Mapping the phenotypic landscape of a transcriptional repressor using Deep Mutational Scanning and Growth-based Quantitative Sequencing

Published

December 2, 2025

Author(s)

Zachary Jansen, Xuan Le, Qiyao Wei, Devon Kulhanek, Nina Alperovich, Olga Vasilyeva, Andrew Gilmour, David Ross, Ross Thyer

Abstract

CymR is a TetR-family transcriptional repressor that recognizes a well-defined operator sequence in the promoter PcymRC. The native ligand cuminic acid and a series of structurally related aromatic acids can bind at an allosteric site and induce a conformational change in CymR, which results in release from the DNA operator and de-repression of the promoter. The amino acid residues that contribute to these core functions have not been mapped in this regulator, nor has the protein been subjected to extensive mutagenesis to modify its function. Here, for the first time, we integrate Deep Mutational Scanning (DMS) with Growth-based Quantitative Sequencing (GROQ-Seq) to evaluate a comprehensive phenotypic landscape of CymR variants, including single amino acid insertions and deletions, spanning a concentration gradient of small molecule inducers. Using this approach, we construct an induction curve for all variants in the library. From this analysis, we identify amino acids throughout the protein that are essential for repressor function and discover several mutations that improve the sensitivity of CymR to the small molecule ligand perillic acid. In addition, rarely investigated insertion mutants are revealed to be a key driver of novel phenotypes, including several regions of CymR where insertions result in an inverted phenotype and the isolation of a variant exhibiting an unusual band-stop phenotype.

Citation

Nucleic Acids Research

Pub Type

Journals

Keywords

Deep-Mutational-Scanning, CymR, Protein-Engineering, GROQ-Seq, library generation, inverted phenotype

Citation

Jansen, Z. , Le, X. , Wei, Q. , Kulhanek, D. , Alperovich, N. , Vasilyeva, O. , Gilmour, A. , Ross, D. and Thyer, R. (2025), Mapping the phenotypic landscape of a transcriptional repressor using Deep Mutational Scanning and Growth-based Quantitative Sequencing, Nucleic Acids Research (Accessed December 3, 2025)