Biosynthesis and genetic engineering of biologically active natural products from marine ascidian symbionts

Numerous marine natural products are currently being assessed clinically for their pharmacological properties. Marine invertebrates supply the highest percentage of these bioactive metabolites. A major obstacle against pursuing a marine drug lead into clinical trials is ensuring its continuous supply in enough quantities. In the following series of results, I provide alternative solutions to the marine natural products supply problem. Previously, It was shown that two didemnid ascidian metabolites, patellamide A and C, are actually produced by the cyanobacterial symbiont Prochloron didemni. In this work, I developed metagenomic methods to further investigate the biosynthesis of this class of metabolites. I discovered that similar gene clusters are responsible for the biosynthesis of more than 60 metabolites isolated from didemnid ascidians in a diversifying manner never described before. In addition, we found that this class of metabolites is present globally in free-living as well as symbiotic cyanobacteria and is worthy of a separate classification. We named this class of molecules cyanobactins, modified N-to-C terminal cyclic peptides produced by cyanobacteria. I developed universal methods for the prediction, cloning, and heterologous expression of cyanobactins from any environment. One member of cyanobactins, trunkamide, was in preclinical evaluations for its anticancer properties. As part of this work, I discovered the biosynthetic bases for iv trunkamide and many relatives thereof. I also developed a robust system that allows the heterologous production of these highly modified peptides in Escherichia coli. Using genetic engineering, I optimized this system for the production of natural and unnatural trunkamide derivatives for structure-activity-relationship studies and natural products discovery and supply. Marine invertebrates harbor a variable pool of natural products where closely related organisms can have a completely different chemical content. This variability complicates the supply problem of marine natural products and delays drug discovery efforts. Using metagenome sequencing and PCR and chemical analyses, I discovered that biosynthetic pathways are sporadically occurring in symbionts of marine ascidians with no apparent pattern. Within didemnid ascidians, this sporadic distribution contributes directly to the observed chemical differences among very closely related organisms.