Metagenomic mining of regulatory elements enables programmable species-selective gene expression

Robust and predictably performing synthetic circuits rely on the use of well-characterized regulatory parts across different genetic backgrounds and environmental contexts. Here, we report the large-scale metagenomic mining of thousands of natural 5′-regulatory sequences from diverse bacteria and their multiplexed gene expression characterization in industrially-relevant microbes. We identified regulatory sequences with broad and host-specific expression properties that are robust in various growth conditions. We further observed significant differences between species’ capacity to utilize exogenous regulatory sequences. Finally, we demonstrated programmable species-selective gene expression that produces distinct and diverse output patterns in different microbes by leveraging regulatory sequences with pre-defined host-specificities. Together, these findings provide a rich resource of characterized and annotated natural regulatory sequences and a framework to engineer synthetic gene circuits with unique and tunable cross-species functionality and properties.