Development of an autonomous and bifunctional quorum-sensing circuit for metabolic flux control in engineered .

Metabolic engineering seeks to reprogram microbial cells to efficiently and sustainably produce value-added compounds. Since chemical production can be at odds with the cell's natural objectives, strategies have been developed to balance conflicting goals. For example, dynamic regulation modulates gene expression to favor biomass and metabolite accumulation at low cell densities before diverting key metabolic fluxes toward product formation. To trigger changes in gene expression in a pathway-independent manner without the need for exogenous inducers, researchers have coupled gene expression to quorum-sensing (QS) circuits, which regulate transcription based on cell density. While effective, studies thus far have been limited to one control point. More challenging pathways may require layered dynamic regulation strategies, motivating the development of a generalizable tool for regulating multiple sets of genes. We have developed a QS-based regulation tool that combines components of the and QS systems to simultaneously and dynamically up- and down-regulate expression of 2 sets of genes. Characterization of the circuit revealed that varying the expression level of 2 QS components leads to predictable changes in switching dynamics and that using components from 2 QS systems allows for independent tuning capability. We applied the regulation tool to successfully address challenges in both the naringenin and salicylic acid synthesis pathways. Through these case studies, we confirmed the benefit of having multiple control points, predictable tuning capabilities, and independently tunable regulation modules.