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.

Pyrroloquinoline alkaloids have been extensively isolated in the past three decades due to their broad-spectrum bioactivities. Although many total synthesis reports have described the unique chemistry of these tricyclic compounds, the biosynthesis of this well-studied natural product class remains elusive despite their simple structures. In this work, cryptic genes of the previously validated ammosamide biosynthetic gene cluster were subjects of in vitro biochemical investigation. Amm3, the putative FADH2-dependent tryptophan halogenase, was successfully expressed, purified, and screened for optimal crystallization conditions. Amm3 does not appear to bind flavin, nor does it halogenate a free-tryptophan substrate. Amid these experiments, it was reported by Ortega and colleagues that MibH, the Microbispora tryptophan-5-halogenase that is the closest homologue to Amm3, set the first precedent of acting on a peptide substrate. In this vein, the amm6 peptide was found to successfully express under quick and aggressive growth conditions in Escherichia coli, and appears to be a promising substrate candidate for Amm3 halogenation.

Bacterial meroterpenoids are hybrid natural products that partially originate from polyketide synthesis and become highly specialized upon prenylation with terpenoid products. The prenylation event is catalyzed by prenyltransferases (PTases), a class of enzymes that are essential to the biosynthesis of terpenoid compounds such as the napyradiomycins, merochlorins, and marinones. Biochemical assays confirm the prenylation activity of prenyltransferases NapT8 and NapT9 from the napyradiomycin pathway and Mcl23 from the merochlorin pathway in Streptomyces. The study of these PTases was extended to further investigate their structure and specifically focused on studying the substrate promiscuity of NapT8. Results confirmed that NapT8 is highly specific for its prenyl substrate in physiological assay conditions and R58 is speculated to be one of the active site residues responsible for this specificity. Additionally, NapT8 activity has been expanded in the presence of a reducing agent, seemingly producing dechlorinated side products that has not been observed before.

In a parallel study, the activity and specificity of related prenyltransferases CnqP3 and CnqP4 derived from marine Streptomyces sp. CNQ-509 were investigated in the context of a putative neomarinone/marinone gene cluster. Despite confirmation of their prenylation capabilities, results show that CnqP3 and CnqP4 did not produce any observable products with the proposed starter units of the neomarinone pathway or the marinone pathway.

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.

Nature produces a plethora of bioactive secondary metabolites with diverse hydrocarbon scaffolds and bioactivities. These natural products serve as signaling and defense molecules in their physiological settings, and as inspiration for therapeutics in the clinic. Natural product scaffolds are elaborated with functional groups that influence their biological activities. One such chemical functionality found in thousands of natural product molecules is halogenation. Reflective of the structural diversity of halogenated natural products (halometabolites), biology has evolved an equally diverse set of enzyme catalysts using numerous strategies for activating halides for addition onto various oxidation states of carbon. The last two decades have witnessed a renaissance in the discovery of mechanistically diverse halogenating enzymes due to the unprecedented opportunity to study them in the context of halometabolite biosynthetic pathways afforded by advancements in nucleic acid sequencing technologies and computational biology. Despite the fact that the majority of halometabolites are brominated compounds derived from the marine environment, the majority of halogenting enzymes characterized to date are involved in chlorination of terrestrial microbial metabolites. Chapter 2 of this thesis describes the biosynthesis of the highly brominated pyrrole-phenol marine bacterial metabolite pentabromopseudilin by the brominated marine pyrrole/phenol (Bmp) biosynthetic pathway, which revealed the first two examples of brominating enzymes—pyrrole and phenol halogenases—from a confirmed biosynthetic context. Chapter 3 provides an in-depth investigation of regiopromiscuous pyrrole halogenase Bmp2 in contrast to canonical regioselective pyrrole chlorinases. In addition to enzymes involved in the addition of halogen atoms to the aromatic building blocks, the Bmp pathway was found to contain a dehalogenating enzyme (Bmp8) that partially undoes the work of the Bmp2 to allow a free bromopyrrole moiety to participate in the final step of pentabromopseudilin biosynthesis. Chapter 4 describes the activity and mechanism of Bmp8 as the first example of a dehalogenating tailoring enzyme from the confirmed context of a halometabolite biosynthetic pathway. Chapter 5 of this thesis concludes with an exploration of additional opportunities in the study of natural products containing halogenated pyrrole moieties, describing a strategy for the elucidation of the biosynthesis of the bioactive pyrrole-imidazole alkaloid class of sponge secondary metabolites.

Organisms across all domains of life have developed elegant strategies to produce specialized metabolites, also referred to as natural products, for defense, structure, and communication. Natural products provide novel inspiration for pharmaceutical development given their potent biological activities and structural diversity. Recent advancements in DNA sequencing technology and the development of predictive bioinformatics tools have unveiled a vast collection of biosynthetic machinery responsible for generating this structural diversity that may be harnessed by synthetic chemists. Many of the enzymes encoded by biosynthetic genes catalyze chemical reactions that are difficult to replicate with the same selectivity by traditional synthetic methods. An example of this discrepancy is illustrated by the current enzymatic and chemical methods known to generate a highly reactive ortho-quinone methide (o-QM) intermediate. The utility of o-QMs in total synthesis has been hampered by complications that surround the preparation of their precursors, the harsh generation methods, and poor chemo-, regio-, and stereoselective control. In contrast, multiple enzymes have been reported to catalyze o-QM formation under mild conditions with remarkable selectivity. Chapter 1 of this dissertation examines several different enzymatic strategies developed to generate o-QMs. Chapter 2 describes progress towards the biomimetic total synthesis of (–)-chlorizidine A that aims to synthetically replicate the final biosynthetic intramolecular cyclization via an o-QM intermediate. Chapter 3 reports the discovery of tetrachlorizine, a novel tetrachlorinated marine natural product, and the identification of its associated biosynthetic gene cluster. Biochemical characterization of a pivotal flavin-dependent oxidase revealed a dehydrogenation reaction that proceeds via an unprecedented o-QM mechanism. This oxidase was then repurposed to generate an isolable o-QM, an extremely rare chemical motif. Chapter 4 investigates the structure-function relationships of two homologous flavin-dependent oxidases with divergent reaction selectivities and highlights their favorable biocatalytic properties. This chapter also discloses the first reported bacterial enzymes capable of performing oxidative cyclization reactions to produce cannabinoids.

Nature communicates using a vast array of chemical compounds known secondary metabolites or natural products. These diverse molecules mediate many critical ecological and developmental processes including predation, reproduction, and defense. For millennia, humans have harnessed the bioactivity of these natural products to improve human health. Many natural products have become or inspired ground-breaking drugs, such as the antiviral acyclovir or the antimalarial artemisinin. Through advancements in DNA sequencing technology, we have discovered that the instructions for making these natural products are encoded within an organism’s genome. Biosynthetic genes encode the production of powerful enzymes which act as nature’s chemists to synthesize the compounds we find in nature. Just as humans have harnessed natural products to address human needs, we have also harnessed these biosynthetic enzymes to produce a vast array of desirable molecules, including the antivirals islatravir and molnupiravir. This process is known as biocatalysis and it is a sustainable way to perform stereo- and regio-selective reactions in mild conditions with minimal byproducts.

Terpenoids are the largest class of natural products to date, with approximately 100,000 terpenoids characterized across all domains of life. Despite the massive structural diversity seen in terpenoids, all terpenoids derive from a small handful of linear polyprenyl diphosphate precursors, which are acted upon by terpene synthase enzymes. Marine sponges are well-known producers of terpenoids, with more than 4000 terpenoids reported from sponges. An unusual group of terpenoids only found in sponges and nudibranchs are the nitrogenous terpenes, which feature a suite of nitrogen-containing functional groups such as isonitriles, isothiocyanates, isocyanates, formamides, and amines. For decades, the biosynthesis of these nitrogenous terpenes remained a mystery, with natural product chemist John D Faulkner once remarking that there is “no more interesting biosynthetic study among the marine natural products.” Herein, I report my investigations into the biosynthesis of nitrogenous terpenes in marine sponges.Chapter 1 serves as an introduction to the dissertation, and includes background information on sponges, marine nitrogenous terpenes, and terpene biosynthesis across the tree of life. Chapter 2 covers the characterization of isonitrile terpenes from a San Diego sponge of the order Bubarida using a chemoselective chlorooxime probe. Thorough investigations into the biosynthesis of nitrogenous terpenes from sponges are detailed in Chapter 3. I report the discovery and characterization of the first terpene synthases from sponges and present evidence that the biosynthetic genes for terpene production are encoded within the sponge animal itself and not a microbial symbiont. Chapter 3 also includes investigations into the origin of the nitrogenous functional groups of nitrogenous terpenes.

Nature is the source of an incredible diversity of complex chemistry. The wide variety of chemical scaffolds produced as secondary metabolites have been the source of many useful medicinal therapies for the treatment of human disease, in addition to providing useful tools for probing biology. The biosynthetic machinery that produces these molecules is encoded in the genomes of organisms, and recent technological advancements in bioinformatics, DNA sequencing, and gene cloning and capture techniques offer researchers new opportunities for natural products discovery. When combined with modern analytical chemistry techniques, these methods are a powerful engine for compound discovery. The marine environment represents a unique well of biodiversity to investigate with this new methodology, with a unique evolutionary history ancient than terrestrial habitats. Chapter 2 of this dissertation describes the in-depth analysis of a collection of 146 marine actinomycetes by mass spectrometry and molecular networking to investigate their biosynthetic capacity. These bacteria were cultured in a variety of media, and their extracts were used to generate approximately 1.8 million mass spectra. Analysis of this large data set was accomplished by the Global Natural Product Social Molecular Networking platform, which allowed for the identification of known metabolites and revealed information about the production of novel metabolites. Chapter 3 of this dissertation presents a more focused study on a single biosynthetic gene cluster from an ocean streptomycete, which produces a suite of glycosylated anthracyclines. In addition to revealing the biosynthetic source of the cosmomycins and describing novel analogs of these compounds, this study describes a novel PCR-independent gene capture method utilizing the recent advances in gene synthesis technology. In Chapter 4 of this dissertation, a phosphonate biosynthetic gene cluster from another marine actinomycete, Salinispora pacifica, was investigated for small molecule production. Analysis of this gene cluster revealed the production of glyphosate degradation products, previously assumed to be anthropogenic pollutants. Bacterial production of these molecules has important implications for environmental study and monitoring of phosphonate pollutants.

Unique marine actinomycetes reside in ocean sediments and represent a resource for drug discovery. Genome sequences can be mined for new biosynthetic genes and their products.