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.
Highlights
Bacteria have evolved diverse mechanisms to sense and adapt to changes in the environment
By searching for motifs in regions located between eilR homologs and divergently aligned eilA homologs in gamma-proteobacteria, we identified a 24-bp consensus motif eilOc, consisting of two conserved, inverted 11-bp sequences separated by two base pairs that is represented twice in these intergenic regions
Using an electrophoretic mobility shift assay (EMSA) to test the affinity of purified EilR to each of the two native DNA sequences and to the consensus operator, we confirmed their function as EilR binding sites
Summary
Bacteria have evolved diverse mechanisms to sense and adapt to changes in the environment. While strong promoters are often required in such applications, gene overexpression causes a general metabolic burden on cells[5], and heterologous expression is often toxic to the host organism[3,6] These stresses result in reduced growth rates and increase the risk of plasmid loss and escape mutants when using constitutive or leaky promoters[7], leading to poor productivities[2,6,8]. Alternative induction strategies include the use of promoters that are activated by cell density-dependent signals[22], by starving cells of an essential nutrient[23], or by dynamic pathway regulation controlled by intermediates[24] These systems do not depend on the addition of inducing compounds, the timing and level of expression are generally difficult to control, and in many instances reduce metabolic activity and require host engineering or stringent media compositions[25,26]. Alluding to the source of EilR from a rainforest bacterium, we named the resulting induction system “Jungle Express” (JEx)
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