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Around weaning, piglets are susceptible to infection by bacterial pathogens, leading to increased morbidity and mortality. We identified isolates of Rothia nasisuis in the upper respiratory tract of weaned healthy piglets that produce valinomycin in vitro and in vivo via a giant multimodule non-ribosomal peptide synthase (NRPS) enzyme complex. Valinomycin is an antiviral and antibiotic ionophore that shuttles potassium ions across membranes and is capable of inflammasome activation and apoptosis in LPS-primed macrophages. R. nasisuis inhibited growth of Streptococcus and Rothia species inhabiting the same niche. To investigate the potential for the valinomycin-producing Rothia nasisuis strain to colonize new-born piglets we performed a colonization study in newborn litters of piglets. Briefly, the newborn piglets of 3 sows farrowing on the same day were randomized between the three sows to avoid genetic bias in the experiment. The sows were housed in separate maternity pens and the piglets marked so they could be identified during the study. 9 of the 12 piglets nursed by sows A and B (further referred to as litters A and B) were orally administered approximately 10exp9 CFU of valinomycin-producing R. nasisuis, using a syringe on d2, d19 and d27 after birth. Three 3 piglets in litters A and B were not inoculated with R. nasisuis to see if they became colonized by horizontal transfer. As a control group all the piglets housed with the sow C (further referred to as litter C) were not inoculated with R. nasisuis.

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. Ch

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. Ch

Asperphenamate is a small peptide natural product that has gained much interest due to its antitumor activity. In the recent years numerous bioactive synthetic asperphenamate analogues have been reported, whereas only a handful of natural analogues either of microbial or plant origin has been discovered. Herein we describe a UHPLC-HRMS/MS and amino acid supplement approach for discovery and design novel asperphenamate analogues. Chemical analysis of Penicillium astrolabium, a prolific producer of asperphenamate, revealed three previously described and two novel asperphenamate analogues produced in significant amounts, suggesting a potential for biosynthesis of further asperphenamate analogues by varying the amino acid availability. Subsequent growth on proteogenic and non-proteogenic amino acid enriched media, revealed a series of novel asperphenamate analogues, including single or double amino acid exchange, as well as benzoic acid exchange for nicotinic acid, with the latter observed from a natural source for the first time. In total, 22 new asperphenamate analogues were characterized by HRMS/MS, with one additionally confirmed by isolation and NMR structure elucidation. This study indicates an extraordinary nonribosomal peptide synthetase (NRPS) flexibility based on substrate availability, and therefore the potential for manipulating and designing novel peptide natural products in filamentous fungi.

Fungi are well known for production of antibiotics and other bioactive secondary metabolites, that can be served as pharmaceuticals, therapeutic agents and industrially useful compounds. However, compared with the characterization of prokaryotic biosynthetic gene clusters (BGCs), less attention has been paid to evaluate fungal BGCs. This is partially because heterologous expression of eukaryotic gene constructs often requires replacement of original promoters and terminators, as well as removal of intron sequences, and this substantially slow down the workflow in natural product discovery. It is therefore of interest to investigate the possibility and effectiveness of heterologous expression and library screening of intact BGCs without refactoring in industrial friendly microbial cell factories, such as the yeast Saccharomyces cerevisiae. Here, we discuss the importance of developing new research directions on library screening of fungal BGCs in yeast without refactoring, followed by outlooking prominent opportunities and challenges for future advancement.

BACKGROUND: Genome mining for biosynthetic gene clusters (BGCs) has become an integral part of natural product discovery. The >200,000 microbial genomes now publicly available hold information on abundant novel chemistry. One way to navigate this vast genomic diversity is through comparative analysis of homologous BGCs, which allows identification of cross-species patterns that can be matched to the presence of metabolites or biological activities. However, current tools are hindered by a bottleneck caused by the expensive network-based approach used to group these BGCs into gene cluster families (GCFs). RESULTS: Here, we introduce BiG-SLiCE, a tool designed to cluster massive numbers of BGCs. By representing them in Euclidean space, BiG-SLiCE can group BGCs into GCFs in a non-pairwise, near-linear fashion. We used BiG-SLiCE to analyze 1,225,071 BGCs collected from 209,206 publicly available microbial genomes and metagenome-assembled genomes within 10 days on a typical 36-core CPU server. We demonstrate the utility of such analyses by reconstructing a global map of secondary metabolic diversity across taxonomy to identify uncharted biosynthetic potential. BiG-SLiCE also provides a "query mode" that can efficiently place newly sequenced BGCs into previously computed GCFs, plus a powerful output visualization engine that facilitates user-friendly data exploration. CONCLUSIONS: BiG-SLiCE opens up new possibilities to accelerate natural product discovery and offers a first step towards constructing a global and searchable interconnected network of BGCs. As more genomes are sequenced from understudied taxa, more information can be mined to highlight their potentially novel chemistry. BiG-SLiCE is available via https://github.com/medema-group/bigslice.

The development and spread of multidrug resistant pathogens have reinforced the urgency to find novel natural products with antibiotic activity. In bacteria, orphan biosynthetic gene clusters (BGCs) far outnumber the BGCs for which chemistry is known, possibly because they are transcriptionally silent under laboratory conditions. A strategy to trigger the production of this biosynthetic potential is to challenge the microorganism with low concentrations of antibiotics, and by using a Burkholderia genetic reporter strain (Seyedsayamdost, Proc Natl Acad Sci 111:7266-7271), we found BGC unsilencing activity for the antimicrobial andrimid, produced by the marine bacterium Vibrio coralliilyticus. Next, we challenged another marine Vibrionaceae, Photobacterium galatheae, carrier of seven orphan BGCs with sub-inhibitory concentrations of andrimid. A combined approach of transcriptional and chemical measurements of andrimid-treated P. galatheae cultures revealed a 10-fold upregulation of an orphan BGC and, amongst others, a 1.6-2.2-fold upregulation of the gene encoding the core enzyme for biosynthesis of holomycin. Also, addition of andrimid caused an increase, based on UV-Vis peak area, of 4-fold in production of the antibiotic holomycin. Transcriptional measurements of stress response related genes in P. galatheae showed a co-occurrence of increased transcript levels of rpoS (general stress response) and andrimid induced holomycin overproduction, while in trimethoprim treated cultures attenuation of holomycin production coincided with a transcriptional increase of recA (SOS stress response). This study shows that using antimicrobial compounds as activators of secondary metabolism can be a useful strategy in eliciting biosynthetic gene clusters and facilitate natural product discovery. Potentially, such interactions could also have ecological relevant implications.

A widely accepted species concept for bacteria has yet to be established. As a result, species designations are inconsistently applied and tied to what can be considered arbitrary metrics. Increasing access to DNA sequence data and clear evidence that bacterial genomes are dynamic entities that include large numbers of horizontally acquired genes have added a new level of insight to the ongoing species concept debate. Despite uncertainties over how to apply species concepts to bacteria, there is clear evidence that sequence-based approaches can be used to resolve cohesive groups that maintain the properties of species. This cohesion is clearly evidenced in the genus Salinispora, where three species have been discerned despite very close relationships based on 16S rRNA sequence analysis. The major phenotypic differences among the three species are associated with secondary metabolite production, which occurs in species-specific patterns. These patterns are maintained on a global basis and provide evidence that secondary metabolites have important ecological functions. These patterns also suggest that an effective strategy for natural product discovery is to target the cultivation of new Salinispora taxa. Alternatively, bioinformatic analyses of biosynthetic genes provide opportunities to predict secondary metabolite novelty and reduce the redundant isolation of well-known metabolites. Although much remains to be learned about the evolutionary relationships among bacteria and how fundamental units of diversity can be resolved, genus and species descriptions remain the most effective method of scientific communication.

A widely accepted species concept for bacteria has yet to be established. As a result, species designations are inconsistently applied and tied to what can be considered arbitrary metrics. Increasing access to DNA sequence data and clear evidence that bacterial genomes are dynamic entities that include large numbers of horizontally acquired genes have added a new level of insight to the ongoing species concept debate. Despite uncertainties over how to apply species concepts to bacteria, there is clear evidence that sequence-based approaches can be used to resolve cohesive groups that maintain the properties of species. This cohesion is clearly evidenced in the genus Salinispora, where three species have been discerned despite very close relationships based on 16S rRNA sequence analysis. The major phenotypic differences among the three species are associated with secondary metabolite production, which occurs in species-specific patterns. These patterns are maintained on a global basis and provide evidence that secondary metabolites have important ecological functions. These patterns also suggest that an effective strategy for natural product discovery is to target the cultivation of new Salinispora taxa. Alternatively, bioinformatic analyses of biosynthetic genes provide opportunities to predict secondary metabolite novelty and reduce the redundant isolation of well-known metabolites. Although much remains to be learned about the evolutionary relationships among bacteria and how fundamental units of diversity can be resolved, genus and species descriptions remain the most effective method of scientific communication.

A widely accepted species concept for bacteria has yet to be established. As a result, species designations are inconsistently applied and tied to what can be considered arbitrary metrics. Increasing access to DNA sequence data and clear evidence that bacterial genomes are dynamic entities that include large numbers of horizontally acquired genes have added a new level of insight to the ongoing species concept debate. Despite uncertainties over how to apply species concepts to bacteria, there is clear evidence that sequence-based approaches can be used to resolve cohesive groups that maintain the properties of species. This cohesion is clearly evidenced in the genus Salinispora, where three species have been discerned despite very close relationships based on 16S rRNA sequence analysis. The major phenotypic differences among the three species are associated with secondary metabolite production, which occurs in species-specific patterns. These patterns are maintained on a global basis and provide evidence that secondary metabolites have important ecological functions. These patterns also suggest that an effective strategy for natural product discovery is to target the cultivation of new Salinispora taxa. Alternatively, bioinformatic analyses of biosynthetic genes provide opportunities to predict secondary metabolite novelty and reduce the redundant isolation of well-known metabolites. Although much remains to be learned about the evolutionary relationships among bacteria and how fundamental units of diversity can be resolved, genus and species descriptions remain the most effective method of scientific communication.