Method for engineering artificial bidirectional promoters

Bidirectional promoters (BDPs) are ubiquitous as they facilitate gene co-expression within functional groups of genes or metabolic pathways. They help to balance enzymatic expression to maintain a stoichiometric relationship and avoid cellular toxicity. While BDPs offer several benefits over single-gene expression cassettes, functionally defined BDPs are scarce and usually unsuited for multi-gene expression in synthetic biology and metabolic engineering efforts. In this paper, we describe the fabrication of BDPs in bacteria using DNA with a random nucleotide composition. We created 230 artificial BDPs using a direct selection method (antibiotic resistance) and 168 gene-specific BDPs using a double fusion protein device (fluorescent reporters along with antibiotic resistance). The method's simplicity, gene-specificity, and universality make it an unprecedented tool for creating artificial BDP libraries representing a wide range of expression levels. This technique addresses the challenges in obtaining balanced co-expression of heterologous genes or pathways in various hosts. Furthermore, it provides insights that help to understand the architecture and features of BDPs.