Ecology and Synthetic Biology as Tools for Natural Products Discovery From Marine Proteobacteria

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Timmermans, Marshall Leo
Natural Products , Microbial Ecology , Synthetic Biology
The discovery and characterization of chemical compounds produced by microorganisms is a crucial step in the development of new drugs, as many pharmaceutical compounds currently in use are derived from bacterial sources. Marine bacteria are prolific producers of fascinating bioactive secondary metabolites with structures and bioactivities that have recently attracted a lot of attention as leads for new pharmaceutical scaffolds. With recent advances in genomic sequencing, genomic analysis, and genetic engineering, it has become clear that only a small portion of the bioactive secondary metabolites that marine bacteria are capable of producing have been characterized. This thesis is an effort to aid in uncovering new chemistries and new biosynthetic pathways among marine proteobacteria, principally guided by genomic analysis and synthetic biology to characterize biosynthetic mechanisms. This thesis will first provide a survey on the current literature on how marine proteobacteria synthesize bioactive compounds in Chapter 2. Chapter 3 describes a methodology for culturing marine proteobacteria that takes advantage of chemical ecology of marine bacteria in order to stimulate silent biosynthetic gene clusters, showing that marine proteobacteria may mediate their ecological relationships through biofilm formation and the production of bioactive secondary metabolites. Chapter 4 describes attempts at characterizing a heretofore uncharacterized biosynthetic transformation, the formation of aromatic sulfonic acid residues in the biosynthesis of the siderophore petrobactin by the marine proteobacterium Marinobacter nauticus through genomics guided genetic engineering. This chapter describes the first example of heterologous production of petrobactin in Escherichia coli and identifies a list of putative sulfonation enzymes. Lastly, Chapter 5 of this thesis describes progress towards using targeted cloning of very large silent biosynthetic gene clusters for the purpose of characterizing uncharacterized secondary metabolites from marine bacteria of the genus Pseudoalteromonas. This targeted cloning has resulted in the successful cloning of a large silent gene cluster predicted to encode a biosynthetic gene cluster for the production of a variant of the highly modified lipopeptide, alterochromide. This thesis represents the culmination of original research in uncovering a portion of the biosynthetic potential of marine bacteria and forming links to the genetic and enzymatic determinants of that biosynthetic potential.
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