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Abstract The commonly used arabinose- and rhamnose-inducible Escherichia coli promoters, PBAD and PRha, 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 PBAD, we confirm known sites and reveal novel binding sites involved in modulating gene expression. In PRha, 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 PBAD and PRha 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 PBAD promoters and no significant increase for PRha. This work expands the genetic parts “toolkit” and increases the understanding of these important commonly used promoters. 

Transcription factor (TF)–promoter pairs have been repurposed from native hosts to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF–promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF–promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library’s distribution characteristics, we elucidate sequence–function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence–function relationship of a σ⁵⁴-dependent, butanol-responsive TF–promoter pair, BmoR-P_BMO derived from Thauera butanivorans, at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized P_BMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in P_BMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ⁵⁴-dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development.