A Natural Product Chemist's Guide to Unlocking Silent Biosynthetic Gene Clusters.

Microbial natural products have provided an important source of therapeutic leads and motivated research and innovation in diverse scientific disciplines. In recent years, it has become evident that bacteria harbor a large, hidden reservoir of potential natural products in the form of silent or cryptic biosynthetic gene clusters (BGCs). These can be readily identified in microbial genome sequences but do not give rise to detectable levels of a natural product. Herein, we provide a useful organizational framework for the various methods that have been implemented for interrogating silent BGCs. We divide all available approaches into four categories. The first three are endogenous strategies that utilize the native host in conjunction with classical genetics, chemical genetics, or different culture modalities. The last category comprises expression of the entire BGC in a heterologous host. For each category, we describe the rationale, recent applications, and associated advantages and limitations.

Bicyclostreptins are radical SAM enzyme-modified peptides with unique cyclization motifs.

Microbial natural products comprise diverse architectures that are generated by equally diverse biosynthetic strategies. In peptide natural products, amino acid sidechains are frequently used as sites of modification to generate macrocyclic motifs. Backbone amide groups, among the most stable of biological moieties, are rarely used for this purpose. Here we report the discovery and biosynthesis of bicyclostreptins-peptide natural products from Streptococcus spp. with an unprecedented structural motif consisting of a macrocyclic ß-ether and a heterocyclic sp3-sp3 linkage between a backbone amide nitrogen and an adjacent α-carbon. Both reactions are installed, in that order, by two radical S-adenosylmethionine (RaS) metalloenzymes. Bicyclostreptins are produced at nM concentrations and are potent growth regulation agents in Streptococcus thermophilus. Our results add a distinct and unusual chemotype to the growing family of ribosomal peptide natural products, expand the already impressive catalytic scope of RaS enzymes, and provide avenues for further biological studies in human-associated streptococci.

Vitamin B3 Triggers Biosynthesis of Secondary Metabolite Dormancy Signals in Streptococcus suis.

Human-associated streptococci have not been viewed as productive sources of natural products. Against expectation, bioinformatic searches recently revealed a large collection of diverse biosynthetic gene clusters coding for ribosomally synthesized and post-translationally modified peptides (RiPPs) in streptococcal genomes. The most abundant of these, the tqq gene cluster, is specific to Streptococcus suis, a burdensome agricultural pathogen and zoonotic agent. Herein, we used high-throughput elicitor screening to identify both small molecule elicitors and products of the tqq cluster. We show that the B3 vitamin niacin effectively elicits the tqq cluster leading to the biosynthesis of a family of RiPP natural products, which we termed threoglucins and characterized structurally. The defining feature of threoglucins is an aliphatic ether bond giving rise to a substituted 1,3-oxazinane heterocycle in the peptide backbone. Isolation of 22 congeners of threoglucins facilitated structure activity relationship studies, demonstrating the requirement for the oxazinane substructure and a Trp-Tyr C-terminal dyad for biological activity, namely antibiotic persistence and allolysis at low and high doses, respectively. Potential therapeutic applications of threoglucins are discussed.

Discovery and Biosynthesis of Streptosactin, a Sactipeptide with an Alternative Topology Encoded by Commensal Bacteria in the Human Microbiome.

Mammalian microbiomes encode thousands of biosynthetic gene clusters (BGCs) and represent a new frontier in natural product research. We recently found an abundance of quorum sensing-regulated BGCs in mammalian microbiome streptococci that code for ribosomally synthesized and post-translationally modified peptides (RiPPs) and contain one or more radical S-adenosylmethionine (RaS) enzymes, a versatile superfamily known to catalyze some of the most unusual reactions in biology. In the current work, we target a widespread group of streptococcal RiPP BGCs and elucidate both the reaction carried out by its encoded RaS enzyme and identify its peptide natural product, which we name streptosactin. Streptosactin is the first sactipeptide identified from Streptococcus spp.; it contains two sequential four amino acid sactionine macrocycles, an unusual topology for this compound family. Bioactivity assays reveal potent but narrow-spectrum activity against the producing strain and its closest relatives that carry the same BGC, suggesting streptosactin may be a long-suspected fratricidal agent of Streptococcus thermophilus. Our results highlight mammalian streptococci as a rich source of unusual enzymatic chemistries and bioactive natural products.

Total Synthesis and Stereochemical Assignment of Streptide.

Streptide (1) is a peptide-derived macrocyclic natural product that has attracted considerable attention since its discovery in 2015. It contains an unprecedented post-translational modification that intramolecularly links the ß-carbon (C3) of a residue 2 lysine with the C7 of a residue 6 tryptophan, thereby forming a 20-membered cyclic peptide. Herein, we report the first total synthesis of streptide that confirms the regiochemistry of the lysine-tryptophan cross-link and provides an unambiguous assignment of the stereochemistry (3R vs 3S) of the lysine-2 C3 center. Both the 3R and the originally assigned 3S lysine diastereomers were independently prepared by total synthesis, and it is the former, not the latter, that was found to correlate with the natural product. The approach enlists a powerful Pd(0)-mediated indole annulation for the key macrocyclization of the complex core peptide, utilizes an underdeveloped class of hypervalent iodine(III) aryl substrates in a palladium-catalyzed C-H activation/ß-arylation reaction conducted on a lysine derivative, and provides access to material with which the role of streptide and related natural products may be examined.

Metabolic model for diversity-generating biosynthesis.

A conventional metabolic pathway leads to a specific product. In stark contrast, there are diversity-generating metabolic pathways that naturally produce different chemicals, sometimes of great diversity. We demonstrate that for one such pathway, tru, each ensuing metabolic step is slower, in parallel with the increasing potential chemical divergence generated as the pathway proceeds. Intermediates are long lived and accumulate progressively, in contrast with conventional metabolic pathways, in which the first step is rate-limiting and metabolic intermediates are short-lived. Understanding these fundamental differences enables several different practical applications, such as combinatorial biosynthesis, some of which we demonstrate here. We propose that these principles may provide a unifying framework underlying diversity-generating metabolism in many different biosynthetic pathways.

Response of Secondary Metabolism of Hypogean Actinobacterial Genera to Chemical and Biological Stimuli.

Microorganisms within microbial communities respond to environmental challenges by producing biologically active secondary metabolites, yet the majority of these small molecules remain unidentified. We have previously demonstrated that secondary metabolite biosynthesis in actinomycetes can be activated by model environmental chemical and biological stimuli, and metabolites can be identified by comparative metabolomics analyses under different stimulus conditions. Here, we surveyed the secondary metabolite productivity of a group of 20 phylogenetically diverse actinobacteria isolated from hypogean (cave) environments by applying a battery of stimuli consisting of exposure to antibiotics, metals, and mixed microbial culture. Comparative metabolomics was used to reveal secondary metabolite responses from stimuli. These analyses revealed substantial changes in global metabolomic dynamics, with over 30% of metabolomic features increasing more than 10-fold under at least one stimulus condition. Selected features were isolated and identified via nuclear magnetic resonance (NMR), revealing several known secondary metabolite families, including the tetarimycins, aloesaponarins, hypogeamicins, actinomycins, and propeptins. One prioritized metabolite was identified to be a previously unreported aminopolyol polyketide, funisamine, produced by a cave isolate of Streptosporangium when exposed to mixed culture. The production of funisamine was most significantly increased in mixed culture with Bacillus species. The biosynthetic gene cluster responsible for the production of funisamine was identified via genomic sequencing of the producing strain, Streptosporangium sp. strain KDCAGE35, which facilitated a deduction of its biosynthesis. Together, these data demonstrate that comparative metabolomics can reveal the stimulus-induced production of natural products from diverse microbial phylogenies.IMPORTANCE Microbial secondary metabolites are an important source of biologically active and therapeutically relevant small molecules. However, much of this active molecular diversity is challenging to access due to low production levels or difficulty in discerning secondary metabolites within complex microbial extracts prior to isolation. Here, we demonstrate that ecological stimuli increase secondary metabolite production in phylogenetically diverse actinobacteria isolated from understudied hypogean environments. Additionally, we show that comparative metabolomics linking stimuli to metabolite response data can effectively reveal secondary metabolites within complex biological extracts. This approach highlighted secondary metabolites in almost all observed natural product classes, including low-abundance analogs of biologically relevant metabolites, as well as a new linear aminopolyol polyketide, funisamine. This study demonstrates the generality of activating stimuli to potentiate secondary metabolite production across diverse actinobacterial genera.

Comparative mass spectrometry-based metabolomics strategies for the investigation of microbial secondary metabolites.

Covering: 2000 to 2016The labor-intensive process of microbial natural product discovery is contingent upon identifying discrete secondary metabolites of interest within complex biological extracts, which contain inventories of all extractable small molecules produced by an organism or consortium. Historically, compound isolation prioritization has been driven by observed biological activity and/or relative metabolite abundance and followed by dereplication via accurate mass analysis. Decades of discovery using variants of these methods has generated the natural pharmacopeia but also contributes to recent high rediscovery rates. However, genomic sequencing reveals substantial untapped potential in previously mined organisms, and can provide useful prescience of potentially new secondary metabolites that ultimately enables isolation. Recently, advances in comparative metabolomics analyses have been coupled to secondary metabolic predictions to accelerate bioactivity and abundance-independent discovery work flows. In this review we will discuss the various analytical and computational techniques that enable MS-based metabolomic applications to natural product discovery and discuss the future prospects for comparative metabolomics in natural product discovery.

Guidelines for metabolomics-guided transposon mutagenesis for microbial natural product discovery.

There is a great discrepancy between the natural product output of cultured microorganisms and their bioinformatically predicted biosynthetic potential, such that most of the molecular diversity contained within microbial reservoirs has yet to be discovered. One of the primary reasons is insufficient expression of natural product biosynthetic gene clusters (BGCs) under standard laboratory conditions. Several methods have been developed to increase production from such "cryptic" BGCs. Among these, we recently implemented mass spectrometry-guided transposon mutagenesis, a forward genetic screen in which mutants that exhibit stimulated biosynthesis of cryptic metabolites, as read out by mass spectrometry, are selected from a transposon mutant library. Herein, we use Burkholderia gladioli as an example and provide guidelines for generating transposon mutant libraries, measuring metabolomic inventories through mass spectrometry, performing comparative metabolomics to prioritize cryptic natural products from the mutant library, and isolating and characterizing novel natural products elicited through mutagenesis. Application of this approach will be useful in both accessing novel natural products from cryptic BGCs and identifying genes involved in their global regulation.

Biosynthesis-guided discovery reveals enteropeptins as alternative sactipeptides containing N-methylornithine.

The combination of next-generation DNA sequencing technologies and bioinformatics has revitalized natural product discovery. Using a bioinformatic search strategy, we recently identified ∼600 gene clusters in otherwise overlooked streptococci that code for ribosomal peptide natural products synthesized by radical S-adenosylmethionine enzymes. These grouped into 16 subfamilies and pointed to an unexplored microbiome biosynthetic landscape. Here we report the structure, biosynthesis and function of one of these natural product groups, which we term enteropeptins, from the gut microbe Enterococcus cecorum. We show three reactions in the biosynthesis of enteropeptins that are each catalysed by a different family of metalloenzymes. Among these, we characterize the founding member of a widespread superfamily of Fe-S-containing methyltransferases, which, together with an Mn2+-dependent arginase, installs N-methylornithine in the peptide sequence. Biological assays with the mature product revealed bacteriostatic activity only against the producing strain, extending an emerging theme of fratricidal or self-inhibitory metabolites in microbiome firmicutes.

MetEx, a Metabolomics Explorer Application for Natural Product Discovery.

Advances in next-generation DNA sequencing technologies, bioinformatics, and mass spectrometry-based metabolite detection have ushered in a new era of natural product discovery. Microbial secondary metabolomes are complex, especially when otherwise silent biosynthetic genes are activated, and there is therefore a need for data analysis software to explore and map the resulting multidimensional datasets. To that end, we herein report the Metabolomics Explorer (MetEx), a publicly available web application for the analysis of parallel liquid chromatography-coupled mass spectrometry (LC-MS)-based metabolomics data. MetEx is a highly interactive application that facilitates visualization and analysis of complex metabolomics datasets, consisting of retention time, m/z, and MS intensity features, as a function of hundreds of conditions or elicitors. The software enables prioritization of leads from three-dimensional maps, extraction of two-dimensional slices from various higher order plots, organization of datasets by elicitor chemotypes, customizable library-based dereplication, and automatically scored lead selection. We describe the application of MetEx to the first UPLC-MS-guided high-throughput elicitor screen in which Burkholderia gladioli was challenged with 750 elicitors, and the resulting profiles were interrogated by UPLC-Qtof-MS and subsequently analyzed with the app. We demonstrate the utility of MetEx by reporting elicitors for several cryptic metabolite groups and by uncovering new natural products that remain to be characterized. MetEx is available at https://mo.princeton.edu/MetEx/.

Quorum Sensing in Streptococcus mutans Regulates Production of Tryglysin, a Novel RaS-RiPP Antimicrobial Compound.

The genus Streptococcus encompasses a large bacterial taxon that commonly colonizes mucosal surfaces of vertebrates and is capable of disease etiologies originating from diverse body sites, including the respiratory, digestive, and reproductive tracts. Identifying new modes of treating infections is of increasing importance, as antibiotic resistance has escalated. Streptococcus mutans is an important opportunistic pathogen that is an agent of dental caries and is capable of systemic diseases such as endocarditis. As such, understanding how it regulates virulence and competes in the oral niche is a priority in developing strategies to defend from these pathogens. We determined that S. mutans UA159 possesses a bona fide short hydrophobic peptide (SHP)/Rgg quorum-sensing system that regulates a specialized biosynthetic operon featuring a radical-SAM (S-adenosyl-l-methionine) (RaS) enzyme and produces a ribosomally synthesized and posttranslationally modified peptide (RiPP). The pairing of SHP/Rgg regulatory systems with RaS biosynthetic operons is conserved across streptococci, and a locus similar to that in S. mutans is found in Streptococcus ferus, an oral streptococcus isolated from wild rats. We identified the RaS-RiPP product from this operon and solved its structure using a combination of analytical methods; we term these RiPPs tryglysin A and B for the unusual Trp-Gly-Lys linkage. We report that tryglysins specifically inhibit the growth of other streptococci, but not other Gram-positive bacteria such as Enterococcus faecalis or Lactococcus lactis We predict that tryglysin is produced by S. mutans in its oral niche, thus inhibiting the growth of competing species, including several medically relevant streptococci.IMPORTANCE Bacteria interact and compete with a large community of organisms in their natural environment. Streptococcus mutans is one such organism, and it is an important member of the oral microbiota. We found that S. mutans uses a quorum-sensing system to regulate production of a novel posttranslationally modified peptide capable of inhibiting growth of several streptococcal species. We find inhibitory properties of a similar peptide produced by S. ferus and predict that these peptides play a role in interspecies competition in the oral niche.

Unlocking Cryptic Metabolites with Mass Spectrometry-Guided Transposon Mutant Selection.

The products of most secondary metabolite biosynthetic gene clusters (BGCs) have yet to be discovered, in part due to low expression levels in laboratory cultures. Reporter-guided mutant selection (RGMS) has recently been developed for this purpose: a mutant library is generated and screened, using genetic reporters to a chosen BGC, to select transcriptionally active mutants that then enable the characterization of the "cryptic" metabolite. The requirement for genetic reporters limits the approach to a single pathway within genetically tractable microorganisms. Herein, we utilize untargeted metabolomics in conjunction with transposon mutagenesis to provide a global read-out of secondary metabolism across large numbers of mutants. We employ self-organizing map analytics and imaging mass spectrometry to identify and characterize seven cryptic metabolites from mutant libraries of two different Burkholderia species. Applications of the methodologies reported can expand our understanding of the products and regulation of cryptic BGCs across phylogenetically diverse bacteria.

Mapping Microbial Response Metabolomes for Induced Natural Product Discovery.

Intergeneric microbial interactions may originate a significant fraction of secondary metabolic gene regulation in nature. Herein, we expose a genomically characterized Nocardiopsis strain, with untapped polyketide biosynthetic potential, to intergeneric interactions via coculture with low inoculum exposure to Escherichia, Bacillus, Tsukamurella, and Rhodococcus. The challenge-induced responses of extracted metabolites were characterized via multivariate statistical and self-organizing map (SOM) analyses, revealing the magnitude and selectivity engendered by the limiting case of low inoculum exposure. The collected inventory of cocultures revealed substantial metabolomic expansion in comparison to monocultures with nearly 14% of metabolomic features in cocultures undetectable in monoculture conditions and many features unique to coculture genera. One set of SOM-identified responding features was isolated, structurally characterized by multidimensional NMR, and revealed to comprise previously unreported polyketides containing an unusual pyrrolidinol substructure and moderate and selective cytotoxicity. Designated ciromicin A and B, they are detected across mixed cultures with intergeneric preferences under coculture conditions. The structural novelty of ciromicin A is highlighted by its ability to undergo a diastereoselective photochemical 12-π electron rearrangement to ciromicin B at visible wavelengths. This study shows how organizing trends in metabolomic responses under coculture conditions can be harnessed to characterize multipartite cultures and identify previously silent secondary metabolism.

Structuring Microbial Metabolic Responses to Multiplexed Stimuli via Self-Organizing Metabolomics Maps.

Secondary metabolite biosynthesis in microorganisms responds to discrete chemical and biological stimuli; however, untargeted identification of these responses presents a significant challenge. Herein we apply multiplexed stimuli to Streptomyces coelicolor and collect the resulting response metabolomes via ion mobility-mass spectrometric analysis. Self-organizing map (SOM) analytics adapted for metabolomic data demonstrate efficient characterization of the subsets of primary and secondary metabolites that respond similarly across stimuli. Over 60% of all metabolic features inventoried from responses are either not observed under control conditions or produced at greater than 2-fold increase in abundance in response to at least one of the multiplexing conditions, reflecting how metabolites encode phenotypic changes in an organism responding to multiplexed challenges. Using abundance as an additional filter, each of 16 known S. coelicolor secondary metabolites is prioritized via SOM and observed at increased levels (1.2- to 22-fold compared with unperturbed) in response to one or more challenge conditions.