Dynamic Knockdown of E. coli Central Metabolism for Redirecting Fluxes of Primary Metabolites

Control of native metabolic enzyme levels is an important part of engineering strains for overproduction of heterologous compounds.  However, for many enzymes involved in central metabolism, static knockdown may result in undesired consequences, such as poor growth and poor expression of recombinant proteins.  In this work, we have developed a strategy for dynamically modulating the abundance of native enzymes within the host cell and applied this to a model system for the production of myo-inositol, an intermediate in the production of glucaric acid.  This system relies on controlled degradation of a key glycolytic enzyme, identified through a simple computational model.  Combining this with tuning of gene expression levels, we have been able to develop an E. coli strain with a “growth mode” very close to wild type and a “production mode” that shows increased yield of myo-inositol and decreased glycolytic flux.  The switch to production mode can be controlled, allowing yield, titer, and productivity trade-offs to be managed through adjustments in induction time.