A Glucose-Sensing Toggle Switch Enables Autonomous Control That Improves Production but Avoids Growth-Toxicity

Currently, most biosynthetic strategies are growth-coupled. This inherently limits product yield, as Escherichia Coli convert ~50% of feedstock carbon (e.g. glucose) to biomass during cell growth. Furthermore, the available gene targets for modification are fundamentally constrained by this growth compatibility requirement.  Genes essential for growth, but detrimental to pathway flux, cannot be removed and production-enzyme levels are limited to prevent growth toxicity and system instability.  Decoupling the growth and production phases enable separate biomass (catalyst) generation and product synthesis phases.  This allows relaxed constraints during production phase to enable essential gene removal and pathway over-expression to reroute substrate flux for increase product yields.  To successfully implement this bi-phasic strategy, we have adopted the bi-stable toggle for bioprocessing. Here we demonstrate the integration of native glucose-sensing into the toggle switch to precisely and autonomously active expression of the PHB operon using smart media design.  The switch remains stably in the new state even after reintroduction of glucose. Our system enables PHB operon expression at levels that improve PHB yield compared to a constitutively expressing PHB strain.  Promisingly, early activation of the PHB operon during growth results in a severe growth-rate reduction (~5 fold lower), indicating the system generates pathway levels incompatible with growth. This result demonstrates the utility of a two-phase production strategy.  While timed induction is common in academic labs, the culture volumes at industrial scale make it infeasible due to expensive chemical inducers.  Our switch is “programmed” to induce simply by media design.  This glucose-sensitive switch allows precise control of genetic programs using only the ingredients necessary for cultivation in rich or minimal media.  It uses native promoters only for initiating the switch, which decouples the activation mechanism from the design of each genetic program.  Nutrient-sensing promoters as the activator of a product pathway are subject to native regulation, often have gradual induction and low activity, and may prevent desired pathway expression levels. This switch frees the production phase from a nutrient-limiting environment that is required for maintaining pathway activity, but is not ideal for production. The glucose toggle switch should be broadly useful to initiate any type of desired genetic program for metabolic engineering applications.