Establishment of Artificial Dynamic Regulatory Network and its Application in Metabolic Engineering

In the past decades, metabolic engineering endows microorganisms with an ability to produce new chemicals. However, a big challenge of metabolic engineering remains on how to maximize the metabolic flux through exogenous pathways while maintaining the balanced state of the microbial host’s native metabolism. Dynamic pathway regulation is a promising alternative to balance endogenous metabolism and the expression of exogenous pathways. However, the current studies mainly focused on dynamically regulating the expression of exogenous pathway genes. Few research was reported to dynamically control the endogenous competing pathways, especially the essential pathways for cell growth to rewire the metabolic flux towards the target compunds. To address this issue, in this study, we designed and established an artificial dynamic regulatory network and demonstrated its effectiveness in improving the biosynthesis of a widely-used platform chemical muconic acid in Escherichia coli. The system was based on a promoter-regulator system in combination with interference antisense RNA responsive to the generated muconic acid and achieved simultaneous up- and down-regulation in an orthogonal manner. With this system, the titer of muconic acid was elevated to the highest level so far for this pathway. This artificial dynamic regulatory network provided a valuable framework to autonomously regulate the desirable pathway genes and the native competing pathway genes responsive to the changing cellular physiological state for optimal microbial production.