Construction and improvement of industrial strains play a central role in the commercial development of microbial fermentation processes. L-tryptophan producers have usually been developed by classical random mutagenesis due to its complicated metabolic network and regulatory mechanism. However, in the present study, an L-tryptophan overproducing Escherichia coli strain was developed by defined genetic modification methodology. Feedback inhibitions of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroF) and anthranilate synthase (TrpED) were eliminated by site-directed mutagenesis. Expression of deregulated AroF and TrpED was achieved by using a temperature-inducible expression plasmid pSV. Transcriptional regulation of trp repressor was removed by deleting trpR. Pathway for L-Trp degradation was removed by deleting tnaA. L-phenylalanine and L-tyrosine biosynthesis pathways that compete with L-tryptophan biosynthesis were blocked by deleting their critical genes (pheA and tyrA). The final engineered E. coli can produce 13.3 g/l of L-tryptophan. Fermentation characteristics of the engineered strains were also analyzed.