Evolution of enzymes with new specificity by high-throughput screening using DmpR-based genetic circuits and multiple flow cytometry rounds.

ABSTRACT: Genetic circuit-based biosensors are useful in detecting target metabolites or in vivo enzymes using transcription factors (Tx) as a molecular switch to express reporter signals, such as cellular fluorescence and antibiotic resistance. Herein, a phenol-detecting Tx (DmpR) was employed as a critical tool for enzyme engineering, specifically for the rapid analysis of numerous mutants with multiple mutations at the active site of tryptophan-indole lyase (TIL, EC 4.1.99.1). Cellular fluorescence was monitored cell-by-cell using flow cytometry to detect the creation of phenolic compounds by a new tyrosine-phenol-lyase (TPL, EC 4.1.99.2). In the TIL scaffold, target amino acids near the indole ring (Asp¹³⁷, Phe³⁰⁴, Val³⁹⁴, Ile³⁹⁶ and His⁴⁶³)

SUBMITTER: Kwon KK 

PROVIDER: S-EPMC5805759 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature