UC San Diego

Microorganisms are prolific producers of natural product small molecules, many of which have been developed into life-saving human drugs. Over the last century, naturally produced chemical compounds have been isolated and characterized from all kingdoms of life, but only recently have we begun to understand the genetic underpinnings of natural product biosynthesis in living cells, an endeavor which has been particularly fruitful in bacteria. Bacteria have relatively simpler genomes compared to higher organisms; furthermore, bacteria tend to cluster elements involved in production of specialized small molecules along their genomes in ‘biosynthetic gene clusters,’ or BGCs. BGCs encoding non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) assembly line biosynthetic machineries have become easy to detect in sequenced bacterial genomes using automated bioinformatic tools, leading to the discovery of many more uncharacterized or ‘cryptic’ BGCs than isolated natural products identified from laboratory culture. Therefore, the work described in this dissertation focuses on the development of tools to characterize cryptic BGCs through heterologous expression and genetic manipulation. While initial efforts focused on terrestrial sources due to ease of access and historical precedence, pioneering work has led to the recognition that marine bacteria are also a rich source of bioactive compounds. Chapter 2 of this dissertation describes the development of a Salinispora tropica heterologous host, S. tropica CNB-4401, which represents the first marine actinobacterial host for natural product BGC expression. Alternatively, chapter 3 describes the development of a broad-host-range expression vector compatible with Gram-negative proteobacterial hosts, using the violacein BGC from the marine γ-proteobacterium Pseudoalteromonas luteoviolacea 2ta16 as a proof-of-principle. This work not only introduces a new genetic platform for cloning and heterologous expression of BGCs in Gram-negative hosts, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility. Finally, chapter 4 describes the thorough interrogation of two avant-garde hybrid NRPS/PKS assembly line biosynthetic pathways from marine α-protoebacteria responsible for producing a diverse group of immunosuppressive cyclic lipodepsipeptides called the thalassospiramides. This work leverages newly developed tools for cloning, expression, and precise genetic manipulation of large BGCs in a Pseudomonas heterologous host.