Engineering of microbial cell factories requires a simultaneous optimization of several criteria such as productivity, yield, titer, stress tolerance, all the while retaining the efficient, cost-effective and robust process. One of the most prominent examples where a rational metabolic engineering strategy played a key role is in the production of 1,4-butanediol (BDO) in E. coli. In this study, we used the ORACLE (Optimization and Risk Analysis of Complex Living Entities) framework to analyze the physiology of metabolically engineered E. coli producing 1,4-butanediol (BDO) and to identify potential strategies for improved production of BDO. The framework allowed us to integrate data across multiple levels and to construct a population of large-scale kinetic models despite the lack of available information about kinetic properties of every enzyme in the metabolic pathways. We analyzed the population of these models and we found that the enzymes that primarily control the fluxes leading to BDO production are part of central glycolysis, the lower branch of tricarboxylic acid cycle and the novel BDO production route. We also identified targets for metabolic engineering for improved BDO production and yield, while constraining other intracellular states within desired bounds. We further analyzed these models to predict the effects of changes of the target enzymes on other intracellular states like energy charge, cofactor levels, redox state, cellular growth and byproduct formation. The conclusions of the performed analysis are consistent with the experimentally tested designs, and these results demonstrate the potential and effectiveness of ORACLE for the design of microbial cell factories.