Biosynthesis of natural products in marine bacteria : studies in molecular genetics, phylogeny and structural elucidation

Chapter 1 introduces marine natural products and discusses the symbiosis of bacteria and marine invertebrates as a source of these molecules. Indications for a bacterial origin of compounds isolated from invertebrates are presented and the major types of bacterial natural product biosynthesis pathways are introduced. The study of biosynthesis opens the possibility of solving the supply issue that often prevents the development of drugs from marine natural products. It also yields novel biochemical reactions and allows insight into the evolution of biosynthetic pathways. Chapter 2 explores the biosynthetic genes responsible for the production of the anticancer compound bryostatin. Bryostatin was isolated from a marine bryozoan; here a putative biosynthetic pathway from a symbiotic bacterium via a modular polyketide synthase (PKS) mechanism is presented. Symbiont DNA was enriched using ultracentrifugation techniques and lambda phage library constructed. For homology screening initially a DNA fragment was used specific for a symbiont PKS derived in earlier work by degenerate PCR Subsequently the ends of the isolated lambda fragments were used in a series of screenings. Short gaps were bridged by PCR. This chromosome walking approach yielded the entire 80 kb bry cluster. The cluster contains all the major elements needed for the production of bryostatin. A putative biosynthetic scheme is described. Chapter 3 investigates the phylogeny of Prochloron sp., a cyanobacterial symbiont of ascidians, based on 16S rRNA gene and 16S-23S internally transcribed spacer region sequences as well as denaturing gradient gel electrophoresis. Prochloron diversity is very low and there is no evidence for cospeciation with the ascidian host. This is in contrast to the vertical transmission of symbionts reported for some ascidians. Prochloron is the source of the patellamides, the relationship between phylogenetic and biosynthetic diversity is discussed. Chapter 4 presents a novel cyclic peptide, trichamide, from the free-living cyanobacterium Trichodesmium erythraeum. A gene cluster related to the patellamide biosynthetic genes from Prochloron sp. was identified by homology in T. erythraeum sequence data available in Genbank. The structure of trichamide was proposed based on sequence information and could be verified by Fourier-transfer mass spectrometr