Metagenomic Mining of Natural Prokaryotic Regulatory Elements for Construction of of Trans-Species Genetic Circuits

Synthetic biologists rely on well-characterized genetic components to modularly assemble increasingly sophisticated gene circuits with specified function. Recent advances in high-throughput DNA sequencing and synthesis have greatly increased our ability to generate and test new genetic parts. Natural enzymes and regulatory proteins have been systematically screened for new functionality, while non-coding cis-regulatory elements have been characterized to better understand biophysical parameters, parts composability, contextual robustness and regulatory logic for building more complex gene circuits. Most commonly used bacterial regulatory components (i.e. promoters, ribosome binding sites) are derived from mutational variants derived from a few natural E. coli seed sequences of limited genetic diversity. Efforts to utilize these sequences in new host contexts have often failed and we have limited mechanistic understanding of how bacteria vary in terms of activating gene expression. For many industrially useful microbes, this lack of regulatory sequence reusability has necessitated creation of host-specific parts, which often have limited range and tunability of expression and have not been robustly characterized in new sequences contexts. Development of truly standardized regulatory parts with predictable activity levels in diverse hosts would greatly accelerate our ability to engineer new organisms and entire microbial communities.

Here, we report the mining of 184 phylogentically diverse microbial genomes to yield a library of ~30,000 natural 5’ regulatory elements for which we systematically quantified their transcription and translation levels across different bacterial species and growth conditions using high throughput DNA synthesis and sequencing. We identified >1000 regulatory sequences that were active in both gram positive and gram negative species, as well those with more defined host ranges. We identified host and promoter GC content as a key factors influencing activation of exogenous regulatory sequences. Based on these results, we developed “trans-species gene circuits” with preprogrammed host-ranges to generate unique patterns of outputs in different hosts. This systems and synthetic approach highlights that large-scale mining of natural cis-activating regulatory sequences constitute an important strategy for expanding our repository of functional genetic elements to build synthetic circuits with new layers of sophistication in multi-species bacterial communities.