Regulatory, genetic, and genomic investigations of natural products biosynthesis in marine cyanobacteria

Natural products from the marine environment constitute one of the richest and most reliable sources of bioactive molecules for drug discovery and biotechnology. Filamentous marine cyanobacteria are among the most prolific natural product producers, and with significant advances in genetics and genomics over the past decade are gaining increasing recognition for unusual or unprecedented biochemistry involved in natural product biosynthesis. However, the further development of several of these cyanobacterial secondary metabolites has not been accomplished because of low yields from field collections, slow growth of these organisms in laboratory cultures, and an absence of any genetic techniques for DNA manipulation.The aim of the research presented in this dissertation is to study the biosynthesis of natural products from the marine filamentous cyanobacterium Lyngbya majuscula to investigate how access to these compounds can be improved and the full potential of cyanobacterial strains can be realized. A variety of experiments are described herein, including: 1) A transcriptional analysis of the neurotoxin jamaicamide gene cluster from Lyngbya majuscula, which employed RT-PCR and reporter gene assays to better understand how cyanobacterial biosynthetic pathways are transcribed; 2) Investigations into jamaicamide regulation, which included a) protein pulldown assays to isolate possible light related regulatory proteins for this pathway and b) the development of a novel mass spectrometry approach to monitor natural product biosynthetic turnover, the effect of environmental parameters on this turnover, and confirm the impact of light on jamaicamide production; 3) Evaluating different methods for transferring DNA into Lyngbya filaments and taking the first steps in heterologously expressing portions of a Lyngbya gene cluster in the actinomycete Streptomyces coelicolor, and 4) Sequencing the genome of Lyngbya majuscula 3L, thereby revealing its natural product potential, a complex gene regulatory network and a surprising inability to fix atmospheric nitrogen. Collectively, these experiments provide new perspective on natural product biosynthesis in Lyngbya strains and direction on how these organisms and their natural products can be harnessed for biomedical and biotechnological applications