Dynamic Regulation of Metabolic Flux Using Orthogonal Quorum Sensing

Abstract

Dynamic regulation allows engineers to direct metabolic flux and cellular resources towards target pathways, improving production of value-added chemicals. One dynamic regulation strategy is quorum sensing (QS), a cell-to-cell communication that allows populations of cells to function as a collective. By applying QS to engineered pathways, the diversion of metabolic resources can be coupled to the population of the culture, thereby ensuring sufficient growth is achieved. These circuits can then be layered to allow for fine-tuned control of the cell.

Previous research has focused on QS systems that utilize acyl homoserine lactones (AHL) as signaling molecules. These systems are well characterized, but pairing them in layered systems is difficult due to similarities in signals, which can cause unintended switching of the opposing control system. Here, we identified orthogonal AHL systems for an independently-controlled, multi-layered regulation circuit, which was then applied to increase the production of the valuable natural products of naringenin and bisnoryangonin in Escherichia coli. To our knowledge, the resulting regulations led to the highest extracellular titers at the flask scale with a final naringenin titer of 1251.2 +/- 59.6 mg/L and a bisnoryangonin titer of 597.7 +/- 18.3 mg/L in naringenin equivalence.

In a parallel effort to obtain orthogonal QS-based regulations, we focused on expanding the available QS systems for the model organism E. coli. Specifically, the Gram-positive QS systems of Agr from Staphylococcus aureus and Com from Bacillus subtilis were implemented and subsequently improved for functionality in E. coli. These systems have tight control of expression, which was demonstrated by dynamic downregulation of the aromatic amino acid pathways via CRISPRi. The efficacy of these systems in synthetic biology was further illustrated by using T7 RNA polymerase to amplify the expression output of an Agr-controlled circuit.

Overall, this work developed and applied QS-based regulation systems to improve microbial production of value-added chemicals in E. coli.