Engineering microorganisms to overproduce interested products is an important practice in metabolic engineering. In the successful examples, overexpressing key genes of metabolic pathways is a widely used strategy for achieving overproduction (Ajikumar et al., 2010, Alper et al., 2005). The purpose is to up-regulate the flux for substrate synthesis or to intensify the shunt at key metabolic nodes toward an improved flux to targeted metabolites. Since the overproduction of natively synthesized metabolites is usually achieved by genetically manipulating metabolic pathways, identifying the key pathways and gene targets is a key step to determine gene overexpression strategies for consequential manipulations. Traditionally, completion of such tasks was largely relying on the experience of metabolic pathways and enzymatic kinetics. However, with the increasing practices of metabolic engineering in overproducing fuels, chemicals and natural products (Stephanopoulos, 2012), empirical predictions have been hardly satisfying the analysis of sophisticated pathways, such as the multiple-repeated pathways in fatty acid synthesis and the rarely explored secondary metabolite biosynthesis. Therefore, it is critical to establish a standard procedure for identifying gene overexpression strategies.