Unlocking the strength of inducible promoters in Gram‐negative bacteria

Inducible bacterial promoters are ubiquitous biotechnology tools that have a consistent architecture including two key elements: the operator region recognized by the transcriptional regulatory proteins, and the −10 and −35 consensus sequences required to recruit the sigma (σ) 70 subunits of RNA polymerase to initiate transcription. Despite their widespread use, leaky transcription in the OFF state remains a challenge. We have updated the architecture of the lac and tet promoters to improve their strength, control and portability by the adaptation of the consensus −10 and −35 sequence boxes strongly targeted by σ(70), incorporation of a strong ribosome binding site recognized broadly by Gram‐negative bacteria, and independent control of the transcriptional regulators by constitutive promoters. To test the promoters, we use the far‐red fluorescent protein mCardinal, which significantly improves the signal‐to‐background ratio of promoter measurements over widely utilized green fluorescent proteins. We validate the improvement in OFF state control and inducibility by demonstrating production of the toxic and aggregate‐prone cocaine esterase enzyme CocE. We further demonstrate portability of the promoters to additional Gram‐negative species Pseudomonas putida and Vibrio natriegens. Our results represent a significant improvement over existing protein expression systems that will enable advances in protein production for various biotechnology applications.