Evolution-guided engineering of small-molecule biosensors

Abstract

Allosteric transcription factors (aTFs) have proven widely applicable for biotechnology and synthetic biology as ligand-specific biosensors enabling real-time monitoring, selection and regulation of cellular metabolism. However, both the biosensor specificity and the correlation between ligand concentration and biosensor output signal, also known as the transfer function, often needs to be optimized before meeting application needs. Here, we present a versatile and high-throughput method to evolve prokaryotic aTF specificity and transfer functions in a eukaryote chassis, namely baker's yeast Saccharomyces cerevisiae. From a single round of mutagenesis of the effector-binding domain (EBD) coupled with various toggled selection regimes, we robustly select aTF variants of the cis,cis-muconic acid-inducible transcription factor BenM evolved for change in ligand specificity, increased dynamic output range, shifts in operational range, and a complete inversion-of-function from activation to repression. Importantly, by targeting only the EBD, the evolved biosensors display DNA-binding affinities similar to BenM, and are functional when ported back into a prokaryotic chassis. The developed platform technology thus leverages aTF evolvability for the development of new host-agnostic biosensors with user-defined small-molecule specificities and transfer functions.

High-Resolution Scanning of Optimal Biosensor Reporter Promoters in Yeast

“…While there is limited mechanistic evidence on how prokaryotic transcriptional activators function in eukaryotes 34 , aTF members of the LTTR subfamily exert transcriptional regulation in prokaryotes by binding as tetramers to two operators, the regulatory binding site (RBS) and activating binding site (ABS), simultaneously 22 . In the OFF state this causes DNA bending, thereby preventing access of the RNA polymerase to specific sequences of the target promoter called UP, whereas in the ON state the aTF is displaced from the ABS which subsequently causes relaxation of DNA bending and closer proximity of the RNA polymerase to UP, and ultimately promotes transcription 35,36 .…”

Rapid, Low-Cost Detection of Water Contaminants Using RegulatedIn VitroTranscription

“…As an example of this, we were able to use the ROSALIND platform to rapidly validate a putative aTF, CtcS, by showing its ability to bind to an identified operator site and induce with chlortetracycline. In addition, the platform should be easily extensible through further engineering of the aTF or operator sequences to enhance sensitivity, modify specificity, and otherwise tune system performance [36,[72][73][74].…”

Transcriptional factor engineering in microbes for industrial biotechnology

“…Besides, the mutagenesis targeting the EBD domains led to the emergence of DNA binding activity in prokaryotes. 41 In the benzoate-degrading soil, bacterium Acinetobacter baylyi ADP, TF paralog BenM and CatM had overlapping but distinct functions. Tumen-Velasquez et al carried out a structure-based protein design and demonstrated that the capability of responding to multiple signals could be integrated.…”