Assembling NRPS modules in e. coli to establish a platform for rational design of biologically active compounds

UnrestrictedNonribosomal peptides (NRPs) are synthesized by modular mega–enzyme called NRP synthetase (NRPS) that catalyze a peptide bond forming reaction using natural amino acid as substrate. The majority of compounds from this class exhibit crucial biological activities such as antibiotic, immunosuppressive, antiviral, and antitumor activities. However, several of these sought-after natural products are often difficult to isolate in adequate amounts from its natural sources due to low production levels or the producing organism's stringent need for expensive and atypical culture apparatus. As we know, enzymes capable of synthesizing these natural products including essential or nonessential secondary metabolites are encoded by biosynthetic genes located on either chromosomal or plasmid DNA.; An alternative and straightforward approach to obtain these compounds is to express the entire biosynthetic gene cluster for an NRPS responsible for producing the intact molecule using E. coli as a heterologous host was developed in our laboratory. Production of triostin A (2) using small shake flask was optimized to provide a quantitative yield rivaling levels isolated from S. triostinicus. Addition of QXC, the priming unit for 2, helped to increase its production and identify the bottleneck in our system. Knock-down of two M-domain in Ecm7 provided modest amounts of 8, an unnatural natural product. A novel compound, 9, was also identified from our culture which will require additional characterization.; This study has ascertained a novel platform for creating a peptide library by means of premeditated engineering of biosynthetic domain(s) to provide a customized macromolecule