Amino acids producing strains have traditionally been developed by random mutation and selection process. These approaches are now in transition towards rational metabolic engineering, in which the entire metabolic and regulatory networks are purposefully engineered. Here, this thesis focuses on the rational metabolic engineering of Escherichia coli for L-isoleucine production. First, feedback inhibitions of aspartokinase I, III and threonine dehydratase by L-threonine, L-lysine and L-isoleucine, respectively, were removed, and the native promoter containing the transcriptional attenuator leader region of the thrABC operon was replaced with the tac promoter. The metA, lysA, tdh and iclR genes were deleted to make more precursors available for L-isoleucine formation. The native promoter of the ppc gene was replaced with the trc promoter in the chromosome to increase the pool of oxaloacetate, a starting precursor of L-isoleucine biosynthesis. This strain was further engineered to remove all the negative regulations and overexpress the ilvA, ilvIH, ilvCED genes constituting an L-isoleucine biosynthesis. Then, branched-chain amino acid exporter encoded by ygaZH was amplified by plasmid-based overexpression, and global regulator encoded by lrp gene was amplified by chromosomal replacement in order to further improve the strain’s performance. The final engineered E. coli strain was able to produce L-isoleucine with a yield of 0.14 g L-isoleucine per g glucose, and 9.46 g/L L-isoleucine by fed-batch culture. The approaches described in this study are a good example of constructing 100% genetically defined microorganisms by rational metabolic engineering for the production of L-isoleucine, and they can also be generally applicable to development of strains for the efficient production of other valuable bioproducts.