(507d) Flux Regulation At a Primary Metabolic Node: Lessons for Acetyl-CoA Derived Products

Managing carbon flux in cellular metabolism is a critical problem in metabolic engineering. Of particular interest is the acetyl-CoA node, as it is the precursor for many important bio-products.  Past methods have focused on improving product yield by reducing acetate overflow through gene deletion, electron carrier manipulation, pathway enzyme overexpression and C-limited fed-batch cultivations. However, little work has directly investigated how flux distribution changes in the context of pathway engineering. Understanding how flux changes in response to heterologous pathways is an important step toward improving product yields through metabolic engineering. 

We have estimated intracellular flux distributions over a wide range of heterologous pathway expression levels, growth rates, and media compositions to better understand how acetyl-CoA consuming pathways are regulated.  We have engineered strains of E. coli with various copy numbers of the polyhydroxybutyrate (PHB) pathway using chemically-induced chromosomal engineering (CIChE). The PHB pathway uses acetyl-CoA as a precursor and therefore competes with native acetyl-CoA consuming reactions. We tested both N-limited and C-limited conditions in chemostats and measured exchange fluxes.  Using these measured fluxes, we estimated internal fluxes using flux balance analysis (FBA). These results suggest a highly integrated tradeoff between PHB, acetate, and lactate that is constrained by ATP and NADH balances in the cell. 

Our study highlights the interdependence of heterologous pathways on global ATP and NADH levels and constrains the strategies that will be effective in improving product flux.  FBA was essential to interpret observed metabolic regulation and formulate strategies for improving production.  The results should be useful to others engineering acetyl-CoA derived products, and the strategy should be of general use in metabolic engineering.