Expanding the σ54-dependent transcription process with orthogonal designs

The significance of orthogonal gene expression lies in its ability to ensure consistent and predictable operation of genetic pathways in synthetic biology. In bacteria, σ factors are responsible for promoter recognition, where the recognition pattern of σ54 is distinct from that of σ70. Moreover, σ54-dependent promoters require bacterial enhancer-binding proteins (bEBPs) for transcription initiation, which are stringently regulated and strongly activated. Thus, σ54 appears to be a promising candidate for orthogonal designs. In this study, through knowledge-based screening and rewiring of the RpoN box in σ54, together with its partnered promoter, we identified three sets of orthogonal expression systems based on σ54-R456H, R456Y, and R456L, with different promoter preferences and ideal mutual orthogonality toward each other and the native σ54. The orthogonality is transferable, as specific transcription via σ54-R456H was demonstrated in three non-model bacteria. When combined with different bEBPs, the system can be employed to control orthogonal downstream output in response to environmental or chemical signals. The orthogonal σ54 factors proved to be capable of orthogonalizing complex biological pathways and genetic circuits. Therefore, the orthogonal transcription system will contribute to the expansion of synthetic biology toolkits, thereby providing reliable and diversified gene expression in a wide range of hosts.