PROJECT SUMMARY Macrocyclic peptides are effective scaffolds for cancer drug discovery because they can combine the metabolic stability and target specificity of biologics with oral bioavailability and membrane permeability of small molecules. The bicyclic octapeptide moroidin is a promising lead structure for anticancer therapy. Moroidin is characterized by an N-terminal pyroglutamate and two tryptophan side-chain cross-linkages. Due to low-yield isolation from source-plant material and synthetic challenges toward macrocyclic complexity and chirality, an optimized biosynthetic route is needed to diversify and produce moroidin analogs in a heterologous expression system. Moroidin is a ribosomally synthesized and post-translationally modified peptide derived from autocatalytic Japanese kerria peptide cyclase KjaBURP, which installs the tryptophan-crosslinks to a leucine and a histidine side chain in a copper-dependent reaction. KjaBURP encodes four moroidin core peptide motifs attached to the C-terminal catalytic BURP domain. Based on preliminary work, I hypothesize that the second moroidin ring can be exploited to generate hundreds of analogs with chemopreventative potential. In Specific Aim 1, metabolic engineering of the moroidin pathway via heterologous expression of moroidin cyclase constructs such as KjaBURP in Nicotiana benthamiana will maximize biosynthetic access to natural moroidins and its analogs. Specific Aim 2 seeks to generate a small library of semi-pure moroidin analogs in transgenic tobacco by engineered KjaBURP, test their cytotoxic activity against cancer cell lines, and use various high-throughput scoring methods to propose a mechanism of action for dose-responsive peptides. The proposed research of moroidin pathway engineering is an approach to generate and biologically screen new plant peptide libraries for utility in cancer drug discovery.