Mining Phage Genomes for Regulatory Elements to Develop a SynBio Toolbox for Non-Model Pseudomonads

Bacteriophages represent a largely untapped reservoir of regulatory elements that can expand the genetic toolbox for engineering non-model hosts. Using ONT-cappable-sequencing, we mapped full-length primary transcripts across five Pseudomonas phages and identified 232 transcription start sites (TSSs) and 176 transcription termination sites (TTSs), including 38 intrinsic factor-independent terminators. Motif discovery revealed 59 σ70-like promoters in KIL5, 37 in KIL3b, and unique phage-encoded RNA polymerase (RNAP) promoter motifs for KNP, ϕ2, and pphageB21, enabling discrimination between host- and phage-dependent transcriptional control. To validate these identifications, a subset of ten promoters and five terminators was quantitatively characterized in vivo in Pseudomonas fluorescens strain GL-S-306, showing promoter activities spanning a large dynamic range and terminator efficiencies allowing complete transcriptional termination. These data establish the first experimentally validated, phage-derived library of promoters and terminators for P. fluorescens and Pseudomonas syringae, providing chassis-specific regulatory parts that enable more finetuned gene-expression than current cross-species SynBio tools. Our results position phage transcriptomics as a scalable strategy for mining regulatory elements tailored to non-model bacterial hosts, while also supporting phage design and engineering efforts and accelerating the development of new synthetic biology platforms.