlie E M StraussPublished in: ACS synthetic biology (2020)
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 the organophosphate hydrolase enzyme paraoxonase 1 (PON1) were rapidly screened via an engineered positive-feedback circuit: a p-nitrophenol (PNP)-specific transcription factor (TF) regulated expression of PON1, which catalyzed paraoxon breakdown and PNP production. Rare active mutant colonies, picked by simple visual fluorescence of a PON1-green fluorescent protein (GFP) fusion, were characterized. In a single screening round, high (library-scale) throughput enabled the discovery of enhanced paraoxon degradation activity in PON1, including structurally unexpected mutations.
Keyphrases
- high throughput
- transcription factor
- induced apoptosis
- single molecule
- poor prognosis
- copy number
- cell cycle arrest
- amino acid
- binding protein
- microbial community
- small molecule
- quantum dots
- genome wide
- single cell
- protein protein
- cell death
- room temperature
- endoplasmic reticulum stress
- dna binding
- signaling pathway
- long non coding rna
- energy transfer
- oxidative stress
- wild type
- genome wide identification