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

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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-phenollyase (TPL, EC 4.1.99.2). In the TIL scaffold, target amino acids near the indole ring (Asp(137), Phe(304), Val(394), Ile(396) and His(463)) were mutated randomly to construct a large diversity of specificity variations. Collection of candidate positives by cell sorting using flow cytometry and subsequent shuffling of beneficial mutations identified a critical hit with four mutations (D137P, F304D, V394L, and I396R) in the TIL sequence. The variant displayed one-thirteenth the level of TPL activity, compared with native TPLs, and completely lost the original TIL activity. The findings demonstrate that hypersensitive, Tx-based biosensors could be useful critically to generate new activity from a related template, which would alleviate the current burden to high-throughput screening.
Publisher
NATURE PUBLISHING GROUP
Issue Date
2018-02
Language
English
Article Type
Article
Citation

SCIENTIFIC REPORTS, v.8

ISSN
2045-2322
DOI
10.1038/s41598-018-20943-8
URI
http://hdl.handle.net/10203/240559
Appears in Collection
CBE-Journal Papers(저널논문)
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