Directed evolution of the PobR allosteric transcription factor to generate a biosensor for 4-hydroxymandelic acid

Abstract

4-Hydroxymandelic acid (HMA) is widely applied in pharmaceuticals, food and cosmetics. In this study, we aimed to develop an allosteric transcription factors (aTFs) based biosensor for HMA. PobR, an aTF for HMA analog 4-hydroxybenzoic acid, was used to alter its selectivity and create novel aTFs responsive to HMA by directed evolution. We established a PobR mutant library with a capacity of 550,000 mutants using error-prone PCR and Megawhop PCR. Through our screening, two mutants were obtained with responsiveness to HMA. Analysis of each missense mutation indicating residues 122-126 were involved in its PobR ligand speci city. These results showed the effectiveness of directed evolution in switching the ligand speci city of a biosensor and improving HMA production.

Directed Evolution of 4-Hydroxyphenylpyruvate Biosensors Based on a Dual Selection System

“…As an aTF, PobR (or PobR WT ) drives the promoter PpobA and is highly specific for its native effector 4HB. Previous studies indicate that PobR is very difficult to be engineered using any rational approach [34,42,43]. Therefore, we used the random mutagenesis approach to modify its ligand specificity, aiming to reduce its sensitivity to 4HB but increase its responsiveness to other aromatic compounds.…”

“…An additional reporter gene (red fluorescent protein mCherry) was also added to the selection system (Figure 2). The reporters were expressed when the PobR protein underwent allosteric changes upon binding to a ligand and then activated the PpobA [34]. The red fluorescence intensity of the mCherry protein was proportional to the ligand levels in the medium and could, therefore, be used to determine the binding affinity of the ligand.…”

“…Directed evolution is often employed in the development of novel aTF-based biosensors. The first workflow in directed evolution is to construct a mutant library and then screen it, where the screening usually ought to be high-throughput and fluorescence-activated cell sorting and droplet-sorting screening methods are often employed [31][32][33][34]. However, these methods are equipment-dependent, limiting the development of novel biosensors.…”

Identifying LasR Quorum Sensors with Improved Signal Specificity by Mapping the Sequence–Function Landscape

“…Various methods have been employed to alter ligand specificity of allosteric transcription factors. Directed evolution via random mutagenesis (by error-prone PCR or gene shuffling) of the ligand binding domain or protein coding sequence has been widely used to engineer ligand specificity, including for improved molecular discrimination and for preferential specificity for molecules other than the natural cognate ligand. − While random mutagenesis can introduce numerous mutations and generate sequence diversity, it often suffers from mutational bias that hinders screening all amino acid substitutions and therefore cannot comprehensively determine sequence–function relationships. − Another approach is designing transcription factors using rational design that applies structure-based computational modeling and has also been used to achieve desired ligand specificity, − yet it requires a large amount of prior knowledge and predictions of biophysical interactions that are often not known . Other studies have chosen the middle ground of semirational design, which utilizes available structural and functional information on the transcription factor to choose the target residues and amino acid diversity for protein engineering. − Semirational design can narrow the designed sequence space to a subset within the limits of what can be experimentally tested and predicted to have a greater likelihood for a desired functionality …”

Modulating Sensitivity of an Erythromycin Biosensor for Precise High-Throughput Screening of Strains with Different Characteristics

“…Performance parameters of a biosensor, including specificity, sensitivity, dynamic range, and operating range, can be tailored through many semirational and rational approaches, , but directed evolution may be the most extensively used strategy to engineer biosensor specificity and sensitivity, especially when the crystal structure of the TF protein is unknown. − For example, in the monodispersed whole-cell biosensor chassis such as E. coli, variants of different affinities to the target effector in the random library may demonstrate different sensitivities in the dose–response curves and can be sorted by FACS or plate-based screening for the applications of interest. , Nevertheless, the screening process is usually labor-intensive and time-consuming, and the variant performance cannot be precisely predicted.…”