BR Brooks

C Pitzler

CJ Jeffery

CR Otey

D Milić

D-H Lee

GE Crooks

H Zhao

H-S Park

J Pleiss

JA Gerlt

JB Siegel

KA Datsenko

LMF Mendonça

O Khersonsky

O Trott

PP Cherepanov

RA Chica

RK Jha

RS Phillips

S Lutz

S-G Lee

SD Copley

SL Choi

SY Ku

T Heinisch

T Watanabe

TV Demidkina

Z Chen

Scientific Reports

AbstractGenetic 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) 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.</jats:p

Kil Koang Kwon

Dae-Hee Lee

Su Jin Kim

Su-Lim Choi

Eugene Rha

Soo-Jin Yeom

Bindu Subhadra

Jinhyuk Lee

Ki Jun Jeong

Seung-Goo Lee

Crossref

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

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

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