Obtaining a Panel of Cascade Promoter-5′-UTR Complexes in <i>Escherichia coli</i>

A promoter is one of the most important and basic tools used to achieve diverse synthetic biology goals. Escherichia coli is one of the most commonly used model organisms in synthetic biology to produce useful target products and establish complicated regulation networks. During the fine-tuning of metabolic or regulation networks, the limited number of well-characterized inducible promoters has made implementing complicated strategies difficult. In this study, 104 native promoter-5′-UTR complexes (PUTR) from <i>E. coli</i> were screened and characterized based on a series of RNA-seq data. The strength of the 104 PUTRs varied from 0.007% to 4630% of that of the P_BAD promoter in the transcriptional level and from 0.1% to 137% in the translational level. To further upregulate gene expression, a series of combinatorial PUTRs and cascade PUTRs were constructed by integrating strong transcriptional promoters with strong translational 5′-UTRs. Finally, two combinatorial PUTRs (P_ssrA-UTR_rpsT and P_dnaKJ-UTR_rpsT) and two cascade PUTRs (PUTR_ssrA-PUTR_infC‑rplT and PUTR_alsRBACE-PUTR_infC‑rplT) were identified as having the highest activity, with expression outputs of 170%, 137%, 409%, and 203% of that of the P_BAD promoter, respectively. These engineered PUTRs are stable for the expression of different genes, such as the red fluorescence protein gene and the β-galactosidase gene. These results show that the PUTRs characterized and constructed in this study may be useful as a plug-and-play synthetic biology toolbox to achieve complicated metabolic engineering goals in fine-tuning metabolic networks to produce target products