Metabolic engineering of Escherichia coli was performed to construct a 100% rationally engineered strain capable of overproducing L-isoleucine, an important branched-chain amino acid. The thrABC (encoding L-threonine biosynthetic enzymes), ilvA (encoding feedback-resistant threonine dehydratase), ilvIH (encoding feedback-resistant acetohydroxy acid synthase III), and ygaZH (encoding branched-chain amino acid exporter) genes were amplified by plasmid-based overexpression. The ilvCED (encoding L-isoleucine biosynthetic enzymes) and lrp (encoding global regulator Lrp) genes were also amplified by chromosomal promoter replacement in order to further increase the flux toward L-isoleucine. The final engineered E. coli strain was able to produce 9.46 g/L of L-isoleucine with a yield of 0.14 g/g of glucose by fed-batch culture. The overall design principles described here for the production of highly regulated product should be useful in designing strains for the production of other similar bioproducts.