Chemical, biochemical and molecular genetic interrogation of biosynthetically robust marine cyanobacteria

Marine cyanobacteria exhibit a high production of bioactive and structurally diverse natural products. A number of these secondary metabolites or their derivatives are lead compounds in drug development programs aimed at providing new therapies to treat cancer, bacterial infections, inflammatory responses, and in crop protection to kill harmful microbial pathogens and insects. Isolation and structural analysis of marine and terrestrial cyanobacterial natural products has provided access to a large number of mixed non-ribosomal peptide synthetase/ polyketide synthase (NRPS/PKS) systems. In chapter 1 of this dissertation I provide a brief history of marine natural products, arguing that the discipline was slow to begin but has grown to become a highly valuable and resourceful discipline. Also, I provide background on the genetic architecture of secondary metabolite pathways citing examples of both canonical and less-common catalytic domains in these megasynthases. Chapter 2 profiles the biochemical robustness of the marine cyanobacterium, Lyngbya bouillonii. Due in part to little physiological variation among marine cyanobacteria, I argue that many of the compounds attributed to a Lyngbya sp. or Lyngbya majuscula were likely isolated from L. bouillonii. Chapters 3 and 4 are dedicated to the attempt of isolating the apratoxin A gene cluster from two different L. bouillonii strains. The first of these chapters describes my effort to use traditional molecular genetic techniques on a strain that was not growing in culture. This approach resulted in the isolation of a pathway inconsistent with the predicted biosynthesis of apratoxin A. In my second attempt, described in chapter 4, I used the new and fundamentally necessary approach of single cell isolation and whole genome amplification, together with traditional techniques to successfully target and isolate the apratoxin gene cluster. The project described in chapter 5 provided me the opportunity to investigate another dimension of marine cyanobacteria. I assayed the biochemical activity of the specialized ECH₁, ECH₂ and ER catalytic domains. I was able to show that the jamaicamide enzymes were catalytically active towards the non-chlorinated jam substrat