Nonribosomal peptides (NRPs) are synthesized by modular mega-enzymes called NRP synthetases (NRPSs) that catalyze a peptide bond-forming reaction using natural amino acids as substrates. Most members of this class of natural products exhibit remarkable biological activities, but many of these valuable compounds are often difficult to obtain in sufficient quantities from their natural sources due to low production levels in the producing organisms or difficulty in culturing them. Harnessing recent progress in our genetic and biochemical understanding of the biosynthesis of these nonprimary metabolites, our laboratory has successfully developed an alternative, straightforward approach for obtaining desired natural products by placing the entire biosynthetic gene cluster in our heterologous host of choice, Escherichia coli. This effort led to the first successful de novo production of heterologous bioactive complex NRPs in E. coli. Through developing our heterologous biosynthetic system, we were able to construct a novel platform suitable for generating an NRP library through rational engineering of the natural modular assembly-line array composed of NRPSs and the auxiliary enzymes. This chapter describes the basic concept in establishing an E. coli-based plasmid-borne heterologous NRP biosynthetic system, and gives selected protocols that have been used successfully for engineering NRP biosynthesis.