Jungle Express is a versatile repressor system for tight transcriptional control

Abstract

Tightly regulated promoters are essential for numerous biological applications, where strong inducibility, portability, and scalability are desirable. Current systems are often incompatible with large-scale fermentations due to high inducer costs and strict media requirements. Here, we describe the bottom-up engineering of ‘Jungle Express’, an expression system that enables efficient gene regulation in diverse proteobacteria. This system is guided by EilR, a multidrug-binding repressor with high affinity to its optimized operator and cationic dyes that act as powerful inducers at negligible costs. In E. coli, the engineered promoters exhibit minimal basal transcription and are inducible over four orders of magnitude by 1 µM crystal violet, reaching expression levels exceeding those of the strongest current bacterial systems. Further, we provide molecular insights into specific interactions of EilR with its operator and with two inducers. The versatility of Jungle Express opens the way for tightly controlled and efficient gene expression that is not restricted to host organism, substrate, or scale.

Comparative analysis of inducible promoters in cyanobacteria

“…In contrast to cyanobacteria, there has been ongoing, successful work published for more accessible model organisms such as E. coli . For example, Ruegg et al reported the optimization of a promoter system in E. coli , previously identified in Enterobacter lignolyticus 24 , which responds to a variety of cationic dyes at very low, non-toxic concentrations, including the cheap inducer compound crystal violet, for which they report a dynamic range of four orders of magnitude.…”

Environmental impacts and limitations of third‐generation biobutanol: Life cycle assessment of n‐butanol produced by genetically engineered cyanobacteria

“…However, there is a large uncertainty as to whether such high partitioning factors can be sustained over long cultivations (Clark et al., ). Further, the “metabolic switch” is achieved in the laboratory with high‐cost inducer molecules (Shabestary et al., ), though significantly cheaper options have recently been reported (Ruegg et al., ). Regarding the CO 2 ‐uptake data, it is likely that actual CO 2 ‐uptake rates in a scaled process will be “higher” than those used here, as there are several genetically tractable Synechococcus cyanobacteria strains that assimilate CO 2 at least twofold faster than Synechocystis and exhibit high photosynthetic efficiency across range of light and salinity conditions (Clark et al., ; Jaiswal et al., ; Ungerer et al., ).…”

Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology

“…The creation of multiple DSBs is lethal to bacterial cells; therefore, a strain containing I-CeuI cannot survive unless the expression of I-CeuI is under very tight control. To choose the optimal genetic circuit to express I-CeuI endonuclease for chromosomal degradation, we screened a variety of tight regulatory systems (23,24). The TetR family repressors TetR (pJKR-H-TetR) and EilR (pRH121) (SI Appendix, Table S1) showed very low basal expression when not induced but a high level of expression when induced (23,24).…”

“…To choose the optimal genetic circuit to express I-CeuI endonuclease for chromosomal degradation, we screened a variety of tight regulatory systems (23,24). The TetR family repressors TetR (pJKR-H-TetR) and EilR (pRH121) (SI Appendix, Table S1) showed very low basal expression when not induced but a high level of expression when induced (23,24). Thus, the I-CeuI gene was placed under control of the anhydrotetracycline (ATc) and crystal violet inducible TetR and EilR genetic circuits (SI Appendix, Figs.…”