UC San Diego

For much of nature the complexity and diversity of language is found not in verbal cues, but rather in genetically encoded chemical signals. These small molecules, often referred to as “natural products”, have evolved over millennia to serve as a form of language used by organisms across the tree of life. The ecological roles of these compounds are varied, and still largely unknown, but humans have a long history of using these compounds for our own benefit, most famously as medicines.These metabolites are produced through multi-step enzymatic reactions in which simple metabolic building blocks are assembled to yield a complex molecular structure. In bacteria, the enzymes necessary for these processes are encoded by genes grouped together on contiguous stretches of DNA, forming so-called ‘biosynthetic gene clusters,’ (BGCs). Connecting natural products to the genes responsible for their production is critical for the discovery of new bioactive molecules, and for expanding our knowledge of biochemical reactions which can inform sustainable, biocatalytic routes to therapeutically valuable chemicals. The overarching goal of this dissertation is to explore the biosynthesis of secondary metabolites from marine bacteria using both in vivo and in vitro strategies.

Both molecular families discussed in this thesis, streptophenazines and salinosporamides, incorporate intermediates from the shikimate pathway. Chapter 1, which serves as an introduction to the thesis, is a review of natural products derived from the shikimate pathway. Chapter 2 deals with the issue of silent BGCs and uses an in vivo, genetic engineering strategy to activate a silent BGC. This work linked the streptophenazine BGC to its molecular products for the first time and developed a series of synthetic biology tools to reliably regulate expression of BGCs. Chapter 3 uses an in vitro, biochemical approach to identify and characterize the enzyme responsible for the novel bicyclization reaction that constructs the bicyclized pharmacophore of salinosporamide A, a marine microbial natural product currently in Phase III clinical trials. Finally, Chapter 4 explores the current status of continued work on the salinosporamide pathway. The work discussed here includes both whole pathway in vivo strategies and enzyme engineering in vitro approaches towards generating novel salinosporamides with alternative bioactivities.