Synthetic Quorum Sensing As a Tunable Cell Density Sensor-Regulator for Dynamic Metabolic Engineering

Metabolic engineering have enabled improvements in titer and yield for a variety of chemicals produced naturally in microorganisms, as well as those produced via the synthetic metabolic pathways consist of heterogeneous enzymes. Almost all precursors for the synthetic metabolic pathways are served from the endogenous intermediates in glycolysis, such as phosphoenolpyruvate, pyruvate and acetyl-CoA. However, endogenous central metabolism also requires these key intermediates for cell growth. Accordingly, there is a trade-off relationship between the cell growth and the target chemicals production, which decrease the total productivity of target chemical. To achieve the drastic improvement in productivity, it is necessary to improve both of final cell density and the specific productivity for efficient bioprocess.

For this end, we previously constructed a synthetic genetic circuit in E. coli for control the local metabolic fluxes around acetyl-CoA, which was termed as synthetic metabolic toggle switch (MTS). The MTS was designed to redirect the metabolic flux from TCA cycle toward target chemical production in the response to the addition of the exogenous inducer IPTG. This circuit succeeded to improve titer and yield of isopropanol production with ensuring the final cell density. However, it required strict optimization of timing for the addition of IPTG in order to achieve the adequate cell density and the improvement in productivity. To solve such problem, it is desired that the engineered E. coli would sense their population and self-trigger the function of the genetic circuit in the response to appropriate cell density.

Here, we reconstructed Vibrio fischeri’s quorum sensing (QS) system, lux system, in E. coli so that it may work as a tunable cell density sensor-regulator. In native lux system, the gene expression under the QS promoter (P_lux) would be activated by transcription factor LuxR in the response to the concentration of auto-inducer AHL reaching to threshold level. AHL is produced by LuxI under control of P_lux promoter. Even though the lux system is the most well studied model QS, there is no report for its application to the microbial chemical production because the cell density upon the self-induction dependent on the QS (threshold cell density) is too much low to induce the genes’ expression for the chemical production. To elevate the threshold cell density, we constructed a synthetic lux system in E. coli by using a synthetic lux promoter (P_luxlacO) which has dual responsiveness to the natural QS activation and the repression by LacI. In this system, the self-induction of target gene expression is driven by QS-signal, and its threshold cell density can be tuned depending on the concentration of IPTG which was added at the beginning of the fermentation. We demonstrated the self-redirection of metabolic flux by the MTS integrated with the synthetic lux system at a desired cell density, which resulted in a significant increase in isopropanol production. This study demonstrated that the dynamic gene expression profiles which is designed by using the genetic circuit allow regulating the microbial phenotype plastically and can help to manage the trade-offs between the growth and the production.