Publications
At Princeton University:
2022
Great progress has been made in understanding gut microbiomes’ products and their effects on health and disease. Less attention, however, has been given to the inputs that gut bacteria consume. Here, we quantitatively examine inputs and outputs of the mouse gut microbiome, using isotope tracing. The main input to microbial carbohydrate fermentation is dietary fiber and to branched-chain fatty acids and aromatic metabolites is dietary protein. In addition, circulating host lactate, 3-hydroxybutyrate, and urea (but not glucose or amino acids) feed the gut microbiome. To determine the nutrient preferences across bacteria, we traced into genus-specific bacterial protein sequences. We found systematic differences in nutrient use: most genera in the phylum Firmicutes prefer dietary protein, Bacteroides dietary fiber, and Akkermansia circulating host lactate. Such preferences correlate with microbiome composition changes in response to dietary modifications. Thus, diet shapes the microbiome by promoting the growth of bacteria that preferentially use the ingested nutrients.
Since the discovery of the kahalalide family of marine depsipeptides in 1993, considerable work has been done to develop these compounds as new and biologically distinct anti-cancer agents. Clinical trials and laboratory research have yielded a wealth of data that indicates tolerance of kahalalides in healthy cells and selective activity against diseased cells. Currently, two molecules have attracted the greates level of attention, kahalalide F (KF) and isokahalalide F (isoKF, Irvalec, PM 02734, elisidepsin). Both compounds were originally isolated from the sarcoglossan mollusk Elysia rufescens but due to distinct structural characteristics it has been hypothesized and recently shown that the ultimate origin of the molecules is microbial. The search for their true source has been a subject of considerable research in the anticipation of finding new analogs and a culturable expression system that can produce sufficient material through fermentation to be industrially relevant.
The gut microbiome contributes to host physiology and nutrition metabolism. The interaction between nutrition components and the gut microbiota results in thousands of metabolites that can contribute to various health and disease outcomes. In parallel, the interactions between foods and their toxicants have captured increasing interest due to their impact on human health. Taken together, investigating dietary interactions with endogenous and exogenous factors and detecting interaction biomarkers in a specific and sensitive manner is an important task. The present study sought to identify for the first time the metabolites produced during the interaction of diet-derived toxicants e.g., N-nitrosamines with green tea polyphenols, using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). In addition, the metabolic products resulting from the incubation of green tea with a complex gut microbiome in the presence of N-nitrosamine were assessed in the same manner. The quinone products of (epi)catechin, quercetin, and kaempferol were identified when green tea was incubated with N-nitrosamine only; whereas, incubation of green tea with N-nitrosamine and a complex gut microbiome prevented the formation of these metabolites. This study provides a new perspective on the role of gut microbiome in protecting against potential negative interactions between food-derived toxicants and dietary polyphenols.
2021
The human microbiome encodes a large repertoire of biochemical enzymes and pathways, most of which remain uncharacterized. Here, using a metagenomics-based search strategy, we discovered that bacterial members of the human gut and oral microbiome encode enzymes that selectively phosphorylate a clinically used antidiabetic drug, acarbose1,2, resulting in its inactivation. Acarbose is an inhibitor of both human and bacterial α-glucosidases3, limiting the ability of the target organism to metabolize complex carbohydrates. Using biochemical assays, X-ray crystallography and metagenomic analyses, we show that microbiome-derived acarbose kinases are specific for acarbose, provide their harbouring organism with a protective advantage against the activity of acarbose, and are widespread in the microbiomes of western and non-western human populations. These results provide an example of widespread microbiome resistance to a non-antibiotic drug, and suggest that acarbose resistance has disseminated in the human microbiome as a defensive strategy against a potential endogenous producer of a closely related molecule.
Plasmids are autonomous genetic elements that can be exchanged between microorganisms via horizontal gene transfer (HGT). Despite the central role they play in antibiotic resistance and modern biotechnology, our understanding of plasmids’ natural ecology is limited. Recent experiments have shown that plasmids can spread even when they are a burden to the cell, suggesting that natural plasmids may exist as parasites. Here, we use mathematical modeling to explore the ecology of such parasitic plasmids. We first develop models of single plasmids and find that a plasmid’s population dynamics and optimal infection strategy are strongly determined by the plasmid’s HGT mechanism. We then analyze models of co-infecting plasmids and show that parasitic plasmids are prone to a “tragedy of the commons” in which runaway plasmid invasion severely reduces host fitness. We propose that this tragedy of the commons is averted by selection between competing populations and demonstrate this effect in a metapopulation model. We derive predicted distributions of unique plasmid types in genomes-comparison to the distribution of plasmids in a collection of 17,725 genomes supports a model of parasitic plasmids with positive plasmid-plasmid interactions that ameliorate plasmid fitness costs or promote the invasion of new plasmids.
Pseudomonas aeruginosa is a significant threat in both healthcare and industrial biofouling. Surface attachment of P. aeruginosa is particularly problematic as surface association induces virulence and is necessary for the ensuing process of biofilm formation, which hampers antibiotic treatments. Previous efforts have searched for dispersal agents of mature biofilm collectives, but there are no known factors that specifically disperse individual surface-attached P. aeruginosa. In this study, we develop a quantitative single-cell surface-dispersal assay and use it to show that P. aeruginosa itself produces factors that can stimulate its dispersal. Through bioactivity-guided fractionation, mass spectrometry, and nuclear magnetic resonance, we elucidated the structure of one such factor, 2-methyl-4- hydroxyquinoline (MHQ). MHQ is an alkyl quinolone with a previously unknown activity and is synthesized by the PqsABC enzymes. Pure MHQ is sufficient to disperse P. aeruginosa, but the dispersal activity of natural P. aeruginosa conditioned media requires additional factors. Whereas other alkyl quinolones have been shown to act as antibiotics or membrane depolarizers, MHQ lacks these activities and known antibiotics do not induce dispersal. In contrast, we show that MHQ inhibits the activity of Type IV Pili (TFP) and that TFP targeting can explain its dispersal activity. Our work thus identifies single-cell surface dispersal as a new activity of P. aeruginosa-produced small molecules, characterizes MHQ as a promising dispersal agent, and establishes TFP inhibition as a viable mechanism for P. aeruginosa dispersal.
2020
Microbial natural products constitute a wide variety of chemical compounds, many which can have antibiotic, antiviral, or anticancer properties that make them interesting for clinical purposes. Natural product classes include polyketides (PKs), nonribosomal peptides (NRPs), and ribosomally synthesized and post-translationally modified peptides (RiPPs). While variants of biosynthetic gene clusters (BGCs) for known classes of natural products are easy to identify in genome sequences, BGCs for new compound classes escape attention. In particular, evidence is accumulating that for RiPPs, subclasses known thus far may only represent the tip of an iceberg. Here, we present decRiPPter (Data-driven Exploratory Class-independent RiPP TrackER), a RiPP genome mining algorithm aimed at the discovery of novel RiPP classes. DecRiPPter combines a Support Vector Machine (SVM) that identifies candidate RiPP precursors with pan-genomic analyses to identify which of these are encoded within operon-like structures that are part of the accessory genome of a genus. Subsequently, it prioritizes such regions based on the presence of new enzymology and based on patterns of gene cluster and precursor peptide conservation across species. We then applied decRiPPter to mine 1,295 Streptomyces genomes, which led to the identification of 42 new candidate RiPP families that could not be found by existing programs. One of these was studied further and elucidated as a representative of a novel subfamily of lanthipeptides, which we designate class V. The 2D structure of the new RiPP, which we name pristinin A3 (1), was solved using nuclear magnetic resonance (NMR), tandem mass spectrometry (MS/MS) data, and chemical labeling. Two previously unidentified modifying enzymes are proposed to create the hallmark lanthionine bridges. Taken together, our work highlights how novel natural product families can be discovered by methods going beyond sequence similarity searches to integrate multiple pathway discovery criteria.
The composition of the skin microbiota varies widely among individuals when sampled at the same body site. A key question is which molecular factors determine strain-level variability within sub-ecosystems of the skin microbiota. Here, we used a genomics-guided approach to identify an antibacterial biosynthetic gene cluster in Cutibacterium acnes (formerly Propionibacterium acnes), a human skin commensal bacterium that is widely distributed across individuals and skin sites. Experimental characterization of this biosynthetic gene cluster resulted in identification of a new thiopeptide antibiotic, cutimycin. Analysis of individual human skin hair follicles revealed that cutimycin contributed to the ecology of the skin hair follicle microbiota and helped to reduce colonization of skin hair follicles by Staphylococcus species.
Introduction: Unroasted green coffee bean is an increasingly popular beverage and weight loss supplement that contains higher levels of chlorogenic acid derivatives and lower alkaloid levels than roasted beans. Nonetheless, how the gut microbiome metabolizes green coffee constituents has not been studied.
Objectives: To identify possible biotransformation products of green coffee extract by the human gut microbiome, and the potential implications of this process on its biological effects or fate inside the body.
Methods: Molecular networking via the GNPS platform was employed for the visualization of green coffee metabolite profiles acquired using LC-tandem mass spectrometry post-incubation with an ex vivo culture of the human gut microbiome.
Results: 36 Metabolites were annotated including four unreported alkyl cinnamate esters in green coffee along with six novel biotransformation products.
Conclusion: Our finding reveals new biotransformation products of cinnamate esters by the gut microbiome mediated via oxidative reactions such as dehydrogenation and hydroxylation, along with methylation, decarboxylation, and deglycosylation. These findings reveal potential interactions between the gut microbiome and green coffee constituents, and paves the way towards studying the effects of these interactions on both microbiome and the human host.
Keywords: Chlorogenic acid; Green coffee; Gut microbiome; Molecular networking; Prooxidants.
The human gut microbiome harbors hundreds of bacterial species with diverse biochemical capabilities. Dozens of drugs have been shown to be metabolized by single isolates from the gut microbiome, but the extent of this phenomenon is rarely explored in the context of microbial communities. Here, we develop a quantitative experimental framework for mapping the ability of the human gut microbiome to metabolize small molecule drugs: Microbiome-Derived Metabolism (MDM)-Screen. Included are a batch culturing system for sustained growth of subject-specific gut microbial communities, an ex vivo drug metabolism screen, and targeted and untargeted functional metagenomic screens to identify microbiome-encoded genes responsible for specific metabolic events. Our framework identifies novel drug-microbiome interactions that vary between individuals and demonstrates how the gut microbiome might be used in drug development and personalized medicine.
Consumption of glucosinolates, pro-drug-like metabolites abundant in Brassica vegetables, has been associated with decreased risk of certain cancers. Gut microbiota have the ability to metabolize glucosinolates, generating chemopreventive isothiocyanates. Here, we identify a genetic and biochemical basis for activation of glucosinolates to isothiocyanates by Bacteroides thetaiotaomicron, a prominent gut commensal species. Using a genome-wide transposon insertion screen, we identified an operon required for glucosinolate metabolism in B. thetaiotaomicron. Expression of BT2159–BT2156in a non-metabolizing relative, Bacteroides fragilis, resulted in gain of glucosinolate metabolism. We show that isothiocyanate formation requires the action of BT2158 and either BT2156 or BT2157 in vitro. Monocolonization of mice with mutant BtΔ2157 showed reduced isothiocyanate production in the gastrointestinal tract. These data provide insight into the mechanisms by which a common gut bacterium processes an important dietary nutrient.
The human microbiome is an important contributor to human health and disease. Although the molecular mechanisms governing most of these contributions are yet to be discovered, several characterized examples were shown to involve the production of a biologically active small molecule by one or more members of the microbiome. Along the same lines, recent systematic analyses revealed a vast, diverse, and largely unexplored potential of the human microbiome to produce such molecules, providing a direct route towards investigating microbe-host and microbe-microbe interactions at the level of molecular mechanism. Since several papers have previously reviewed the role of microbiome-derived small molecules in mediating microbe-host interactions in the context of human health and disease, we will here explore their role in mediating microbe-microbe interactions instead, with a focus on two important processes in microbial chemical ecology: competition and cooperation. We will discuss recent advances in discovering small molecules from the human microbiome with potential roles in microbial ecology, and efforts to define these roles in a biologically relevant context. Since each site on the human body represents its own ecological niche, and harbors a largely unique set of microbial inhabitants, we will organize the molecules and interactions based on the human body site where they are located.
2019
Extensive progress has been made in determining the effects of the microbiome on human physiology and disease, but the underlying molecules and mechanisms governing these effects remain largely unexplored. Here, we combine a new computational algorithm with synthetic biology to access biologically active small molecules encoded directly in human microbiome–derived metagenomic sequencing data. We discover that members of a clinically used class of molecules are widely encoded in the human microbiome and that they exert potent antibacterial activities against neighboring microbes, implying a possible role in niche competition and host defense. Our approach paves the way toward a systematic unveiling of the chemical repertoire encoded by the human microbiome and provides a generalizable platform for discovering molecular mediators of microbiome-host and microbiome-microbiome interactions.
Chemical defense against predators is widespread in natural ecosystems. Occasionally, taxonomically distant organisms share the same defense chemical. Here, we describe an unusual tripartite marine symbiosis, in which an intracellular bacterial symbiont (“Candidatus Endobryopsis kahalalidefaciens”) uses a diverse array of biosynthetic enzymes to convert simple substrates into a library of complex molecules (the kahalalides) for chemical defense of the host, the alga Bryopsis sp., against predation. The kahalalides are subsequently hijacked by a third partner, the herbivorous mollusk Elysia rufescens, and employed similarly for defense. “Ca. E. kahalalidefaciens” has lost many essential traits for free living and acts as a factory for kahalalide production. This interaction between a bacterium, an alga, and an animal highlights the importance of chemical defense in the evolution of complex symbioses.
Marine sponges often house small-molecule-producing symbionts extracellularly in their mesohyl, providing the host with a means of chemical defence against predation and microbial infection. Here, we report an intriguing case of chemically mediated symbiosis between the renieramycin-containing sponge Haliclona sp. and its herein discovered renieramycin-producing symbiont Candidatus Endohaliclona renieramycinifaciens. Remarkably, Ca. E. renieramycinifaciens has undergone extreme genome reduction where it has lost almost all necessary elements for free living while maintaining a complex, multi-copy plasmid-encoded biosynthetic gene cluster for renieramycin biosynthesis. In return, the sponge houses Ca. E. renieramycinifaciens in previously uncharacterized cellular reservoirs (chemobacteriocytes), where it can acquire nutrients from the host and avoid bacterial competition. This relationship is highly specific to a single clade of Haliclona sponges. Our study reveals intracellular symbionts as an understudied source for defence chemicals in the oldest-living metazoans and paves the way towards discovering similar systems in other marine sponges.
Media
Maria Diarey Tianero-McIntosh’s “Behind the Paper” article, here.
Janie Kim’s Small Things Considered blog post, here.
2017
The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut bacteria. Here, by expressing a subset of these clusters in Escherichia coli or Bacillus subtilis, we show that they encode pyrazinones and dihydropyrazinones. At least one of the 47 clusters is present in 88% of the National Institutes of Health Human Microbiome Project (NIH HMP) stool samples, and they are transcribed under conditions of host colonization. We present evidence that the active form of these molecules is the initially released peptide aldehyde, which bears potent protease inhibitory activity and selectively targets a subset of cathepsins in human cell proteomes. Our findings show that an approach combining bioinformatics, synthetic biology, and heterologous gene cluster expression can rapidly expand our knowledge of the metabolic potential of the microbiota while avoiding the challenges of cultivating fastidious commensals.
2015
Marine natural products are an important source of lead compounds against many pathogenic targets. Herein, we report the discovery of lobosamides A-C from a marine actinobacterium, Micromonospora sp., representing three new members of a small but growing family of bacterially produced polyene macrolactams. The lobosamides display growth inhibitory activity against the protozoan parasite Trypanosoma brucei (lobosamide A IC50 = 0.8 μM), the causative agent of human African trypanosomiasis (HAT). The biosynthetic gene cluster of the lobosamides was sequenced and suggests a conserved cluster organization among the 26-membered macrolactams. While determination of the relative and absolute configurations of many members of this family is lacking, the absolute configurations of the lobosamides were deduced using a combination of chemical modification, detailed spectroscopic analysis, and bioinformatics. We implemented a “molecules-to-genes-to-molecules” approach to determine the prevalence of similar clusters in other bacteria, which led to the discovery of two additional macrolactams, mirilactams A and B from Actinosynnema mirum. These additional analogs have allowed us to identify specific structure-activity relationships that contribute to the antitrypanosomal activity of this class. This approach illustrates the power of combining chemical analysis and genomics in the discovery and characterization of natural products as new lead compounds for neglected disease targets.
Developments in the use of genomics to guide natural product discovery and a recent emphasis on understanding the molecular mechanisms of microbiota-host interactions have converged on the discovery of small molecules from the human microbiome. Here, we review what is known about small molecules produced by the human microbiota. Numerous molecules representing each of the major metabolite classes have been found that have a variety of biological activities, including immune modulation and antibiosis. We discuss technologies that will affect how microbiota-derived molecules are discovered in the future and consider the challenges inherent in finding specific molecules that are critical for driving microbe-host and microbe-microbe interactions and understanding their biological relevance.
A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.
The capsule from Bacteroides, a common gut symbiont, has long been a model system for studying the molecular mechanisms of host-symbiont interactions. The Bacteroides capsule is thought to consist of an array of phase-variable polysaccharides that give rise to subpopulations with distinct cell surface structures. Here, we report the serendipitous discovery of a previously unknown surface structure in Bacteroides thetaiotaomicron: a surface layer composed of a protein of unknown function, BT1927. BT1927, which is expressed in a phase-variable manner by ~1:1,000 cells in a wild-type culture, forms a hexagonally tessellated surface layer. The BT1927-expressing subpopulation is profoundly resistant to complement-mediated killing, due in part to the BT1927-mediated blockade of C3b deposition. Our results show that the Bacteroides surface structure is capable of a far greater degree of structural variation than previously known, and they suggest that structural variation within a Bacteroides species is important for productive gut colonization. IMPORTANCE: Many bacterial species elaborate a capsule, a structure that resides outside the cell wall and mediates microbe-microbe and microbe-host interactions. Species of Bacteroides, the most abundant genus in the human gut, produce a capsule that consists of an array of polysaccharides, some of which are known to mediate interactions with the host immune system. Here, we report the discovery of a previously unknown surface structure in Bacteroides thetaiotaomicron. We show that this protein-based structure is expressed by a subset of cells in a population and protects Bacteroides from killing by complement, a component of the innate immune system. This novel surface layer protein is conserved across many species of the genus Bacteroides, suggesting an important role in colonization and host immune modulation.
Genetic tools to engineer a prominent member of the human gut microbiome represent initial steps toward cell-based diagnostics and therapeutics.
Before Princeton University:
2014
Several recent studies describe the influence of the gut microbiota on host brain and behavior. However, the mechanisms responsible for microbiota-nervous system interactions are largely unknown. Using a combination of genetics, biochemistry, and crystallography, we identify and characterize two phylogenetically distinct enzymes found in the human microbiome that decarboxylate tryptophan to form the β-arylamine neurotransmitter tryptamine. Although this enzymatic activity is exceedingly rare among bacteria more broadly, analysis of the Human Microbiome Project data demonstrate that at least 10% of the human population harbors at least one bacterium encoding a tryptophan decarboxylase in their gut community. Our results uncover a previously unrecognized enzymatic activity that can give rise to host-modulatory compounds and suggests a potential direct mechanism by which gut microbiota can influence host physiology, including behavior.
Natural products (secondary metabolites) found in marine invertebrates are often thought to be produced by resident symbiotic bacteria, and these products appear to play a major role in the symbiotic interaction of bacteria and their hosts. In these animals, there is extensive variation, both in chemistry and in the symbiotic bacteria that produce them. Here, we sought to answer the question of what factors underlie chemical variation in the ocean. As a model, we investigated the colonial tunicate Lissoclinum patella because of its rich and varied chemistry and its broad geographic range. We sequenced mitochondrial cytochrome c oxidase 1 (COXI) genes, and found that animals classified as L. patella fall into three phylogenetic groups that may encompass several cryptic species. The presence of individual natural products followed the phylogenetic relationship of the host animals, even though the compounds are produced by symbiotic bacteria that do not follow host phylogeny. In sum, we show that cryptic populations of animals underlie the observed chemical diversity, suggesting that the host controls selection for particular secondary metabolite pathways. These results imply novel approaches to obtain chemical diversity from the oceans, and also demonstrate that the diversity of marine natural products may be greatly impacted by cryptic local extinctions.
In complex biological systems, small molecules often mediate microbe-microbe and microbe-host interactions. Using a systematic approach, we identified 3,118 small-molecule biosynthetic gene clusters (BGCs) in genomes of human-associated bacteria and studied their representation in 752 metagenomic samples from the NIH Human Microbiome Project. Remarkably, we discovered that BGCs for a class of antibiotics in clinical trials, thiopeptides, are widely distributed in genomes and metagenomes of the human microbiota. We purified and solved the structure of a thiopeptide antibiotic, lactocillin, from a prominent member of the vaginal microbiota. We demonstrate that lactocillin has potent antibacterial activity against a range of Gram-positive vaginal pathogens, and we show that lactocillin and other thiopeptide BGCs are expressed in vivo by analyzing human metatranscriptomic sequencing data. Our findings illustrate the widespread distribution of small-molecule-encoding BGCs in the human microbiome, and they demonstrate the bacterial production of drug-like molecules in humans.
2013
The switch from culture-based enumeration to deep sequencing has enabled microbial community composition to be profiled en masse. In a new article, Maurice et al., 2013 report the use of fluorescence-activated cell sorting (FACS) to perform a high-throughput analysis of gut microbiota community function.
- Marcobal, A, et al. “A metabolomic view of how the human gut microbiota impacts the host metabolome using humanized and gnotobiotic mice.”. ISME J 710 (2013): , 7, 10, 1933-43. Web.
Defining the functional status of host-associated microbial ecosystems has proven challenging owing to the vast number of predicted genes within the microbiome and relatively poor understanding of community dynamics and community-host interaction. Metabolomic approaches, in which a large number of small molecule metabolites can be defined in a biological sample, offer a promising avenue to ‘fingerprint’ microbiota functional status. Here, we examined the effects of the human gut microbiota on the fecal and urinary metabolome of a humanized (HUM) mouse using an optimized ultra performance liquid chromatography-mass spectrometry-based method. Differences between HUM and conventional mouse urine and fecal metabolomic profiles support host-specific aspects of the microbiota’s metabolomic contribution, consistent with distinct microbial compositions. Comparison of microbiota composition and metabolome of mice humanized with different human donors revealed that the vast majority of metabolomic features observed in donor samples are produced in the corresponding HUM mice, and individual-specific features suggest ‘personalized’ aspects of functionality can be reconstituted in mice. Feeding the mice a defined, custom diet resulted in modification of the metabolite signatures, illustrating that host diet provides an avenue for altering gut microbiota functionality, which in turn can be monitored via metabolomics. Using a defined model microbiota consisting of one or two species, we show that simplified communities can drive major changes in the host metabolomic profile. Our results demonstrate that metabolomics constitutes a powerful avenue for functional characterization of the intestinal microbiota and its interaction with the host.
2012
Secondary metabolites are ubiquitous in bacteria, but by definition, they are thought to be nonessential. Highly toxic secondary metabolites such as patellazoles have been isolated from marine tunicates, where their exceptional potency and abundance implies a role in chemical defense, but their biological source is unknown. Here, we describe the association of the tunicate Lissoclinum patella with a symbiotic α-proteobacterium, Candidatus Endolissoclinum faulkneri, and present chemical and biological evidence that the bacterium synthesizes patellazoles. We sequenced and assembled the complete Ca. E. faulkneri genome, directly from metagenomic DNA obtained from the tunicate, where it accounted for 0.6% of sequence data. We show that the large patellazoles biosynthetic pathway is maintained, whereas the remainder of the genome is undergoing extensive streamlining to eliminate unneeded genes. The preservation of this pathway in streamlined bacteria demonstrates that secondary metabolism is an essential component of the symbiotic interaction.
Ascidians (tunicates) are rich sources of structurally elegant, pharmaceutically potent secondary metabolites and, more recently, potential biofuels. It has been demonstrated that some of these compounds are made by symbiotic bacteria and not by the animals themselves, and for a few other compounds evidence exists supporting a symbiotic origin. In didemnid ascidians, compounds are highly variable even in apparently identical animals. Recently, we have explained this variation at the genomic and metagenomic levels and have applied the basic scientific findings to drug discovery and development. This review discusses what is currently known about the origin and variation of symbiotically derived metabolites in ascidians, focusing on the family Didemnidae, where most research has occurred. Applications of our basic studies are also described.
The cyanobactin ribosomal peptide (RP) natural product pathway was manipulated to incorporate multiple tandem mutations and non-proteinogenic amino acids, using eight heterologous components simultaneously expressed in Escherichia coli . These studies reveal the potential of RPs for the rational synthesis of complex, new small molecules over multiple-step biosynthetic pathways using simple genetic engineering.
2011
Tiny marine animals represent an untapped reservoir for undiscovered, bioactive natural products. However, their small size and extreme chemical variability preclude traditional chemical approaches to discovering new bioactive compounds. Here, we use a metagenomic method to directly discover and rapidly access cyanobactin class natural products from these variable samples, and provide proof-of-concept for genome-based discovery and supply of marine natural products. We also address practical optimization of complex, multistep ribosomal peptide pathways in heterologous hosts, which is still very challenging. The resulting methods and concepts will be applicable to ribosomal peptide and other biosynthetic pathways.
The relationship between tunicates and the uncultivated cyanobacterium Prochloron didemni has long provided a model symbiosis. P. didemni is required for survival of animals such as Lissoclinum patella and also makes secondary metabolites of pharmaceutical interest. Here, we present the metagenomes, chemistry, and microbiomes of four related L. patella tunicate samples from a wide geographical range of the tropical Pacific. The remarkably similar P. didemni genomes are the most complex so far assembled from uncultivated organisms. Although P. didemni has not been stably cultivated and comprises a single strain in each sample, a complete set of metabolic genes indicates that the bacteria are likely capable of reproducing outside the host. The sequences reveal notable peculiarities of the photosynthetic apparatus and explain the basis of nutrient exchange underlying the symbiosis. P. didemni likely profoundly influences the lipid composition of the animals by synthesizing sterols and an unusual lipid with biofuel potential. In addition, L. patella also harbors a great variety of other bacterial groups that contribute nutritional and secondary metabolic products to the symbiosis. These bacteria possess an enormous genetic potential to synthesize new secondary metabolites. For example, an antitumor candidate molecule, patellazole, is not encoded in the genome of Prochloron and was linked to other bacteria from the microbiome. This study unveils the complex L. patella microbiome and its impact on primary and secondary metabolism, revealing a remarkable versatility in creating and exchanging small molecules.
The enzymatic basis of ribosomal peptide natural product prenylation has not been reported. Here, we characterize a prenyltransferase, LynF, from the TruF enzyme family. LynF is the first characterized representative of the TruF protein family, which is responsible for both reverse- and forward-O-prenylation of tyrosine, serine, and threonine in cyclic peptides known as cyanobactins. We show that LynF reverse O-prenylates tyrosine in macrocyclic peptides. Based upon these results, we propose that the TruF family prenylates mature cyclic peptides, from which the leader sequence and other enzyme recognition elements have been excised. This differs from the common model of ribosomal peptide biosynthesis, in which a leader sequence is required to direct post-translational modifications. In addition, we find that reverse O-prenylated tyrosine derivatives undergo a facile Claisen rearrangement at ‘physiological’ temperature in aqueous buffers, leading to forward C-prenylated products. Although the Claisen rearrangement route to natural products has been chemically anticipated for at least 40 years, it has not been demonstrated as a route to prenylated natural products. Here, we show that the Claisen rearrangement drives phenolic C-prenylation in at least one case, suggesting that this route should be reconsidered as a mechanism for the biosynthesis of prenylated phenolic compounds.
Ribosomal peptide natural products are ubiquitous, yet relatively few tools exist to predict structures and clone new pathways. Cyanobactin ribosomal peptides are found in ~30% of all cyanobacteria, but the connection between gene sequence and structure was not defined, limiting the rapid identification of new compounds and pathways. Here, we report discovery of four orphan cyanobactin gene clusters by genome mining and an additional pathway by targeted cloning, which represented a tyrosine O-prenylating biosynthetic pathway. Genome mining enabled discovery of five cyanobactins, including peptide natural products from Spirulina supplements. A phylogenetic model defined four cyanobactin genotypes, which explain the synthesis of multiple cyanobactin structural classes and help direct pathway cloning and structure prediction efforts. These strategies were applied to DNA isolated from a mixed cyanobacterial bloom containing cyanobactins.
The complex evolution of secondary metabolism is important in biology, drug development, and synthetic biology. To examine this problem at a fine scale, we compared the genomes and chemistry of 24 strains of uncultivated cyanobacteria, Prochloron didemni, that live symbiotically with tropical ascidians and that produce natural products isolated from the animals. Although several animal species were obtained along a >5500 km transect of the Pacific Ocean, P. didemni strains are >97% identical across much of their genomes, with only a few exceptions concentrated in secondary metabolism. Secondary metabolic gene clusters were sporadically present or absent in identical genomic locations with no consistent pattern of co-occurrence. Discrete mutations were observed, leading to new chemicals that we isolated from animals. Functional cassettes encoding diverse chemicals are exchanged among a single population of symbiotic P. didemni that spans the tropical Pacific, providing the host animals with a varying arsenal of secondary metabolites.
2010
The cyanobactin biosynthetic pathways pat and tru, isolated from metagenomes of marine animals, lead to diverse natural products containing heterocycles derived from Cys, Ser, and Thr. Previous work has shown that PatD and TruD are extremely broad-substrate heterocyclase enzymes. These enzymes are virtually identical in their N-terminal putative catalytic domains, but only approximately 77% identical in their C-terminal putative substrate-binding domains. Here, we show that these differences allow the enzymes to control regioselectivity of posttranslational modifications, helping to control product chemistry in this hypervariable family of marine natural products.
Ascidians (tunicates; sea squirts) are sources of diverse, bioactive natural products, one of which is an approved drug and many of which are potent drug leads. It has been shown that symbiotic bacteria living with ascidians produce some of the bioactive compounds isolated from whole animals, and indirect evidence strongly implicates symbiotic bacteria in the synthesis of many others. However, for the majority the producing organism has not been identified. In cases where a symbiotic origin has been definitively assigned, the resulting data lead to improved paths to drug discovery and development from marine animals. This review traces evidence for symbiotic production where such evidence exists and describes the strengths and limitations of that evidence.
2009
Deep metagenome mining is a new method for engineering natural product pathways, focusing on examining symbiotic organisms. The method has been applied to a family of compounds known as cyanobactins, which are ribosomally synthesized peptides produced by cyanobacteria. Often, these cyanobacteria live symbiotically with marine animals, leading to production of natural products in whole animal samples. Here, we focus on methods to identify, clone, and study cyanobactin natural product genes from axenic organisms and metagenomic environments. The application to deep metagenome mining is described, along with other potential targets of this methodology.
Ribosomally synthesized bacterial natural products rival the nonribosomal peptides in their structural and functional diversity. The last decade has seen substantial progress in the identification and characterization of biosynthetic pathways leading to ribosomal peptide natural products with new and unusual structural motifs. In some of these cases, the motifs are similar to those found in nonribosomal peptides, and many are constructed by convergent or even paralogous enzymes. Here, we summarize the major structural and biosynthetic categories of ribosomally synthesized bacterial natural products and, where applicable, compare them to their homologs from nonribosomal biosynthesis.
2008
More than 100 cyclic peptides harboring heterocyclized residues are known from marine ascidians, sponges and different genera of cyanobacteria. Here, we report an assembly line responsible for the biosynthesis of these diverse peptides, now called cyanobactins, both in symbiotic and free-living cyanobacteria. By comparing five new cyanobactin biosynthetic clusters, we produced the prenylated antitumor preclinical candidate trunkamide in Escherichia coli culture using genetic engineering.
Two new cyclic hexapeptides, mollamides B (1) and C (2), were isolated from the Indonesian tunicate Didemnum molle along with the known peptide keenamide A (3). The structures were established using 1D and 2D NMR experiments. The relative configuration of mollamide B at the thiazoline moiety was determined using molecular modeling coupled with NMR-derived restraints. Their absolute configuration was determined using Marfey’s method. The new peptides have been evaluated for their antimicrobial, antimalarial, anticancer, anti-HIV-1, anti-Mtb, and anti-inflammatory activities. Keenamide A and mollamide B show cytotoxicity against several cancer cell lines.
2006
Four new manzamine-type alkaloids, 12,28-oxamanzamine E (2), 12,34-oxa-6-hydroxymanzamine E (3), 8-hydroxymanzamine B (5), and 12,28-oxaircinal A (11), were isolated from three collections of an Indonesian sponge of the genus Acanthostrongylophora together with 13 known manzamine alkaloids, ircinal A, ircinol A, xestomanzamine A, manzamines A, E, F, J, and Y, manadomanzamines A and B, neo-kauluamine, 8-hydroxymanzamine A, and manzamine A N-oxide. The structures of the new compounds were elucidated by means of 1D and 2D NMR spectroscopic methods. Three of these compounds (2, 3, and 11) possess a unique manzamine-type aminal ring system generated through an ether linkage between carbons 12-28 or between carbons 12-34. In the case of manzamine B and related metabolites, carbons 11 and 12 of the typical manzamine structure have an epoxide group and add to our growing understanding of manzamine structure-activity relationships (SAR) and metabolism. The bioactivity and SAR for a number of previously reported manzamine-related metabolites against malaria, leishmania, tuberculosis, and HIV-1 are also presented. Manzamine Y (9) showed significant inhibitory activity of GSK3, an enzyme implicated in Alzheimer’s disease pathology. The toxicity of manzamine A and neo-kauluamine was evaluated against both medaka fry and eggs.
A large family of cytotoxic cyclic peptides exemplified by the patellamides has been isolated from ascidians harboring the obligate cyanobacterial symbionts Prochloron spp.. Genome sequence analysis of these symbionts has revealed that Prochloron spp. synthesize patellamides by a ribosomal pathway. To understand how this pathway evolved to produce a suite of related metabolites, we analyzed 46 prochloron-containing ascidians from the tropical Pacific Ocean for the presence of patellamide biosynthetic genes and taxonomic markers. Here, we show that Prochloron spp. generate a diverse library of patellamides using small, hypervariable cassettes within a conserved genetic background. Each symbiont strain contains a single pathway, and mixtures of symbionts within ascidians lead to the accumulation of chemical libraries. We used this information to engineer the production of a new cyclic peptide in Escherichia coli, thereby demonstrating the power of comparative analysis of closely related symbiotic pathways to direct the genetic synthesis of new molecules.