(66a) A Glucose Valve for Pathway Engineering

In engineering metabolic pathways, branch points frequently arise that compete for pathway intermediates, thus limiting productivity. Common solutions of knocking out these competing pathway genes, however, are infeasible when the heterologous pathway competes directly with central metabolism. In this study, we describe the engineering of E. coli to create a platform capable of directing unphosphorylated glucose towards heterologous pathways. We have created a viable E. coli host that decouples glucose transport and phosphorylation allowing us to independently control the flux of glucose. Through the use of glucokinase (glk) antisense RNA constructs, we are able to inhibit glk activity by up to 25% and perturb central metabolism. Further studies showed that these constructs are able to boost specific productivity in a model heterologous glucose-consuming pathway. The use of antisense RNA in this manner allows for fine analog control creating a ?glucose valve' and novel optimization opportunities for pathway development. In this talk, I will highlight some of the successes and limitations of this technology as well as future areas of development.