Unlocking the strength of inducible promoters in Gram‐negative bacteria

Abstract

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 aggregateprone 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.

Rapid, high-titer biosynthesis of melanin using the marine bacterium Vibrio natriegens

“…It has been adopted as a wide-spread standard for laboratories recombinantly expressing proteins in E. coli . Similarly, this system has been adopted in V. natriegens ( Weinstock et al, 2016 ; Tschirhart et al, 2019 ; Carrillo Rincon and Farny, 2023 ). However, a significant drawback of this system is its tunability and leaky expression ( Rosano and Ceccarelli, 2014 ), with even small doses of IPTG resulting in dramatic gene expression, but more so for large scale biomanufacturing is the steep cost of using IPTG as an inducing molecule.…”

A dual-inducible control system for multistep biosynthetic pathways

“…Unfortunately, there is currently no a versatile dual overexpression system that permits the differential expression of distinct sets of genes through two independent inducible promoters, which can be used across various Gram-negative species. We have previously demonstrated the strength and portability of the σ 70 V2TcR and σ 70 V2/3LacI promoters separately in E. coli, P. putida, and V. natriegens Carrillo Rincón and Farny 2023). Thus, to test the independent and concurrent regulation of two proteins simultaneously from these promoters, we introduced two different reporter proteins into the dualinducible σ 70 V2TcR/V2LacI and σ 70 V2TcR/V3LacI expression systems (Fig.…”

“…Total gene expression control is crucial for regulating enzymatic reactions involved in the biosynthesis of natural products, as demonstrated with the production of terpenes presented in this study. The synthetic expression systems evaluated in this study were evaluated in the pJH0204 plasmid which contains the pColE1 origin of replication (Carrillo Rincón and Farny 2023). This plasmid has the advantage to integrate in the chromosome of P. putida and replicate in V. natriegens, therefore offers a platform to use multiple chassis (Carrillo Rincón and Farny 2023).…”

“…The inducible tet and lac promoters, optimized for σ 70 recognition, have shown precise regulation and robust gene overexpression in various Gram-negative bacteria. However, these genetic tools have only been assessed for their efficacy in producing the reporter protein mCardinal and the cocaine esterase CocE (Carrillo Rincón and Farny 2023). Multiple biological processes necessitate the coordinated activity of numerous genes, such as those involved in synthesizing natural products encoded by biosynthetic gene clusters (Jensen 2016, Meyer, Segall-Shapiro et al 2019.…”

“…Adapting the consensus sequences recognized by the σ 70 factor to the lac and tet promoters significantly enhanced their strength, surpassing the efficiency of the pET system in E. coli (Carrillo Rincón and Farny 2023). Nevertheless, the uninduced state of the optimal σ 70 V2LacI expression system was scarcely repressed by its transcriptional regulator LacI, thereby negating the advantages of having an inducible system (Carrillo Rincón and Farny 2023). One strategy to enhance the fold-change and minimize the leaky expression of the target gene, thereby reducing unnecessary consumption of cellular resources required for cell growth, involves the adaptation of toehold switch riboregulators (Hwang, Kim et al 2021).…”