Natural products display impressive activities against a wide range of targets, including viruses, microbes and tumors. However, their clinical use is hampered frequently by their scarcity and undesirable toxicity. Not only can engineering Escherichia coli for plasmid-based pharmacophore biosynthesis offer alternative means of simple and easily-scalable production of valuable yet hard-to-obtain compounds, but also carries a potential for providing a straightforward and efficient means of preparing natural product analogs. Quinomycin family of nonribosomal peptides, including echinomycin and SW-163s, are important secondary metabolites imparting antibiotic antitumor activity via DNA bisintercalation. Previously we have shown successful production of echinomycin in E. coli. Here we present successful engineering of the E. coli echinomycin biosynthetic pathway for the production of a synthetic analog TANDEM by site-specific mutagenesis of an echinomycin biosynthetic gene, and a novel bioactive compound by introduction of a gene from the newly identified SW-163 biosynthetic gene cluster.