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Metabolic engineering requires fine-tuned gene expression for most pathway optimization applications. To develop a suitable suite of promoters, traditional bacterial promoter engineering efforts have focused on modifications to the core region, especially the -10 and -35 regions, of native promoters. Here, we demonstrate an alternate, unexplored route of promoter engineering through randomization of the UP element of the promoter-a region that contacts the alpha subunit carboxy-terminal domain instead of the sigma subunit of the RNA polymerase holoenzyme. Through this work, we identify five novel UP element sequences through library-based searches in Escherichia coli. The resulting elements were used to activate the E. coli core promoter, rrnD promoter, to levels on par and higher than the prevalent strong bacterial promoter, OXB15. These relative levels of expression activation were transferrable when applied upstream of alternate core promoter sequences, including rrnA and rrnH . This work thus presents and validates a novel strategy for bacterial promoter engineering with transferability across varying core promoters and potential for transferability across bacterial species.