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.
Highlights
The recent progress in synthetic biology has encouraged the synthesis of custom-made enzymes and directed the rapid evolution of new specificities[1,2,3]
In the present study, using screening based on phenol-detecting transcription factors (Tx) we tested the large diversities of a focused library with multiple combinations of five mutations near the substrate site of TIL, which were targeted by the comparison of structural homologues and related sequences
The diversity libraries were analysed using flow cytometry, which enables cell sorting based on cellular fluorescence and which is increasingly being used for this sort of analysis[13,14,15]
Summary
The recent progress in synthetic biology has encouraged the synthesis of custom-made enzymes and directed the rapid evolution of new specificities[1,2,3]. The practical problem at this stage is the limited throughput of the screening used to identify positives, which is usually too slow to cover the genetic diversity These rapid profiling and screening issues have become more important with the rise of synthetic biology, which requires the optimisation of multiple amino acids to generate custom-made activities or specificities in target structures[6,7]. In the present study, using screening based on phenol-detecting transcription factors (Tx) we tested the large diversities of a focused library with multiple combinations of five mutations near the substrate site of TIL, which were targeted by the comparison of structural homologues and related sequences. The fluorescence signal is obtained from specific Tx triggering the expression of cellular fluorescence in response to specific metabolites or in vivo enzymes
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