Mohamed S. Donia

A global assembly line for cyanobactins.

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.

Small molecules from the human microbiota

Microbial bioactive molecules Human cells are outnumbered by the microbial cells of our commensals by an order of magnitude. All of these organisms are metabolically active and secrete multiple bioactive molecules. Genomics has unveiled a remarkable array of biosynthetic gene clusters in the human microbiota, which encode diverse metabolites. Donia et al. review how molecules ranging from lantibiotics and microcins to indoxyl sulfate and immunemodulatory oligosaccharides and lipids could affect the health and physiology of the whole organism, depending on the composition of an individuals microbial community. Science, this issue p. 10.1126/science.1254766 BACKGROUND Two developments in distinct fields are converging to create interest in discovering small molecules from the human microbiome. First, the use of genomics to guide natural product discovery has led to the unexpected discovery of numerous biosynthetic gene clusters in genomes of the human microbiota. Second, the microbiome research community is moving from a focus on “who’s there?” to “what are they doing?” with an accompanying emphasis on understanding microbiota-host interactions at the level of molecular mechanism. This merger has sparked a concerted hunt for the mediators of microbe-host and microbe-microbe interactions, including microbiota-derived small molecules. ADVANCES Numerous small molecules are known that are produced by the human microbiota. The microbiota-derived ribosomally synthesized, posttranslationally modified peptides (RiPPs) include widely distributed lantibiotics and microcins; these molecules have narrow-spectrum activity and are presumptive mediators of interactions among closely related species. Another notable RiPP is Escherichia coli heat-stable enterotoxin, a guanylate cyclase 2C agonist from which the recently approved gastrointestinal motility drug linaclotide was derived. Fewer amino acid metabolites are synthesized by the microbiota, but they are produced at very high levels that vary widely among individuals (e.g., indoxyl sulfate at 10 to 200 mg/day). Gut bacterial species convert common dietary amino acids into distinct end products, such as tryptophan to indoxyl sulfate, indole propionic acid, and tryptamine—indicating that humans with the same diet but different gut colonists can have widely varying gut metabolic profiles. Microbially produced oligosaccharides differ from other natural products because they are cell-associated (i.e., nondiffusible) and because many more biosynthetic loci exist for them than for other small molecule classes. Well-characterized examples, such as Bacteroides polysaccharide A, show that oligosaccharides may not simply play a structural role or mediate adhesion; rather, they can be involved in highly specific ligand-receptor interactions that result in immune modulation. Similarly, the (glyco)lipids α-galactosylceramide and mycolic acid can play roles in immune signaling. The most prominent microbiota-derived terpenoids are microbial conversion products of the cholic acid and chenodeoxycholic acid in host bile. These secondary bile acids can reach high concentration (mM) in the gut and vary widely in composition among individuals. Several canonical virulence factors from pathogens are derived from nonribosomal peptides (NRPs) and polyketides (PKs), but less is known about NRPs and PKs from the commensal microbiota. A recent computational effort has identified ~14,000 biosynthetic gene clusters in sequenced genomes from the human microbiota, 3118 of which were present in one or more of the 752 metagenomic sequence samples from the NIH Human Microbiome Project. Nearly all of the gene clusters that were present in >10% of the samples from the body site of origin are uncharacterized, highlighting the potential for identifying the molecules they encode and studying their biological activities. OUTLOOK There are two central challenges facing the field. The first is to distinguish, from among thousands of microbiota-derived molecules, which ones drive a key phenotype at physiologically relevant concentrations. Second, which experimental systems are appropriate for testing the activity of an individual molecule from a complex milieu? Meeting these challenges will require developing new computational and experimental technologies, including a capacity to identify biosynthetic genes and predict the structure and target of their biological activity, and systems in which germ-free mice are colonized by mock communities that differ only by the presence or absence of a biosynthetic gene cluster. Small-molecule–mediated microbe-host and microbe-microbe interactions. Commensal organisms of the human microbiota produce many diverse small molecules with an equally diverse array of targets that can exacerbate or modulate immune responses and other physiological functions in the host. Several act as antibacterials to remove competing organisms, but many other products have unknown targets and effects on commensals and the host. 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.

Ribosomal peptide natural products: bridging the ribosomal and nonribosomal worlds.

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.

A metabolomic view of how the human gut microbiota impacts the host metabolome using humanized and gnotobiotic mice

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.

Complex microbiome underlying secondary and primary metabolism in the tunicate-Prochloron symbiosis

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.

Discovery and characterization of gut microbiota decarboxylases that can produce the neurotransmitter tryptamine.

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.

Genome streamlining and chemical defense in a coral reef symbiosis

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.

Linking Chemistry and Genetics in the Growing Cyanobactin Natural Products Family

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.

Life in cellulose houses: symbiotic bacterial biosynthesis of ascidian drugs and drug leads

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.

Insights into heterocyclization from two highly similar enzymes

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.