Design and implementation of metabolic networks for the improvement of product yields in cofactor-limiting systems in Escherichia coli

Metabolic engineering is the rational alteration of the genetic structure of an organism to make this organism achieve a desired goal. One important aspect of metabolic engineering is the manipulation of metabolic pathways in microorganisms to increase the yield and productivity of cofactor dependent products. When designing a metabolic network to maximize product formation from a substrate, it is crucial to take into consideration cofactor constraint and maintain a proper balance between yield and productivity. The purpose of this study is to design and optimize a metabolic network to increase the yield and productivity of cofactor dependent products taking into consideration cofactor constraint. The production of succinate, a valuable specialty chemical, was used as a model system to explore the effect of manipulating NADH in vivo as well as to study the effect of alleviating cofactor constraint through pathway engineering. Additionally the production of the biodegradable polymer poly-beta-hydroxybutyrate was used as a model system to explore the effect of manipulating NADPH availability in vivo. Currently, the maximum theoretical succinate yield under strictly anaerobic conditions through the fermentative succinate biosynthesis pathway is limited to one mole per mole of glucose due to NADH limitation. In order to surpass the maximum anaerobic theoretical succinate yield from glucose, a genetically engineered E. coli strain was constructed to meet the NADH requirement and carbon demand to produce high quantities and yield of succinate. The implemented strategic design involves a dual succinate synthesis route, which diverts required quantities of NADH through the traditional fermentative pathway and maximizes the carbon converted to succinate by balancing the carbon flux through the fermentative pathway and the glyoxylate pathway (which has a lower NADH requirement). The implementation of this metabolic network to produce succinate in E. coli increases the succinate yield from glucose to 1.6 mol/mol with an average anaerobic productivity rate of 10 mM/h. The final strain demonstrated to be stable and robust in performance. Based on the proposed stoichiometric model, the experimental estimated metabolic fluxes of this strain were in excellent agreement with theoretical optimized fluxes (Cox et al. 2005)