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) 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.

Acclimation of bacterial cell state for high-throughput enzyme engineering using a DmpR-dependent transcriptional activation system

“…The constructed plasmid was then transformed into JM109(DE3) cells harbouring pGESS for analysing the influence of RelA. Thermostable TPL (TTPL) from S. toebii was cloned into the pSHCE plasmid using NdeI and HindIII and transformed into DH5α cells harbouring pGESS (forward primer: 5′-GAT ATC ATA TGC AGC GAC CCT-3′; reverse primer: 5′-ACA GCC AAG CTT AGC TGA TCG GCT CGA AG-3′) 24 . fluorescence analysis.…”

“…We previously reported a genetic enzyme screening system (GESS) wherein the σ 54 -dependent TF DmpR has been used to sense phenolic compounds in Escherichia coli 23,24 . Other than DmpR, GESS consists of the Po promoter of the dmp operon from Pseudomonas sp.…”

“…Other than DmpR, GESS consists of the Po promoter of the dmp operon from Pseudomonas sp. and reporter proteins to facilitate analysis of enzyme-derived catalytic activity 23,24 . Thus, DmpR-GESS is a TF-based biosensor that senses phenolic compounds as products of enzymatic reactions.…”

“…Various substrates that may release phenol or para-nitrophenol could be used for different enzyme activities because these phenolic compounds generated from the enzyme reaction can induce intracellular fluorescent protein expression via phenolic compound-dependent transcriptional activation of DmpR. Indeed, we have reported the successful applications of GESS in enzyme assays and screening 24,25 . However, in enzymatic assays using DmpR-GESS, the effects of the cellular state was not studied in previous studies and culture conditions were tied strictly to the stringent conditions.…”

Development of a Metabolite Sensor for High-Throughput Detection of Aldehydes in Escherichia Coli

“…While a sensing module contains a transcriptional regulator which is activated in the presence of a target ligand, a reporter module consisting of a corresponding cognate promoter which drives transcription of a reporter gene (e.g., lacZ, gfp , and mutant variants) enables the output of measurable signals (Chong and Ching, 2016 ). The utilization of metabolite sensors has recently gained particular interest for evolution of bacterial strains (Morgan et al, 2016 ; Rogers et al, 2016 ; Liu et al, 2017 ) and enzymes (Cheng et al, 2015 ; Kwon et al, 2018 ).…”