A synthetic anhydrotetracycline-controllable gene expression system in Ralstonia eutropha H16

Controllable gene expression systems that are orthogonal to the host's native gene regulation network are invaluable tools for synthetic biology. In Ralstonia eutropha H16, such systems are extremely limited despite the importance of this organism in microbiological research and biotechnological application. Here we developed an anhydrotetracycline (aTc)-inducible gene expression system, which is composed of a synthetic promoter containing the operator tetO, the repressor TetR, and the inducer aTc. Using a reporter-activity based promoter library screen, we first identified the active hybrids between the tetO operators and the R. eutropha native rrsC promoter (PrrsC). Next, we showed that the hybrid promoters are repressable by TetR. To optimize the dynamic range of the system, a high-throughput screening of 300 mutants of R. eutropha phaC1 promoter was conducted to identify suitable promoters to tune the tetR expression level. The final controllable expression system contains the modified PrrsC with two copies of the tetO1 operator integrated and the tetR driven by the mutated PphaC1. The system has decreased basal expression level and can be tuned by different aTc concentrations with greater than 10-fold dynamic range. The system was used to alleviate cellular toxicity caused by AlsS overexpression, which impeded our metabolic engineering work on isobutanol and 3-methyl-1-butanol production in R. eutropha H16.