We have developed a pathway design and optimization scheme that accommodates genetically and/or environmentally derived operational constraints. The full set of theoretically optimal pathways have been expressed in terms of the underlying elementary flux modes and then the sensitivity of the optimal yield to a wide class of physiological perturbations has been examined. We have taken succinate production as our model system. An in-depth study of the anaerobic metabolic fluxes of various mutant strains of Escherichia coli overexpressing the Lactococcus lactis pyruvate carboxylase for the production of succinate was performed.  A metabolic network design that includes an active glyoxylate pathway has been implemented to increase succinate yield from glucose.  The design consists of 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).  Strains were characterized to understand their metabolic response and to determine the significance of the fermentative and the glyoxylate pathways in the production of succinate.  Measured fluxes obtained under batch cultivation conditions were used to estimate intracellular fluxes and identify critical branch point flux split ratios. The comparison of changes in branch point flux split ratios at the OAA and pyruvate-PEP nodes as a result of different mutations revealed the sensitivity of succinate yield to these manipulations. The model predictions compare very favorably with experimental observations.