Unlocking the strength of inducible promoters in gram-negative bacteria

Abstract

Inducible promoters, such as the lac and tet promoters, are ubiquitous biotechnology tools. Inducible bacterial promoters have a consistent architecture including two key elements: the operator region recognized by the transcriptional regulator proteins (e.g., LacI and TetR, and the -10 and -35 consensus sequences required to recruit the sigma ({sigma}) subunits of RNA polymerase to initiate transcription. Despite their widespread use in molecular biology, there remain problems with current inducible expression systems. Leaky transcription in the OFF state remains a particular challenge. Here we have updated the architecture of the lac and tet expression systems to improve their strength, control, and portability. We modified the genetic architecture of the lac and tet expression systems to contain consensus -10 and -35 sequence boxes to be strongly targeted by {sigma}70, to incorporate of a strong ribosome binding site recognized broadly by gram-negative bacteria, and to independently control of the transcriptional regulators by optimized constitutive promoters. To test the promoters, we use the far-red fluorescent protein mCardinal, which we demonstrate significantly improves the signal-to-background ratio of promoter measurement assays 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. SignificanceMany of the latest advances in pharmaceuticals, materials, and foods involve the production of recombinant proteins from bacterial hosts. However, the regulated production of enzymes and functional protein products that are toxic to their microbial hosts remains a challenge. Our work provides new tools that enable tight control over expression of protein products in bacterial host strains. We show that our tools function not only in the broadly utilized Escherichia coli, but also in other gram-negative bacteria including the soil organism Pseudomonas putida and the marine bacterium Vibrio natriegens. Our technology will facilitate more efficient production of a broader range of protein products in diverse microbial hosts.