Over the past decade, bacterial genome sequences have revealed an immense reservoir of biosynthetic gene clusters, sets of contiguous genes that have the potential to produce drugs or drug-like ...molecules. However, the majority of these gene clusters appear to be inactive for unknown reasons prompting terms such as “cryptic” or “silent” to describe them. Because natural products have been a major source of therapeutic molecules, methods that rationally activate these silent clusters would have a profound impact on drug discovery. Herein, a new strategy is outlined for awakening silent gene clusters using small molecule elicitors. In this method, a genetic reporter construct affords a facile read-out for activation of the silent cluster of interest, while high-throughput screening of small molecule libraries provides potential inducers. This approach was applied to two cryptic gene clusters in the pathogenic model Burkholderia thailandensis . The results not only demonstrate a prominent activation of these two clusters, but also reveal that the majority of elicitors are themselves antibiotics, most in common clinical use. Antibiotics, which kill B. thailandensis at high concentrations, act as inducers of secondary metabolism at low concentrations. One of these antibiotics, trimethoprim, served as a global activator of secondary metabolism by inducing at least five biosynthetic pathways. Further application of this strategy promises to uncover the regulatory networks that activate silent gene clusters while at the same time providing access to the vast array of cryptic molecules found in bacteria.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Microbial genomes harbor an abundance of biosynthetic gene clusters, but most are expressed at low levels and need to be activated for characterization of their cognate natural products. In this ...work, we report the combination of high‐throughput elicitor screening (HiTES) with matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) for the rapid identification of cryptic peptide natural products. Application to Streptomyces ghanaensis identified amygdalin as an elicitor of a novel non‐ribosomal peptide, which we term cinnapeptin. Complete structural elucidation revealed cinnapeptin as a cyclic depsipeptide with an unusual 2‐methyl‐cinnamoyl group. Insights into its biosynthesis were provided by whole genome sequencing and gene deletion studies, while bioactivity assays showed antimicrobial activity against Gram‐positive bacteria and fission yeast. MALDI‐HiTES is a broadly applicable tool for the discovery of cryptic peptides encoded in microbial genomes.
A new method, MALDI‐HiTES, is reported for the rapid identification of cryptic peptide natural products. Application to a streptomycete revealed numerous, uncharacterized natural products and led to the discovery of an antimicrobial depsipeptide, cinnapeptin, which harbors an unusual 2‐methylcinnamoyl moiety. Genomic and genetic studies located the responsible gene cluster and provided insights into the biogenesis of cinnapeptin.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Bacterial metabolism is comprised of primary metabolites, the intracellular molecules of life that enable growth and proliferation, and secondary metabolites, predominantly extracellular molecules ...that facilitate a microbe’s interaction with its environment. While our knowledge of primary metabolism and its web of interconnected intermediates is quantitative and holistic, significant knowledge gaps remain in our understanding of the secondary metabolomes of bacteria. In this Perspective, I discuss the main challenges involved in obtaining a global, comprehensive picture of bacterial secondary metabolomes, specifically in biosynthetically “gifted” microbes. Recent methodological advances that can meet these challenges will be reviewed. Applications of these methods combined with ongoing innovations will enable a detailed picture of global secondary metabolomes, which will in turn shed light onto the biology, chemistry, and enzymology underlying natural products and simultaneously aid drug discovery.
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CEKLJ, DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Streptococcal bacteria use peptide signals as a means of intraspecies communication. These peptides can contain unusual post-translational modifications, providing opportunities for expanding our ...understanding of nature's chemical and biosynthetic repertoires. Here, we have combined tools from natural products discovery and mechanistic enzymology to elucidate the structure and biosynthesis of streptide, a streptococcal macrocyclic peptide. We show that streptide bears an unprecedented post-translational modification involving a covalent linkage between two unactivated carbons within the side chains of lysine and tryptophan. The biosynthesis of streptide was addressed by genetic and biochemical studies. The former implicated a new SPASM-domain-containing radical SAM enzyme StrB, while the latter revealed that StrB contains two 4Fe-4S clusters and installs the unusual lysine-to-tryptophan crosslink in a single step. By intramolecularly stitching together the side chains of lysine and tryptophan, StrB provides a new route for biosynthesizing macrocyclic peptides.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UL, UM, UPUK
Selenium is an essential micronutrient in diverse organisms. Two routes are known for its insertion into proteins and nucleic acids, via selenocysteine and 2-selenouridine, respectively1. However, ...despite its importance, pathways for specific incorporation of selenium into small molecules have remained elusive. Here we use a genome-mining strategy in various microorganisms to uncover a widespread three-gene cluster that encodes a dedicated pathway for producing selenoneine, the selenium analogue of the multifunctional molecule ergothioneine2,3. We elucidate the reactions of all three proteins and uncover two novel selenium-carbon bond-forming enzymes and the biosynthetic pathway for production of a selenosugar, which is an unexpected intermediate en route to the final product. Our findings expand the scope of biological selenium utilization, suggest that the selenometabolome is more diverse than previously thought, and set the stage for the discovery of other selenium-containing natural products.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Microbial natural products are genetically encoded by dedicated biosynthetic gene clusters (BGCs). A given BGC usually produces a family of related compounds that share a core but contain variable ...substituents. Though common, the reasons underlying this divergent biosynthesis are in general unknown. Herein, we have addressed this issue using the hydroxyalkylquinoline (HAQ) family of natural products synthesized by Burkholderia thailandensis. Investigations into the detailed functions of two analogs show that they act synergistically in inhibiting bacterial growth. One analog is a nanomolar inhibitor of pyrimidine biosynthesis and at the same time disrupts the proton motive force. A second analog inhibits the cytochrome bc1 complex as well as pyrimidine biogenesis. These results provide a functional rationale for the divergent nature of HAQs. They imply that synergy and target promiscuity are driving forces for the evolution of tailoring enzymes that diversify the products of the HAQ biosynthetic pathway.
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•HMNQ and HQNO, two natural quinolones, act synergistically to inhibit bacterial growth•Bacterial cytological profiles show they inhibit distinct steps in energy production•Primary metabolite analysis reveals that both also inhibit pyrimidine biosynthesis•Our results provide a functional rationale for the structural divergence in quinolones
Secondary metabolism has long been known to be diversity-oriented. Here, Wu and Seyedsayamdost provide an explanation for this phenomenon by demonstrating that two structurally related quinolone natural products from the same biosynthetic pathway have different targets and synergistically inhibit bacterial growth.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The majority of natural product biosynthetic gene clusters in bacteria are silent under standard laboratory growth conditions, making it challenging to uncover any antibiotics that they may encode. ...Herein, bioactivity assays are combined with high‐throughput elicitor screening (HiTES) to access cryptic, bioactive metabolites. Application of this strategy in Saccharopolyspora cebuensis, with inhibition of Escherichia coli growth as a read‐out, led to the identification of a novel lanthipeptide, cebulantin. Extensive NMR spectroscopic analysis allowed the elucidation of the structure of cebulantin. Subsequent bioactivity assays revealed it to be an antibiotic selective for Gram‐negative bacteria, especially against Vibrio species. This approach, referred to as bioactivity‐HiTES, has the potential to uncover cryptic metabolites with desired biological activities that are hidden in microbial genomes.
Cryptic lantibiotic unveiled: A new natural product discovery method, bioactivity‐HiTES, facilitates the identification of cryptic, bioactive metabolites. Using this approach, a lanthipeptide antibiotic, cryptic under normal growth conditions, with selective inhibitory activity against Gram‐negative bacteria, was characterized.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Abstract
Natural products have traditionally served as a dominant source of therapeutic agents. They are produced by dedicated biosynthetic gene clusters that assemble complex, bioactive molecules ...from simple precursors. Recent genome sequencing efforts coupled with advances in bioinformatics indicate that the majority of biosynthetic gene clusters are not expressed under normal laboratory conditions. Termed ‘silent’ or ‘cryptic’, these gene clusters represent a treasure trove for discovery of novel small molecules, their regulatory circuits and their biosynthetic pathways. In this review, we assess the capacity of exogenous small molecules in activating silent secondary metabolite gene clusters. Several approaches that have been developed are presented, including coculture techniques, ribosome engineering, chromatin remodeling and high-throughput elicitor screens. The rationale, applications and mechanisms attendant to each are discussed. Some general conclusions can be drawn from our analysis: exogenous small molecules comprise a productive avenue for the discovery of cryptic metabolites. Specifically, growth-inhibitory molecules, in some cases clinically used antibiotics, serve as effective inducers of silent biosynthetic gene clusters, suggesting that old antibiotics may be used to find new ones. The involvement of natural antibiotics in modulating secondary metabolism at subinhibitory concentrations suggests that they represent part of the microbial vocabulary through which inter- and intraspecies interactions are mediated.
Microbial secondary metabolites represent a significant source of potential drug leads; however, the majority of the corresponding biosynthetic genes are not expressed under normal laboratory conditions. In this review, we assess the capacity of exogenous small molecules, especially antibiotics, to activate these silent 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.
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