(24a) Nucleotide-Level Characterization and Improvement of Inducible Transcription Factor Promoters Using a High-Throughput Approach

Bacterial transcription factor-promoter pairs exist in nature to allow dynamic regulation of gene expression based on the cellular environment, including carbon and energy sources. This capability can be useful in the development of in vivo biosensors and synthetic regulons. Transcription factor-promoter pairs are the basis for microbial logic systems, but have often suffered from low basal expression (leakiness) or low inducibility (dynamic range), both of which increase the noise to signal ratio. Additionally, operator sequences are often annotated imprecisely and with no nucleotide-level information on which residues are more important for mediation of the interaction. These transcription factor-promoter interactions are often not optimized for biotechnological applications and require further investigation. Protein−DNA binding interactions can be elucidated and ultimately manipulated by quantifying the sequence−function relationship of promoter DNA. A method for elucidating sequence−function relationships employs fluorescence-activated cell sorting (FACS) and high-throughput sequencing (called “sort-seq”).

The commonly used arabinose- and rhamnose-inducible Escherichia coli promoters, P_BAD and P_Rha, exhibit tight regulation through activation via their respective transcription factors, AraC and RhaS, alongside the cyclic AMP receptor protein. The mechanisms of these promoters have been characterized on a parts level, but nucleotide-level analysis has yet to be elucidated. Therefore, we describe here a massively parallel reporter assay that maps regulatory sites at the nucleotide level. The relative importance of nucleotides in each binding site is revealed, including loci not included in previous annotations. For P_BAD, we confirm known sites and reveal novel binding sites involved in modulating gene expression. In P_Rha, we refine the length and sequence specificity of rhaI half-sites, updating previous annotations and providing nucleotide level insights into RhaS-mediated regulation. Mutations that lead to increased promoter strength, wider dynamic range, and altered basal expression are identified for both promoters. Engineered versions of P_BAD and P_Rha promoters based on this data show improvements in dynamic range alongside a seven- and three-fold increase in promoter strength, respectively, with a slight increase in basal expression for the P_BAD promoters and no significant increase for P_Rha. This work expands the genetic parts “toolkit” and increases the understanding of these important commonly used promoters.

Additionally, we will discuss the application of this method to other transcription factor-promoter pairs including those from non-model organisms, including those from Clostridium.