A four carbon linear chain diamine, putrescine (1,4-diaminobutane), is an important platform chemical having a wide range of applications in chemical industry. Biotechnological production of putrescine from renewable feedstock is a promising alternative to the chemical synthesis that originates from non-renewable petroleum. Here we report development of a metabolically engineered strain of Escherichia coli that produces putrescine at high titer in glucose mineral salts medium. First, a base strain was constructed by inactivating the putrescine degradation and utilization pathways, and deleting the ornithine carbamoyltransferase chain I gene argI to make more precursors available for putrescine synthesis. Next, ornithine decarboxylase, which converts ornithine to putrescine, was amplified by a combination of plasmid-based and chromosome-based overexpression of the coding genes under the strong tac or trc promoter. Furthermore, the ornithine biosynthetic genes (argC-E) were overexpressed from the trc promoter, which replaced the native promoter in the genome, to increase the ornithine pool. Finally, strain performance was further improved by the deletion of the stress responsive RNA polymerase sigma factor RpoS, a well-known global transcription regulator that controls the expression of ca. 10% of the E. coli genes. The final engineered E. coli strain was able to produce 1.68 g L−1 of putrescine with a yield of 0.168 g g−1 glucose. Furthermore, high cell density cultivation allowed production of 24.2 g L−1 of putrescine with a productivity of 0.75 g L−1 h−1. The strategy reported here should be useful for the bio-based production of putrescine from renewable resources, and also for the development of strains capable of producing other diamines, which are important as nitrogen-containing platform chemicals. Biotechnol. Bioeng. 2009; 104: 651–662 © 2009 Wiley Periodicals, Inc.