Draft genome of Omphalotus olearius provides a predictive framework for sesquiterpenoid natural product biosynthesis in Basidiomycota

The secondary metabolome of Basidiomycota represents a largely uncharacterized source of pharmaceutically relevant natural products. Terpenoids are the primary class of bioactive compounds isolated from mushrooms. The Jack O'Lantern mushroom Omphalotus olearius was identified 50 years ago as a prolific producer of anticancer illudin sesquiterpenoids; however, to date there have been exceptionally few studies into the biosynthesis of these important compounds. Here, we report the draft genome sequence of O. olearius, which reveals a diverse network of sesquiterpene synthases and two metabolic gene clusters associated with illudin biosynthesis. Characterization of the sesquiterpene synthases enabled a comprehensive survey of all currently available Basidiomycota genomes, thereby creating a predictive resource for terpenoid natural product biosynthesis in these organisms. Our results will facilitate discovery and biosynthetic production of unique pharmaceutically relevant bioactive compounds from Basidiomycota.     

Biodiversity of Secondary Metabolites Compounds Isolated from Phylum Actinobacteria and Its Therapeutic Applications

The current review aims to summarise the biodiversity and biosynthesis of novel secondary metabolites compounds, of the phylum Actinobacteria and the diverse range of secondary metabolites produced that vary depending on its ecological environments they inhabit. Actinobacteria creates a wide range of bioactive substances that can be of great value to public health and the pharmaceutical industry. The literature analysis process for this review was conducted using the VOSviewer software tool to visualise the bibliometric networks of the most relevant databases from the Scopus database in the period between 2010 and 22 March 2021. Screening and exploring the available literature relating to the extreme environments and ecosystems that Actinobacteria inhabit aims to identify new strains of this major microorganism class, producing unique novel bioactive compounds. The knowledge gained from these studies is intended to encourage scientists in the natural product discovery field to identify and characterise novel strains containing various bioactive gene clusters with potential clinical applications. It is evident that Actinobacteria adapted to survive in extreme environments represent an important source of a wide range of bioactive compounds. Actinobacteria have a large number of secondary metabolite biosynthetic gene clusters. They can synthesise thousands of subordinate metabolites with different biological actions such as anti-bacterial, anti-parasitic, anti-fungal, anti-virus, anti-cancer and growth-promoting compounds. These are highly significant economically due to their potential applications in the food, nutrition and health industries and thus support our communities’ well-being.     

Synergizing the potential of bacterial genomics and metabolomics to find novel antibiotics

Antibiotic development based on natural products has faced a long lasting decline since the 1970s, while both the speed and the extent of antimicrobial resistance (AMR) development have been severely underestimated. The discovery of antimicrobial natural products of bacterial and fungal origin featuring new chemistry and previously unknown mode of actions is increasingly challenged by rediscovery issues. Natural products that are abundantly produced by the corresponding wild type organisms often featuring strong UV signals have been extensively characterized, especially the ones produced by extensively screened microbial genera such as streptomycetes. Purely synthetic chemistry approaches aiming to replace the declining supply from natural products as starting materials to develop novel antibiotics largely failed to provide significant numbers of antibiotic drug leads. To cope with this fundamental issue, microbial natural products science is being transformed from a ‘grind-and-find’ study to an integrated approach based on bacterial genomics and metabolomics. Novel technologies in instrumental analytics are increasingly employed to lower detection limits and expand the space of detectable substance classes, while broadening the scope of accessible and potentially bioactive natural products. Furthermore, the almost exponential increase in publicly available bacterial genome data has shown that the biosynthetic potential of the investigated strains by far exceeds the amount of detected metabolites. This can be judged by the discrepancy between the number of biosynthetic gene clusters (BGC) encoded in the genome of each microbial strain and the number of secondary metabolites actually detected, even when considering the increased sensitivity provided by novel analytical instrumentation. In silico annotation tools for biosynthetic gene cluster classification and analysis allow fast prioritization in BGC-to-compound workflows, which is highly important to be able to process the enormous underlying data volumes. BGC prioritization is currently accompanied by novel molecular biology-based approaches to access the so-called orphan BGCs not yet correlated with a secondary metabolite. Integration of metabolomics, in silico genomics and molecular biology approaches into the mainstream of natural product research will critically influence future success and impact the natural product field in pharmaceutical, nutritional and agrochemical applications and especially in anti-infective research.

Antimicrobial resistance is a major public concern and novel antibiotics are largely based on natural products. We summarize recent analytical and genome based technological developments that gain increasing importance in the natural products field.     

Discovery of small molecule protease inhibitors by investigating a widespread human gut bacterial biosynthetic pathway

Natural products from the human microbiota may mediate host health and disease. However, discovery of the biosynthetic gene clusters that generate these metabolites has far outpaced identification of the molecules themselves. Here, we used an isolation-independent approach to access the probable products of a nonribosomal peptide synthetase-encoding gene cluster from Ruminococcus bromii, an abundant gut commensal bacterium. By combining bioinformatics with in vitro biochemical characterization of biosynthetic enzymes, we predicted that this pathway likely generates an N-acylated dipeptide aldehyde (ruminopeptin). We then used chemical synthesis to access putative ruminopeptin scaffolds. Several of these compounds inhibited Staphylococcus aureus endoproteinase GluC (SspA/V8 protease). Homologs of this protease are found in gut commensals and opportunistic pathogens as well as human gut metagenomes. Overall, this work reveals the utility of isolation-independent approaches for rapidly accessing bioactive compounds and highlights a potential role for gut microbial natural products in targeting gut microbial proteases.     

Some structural systematics in metal carbonyl cluster chemistry

The recently developed electron-counting rules for condensed polyhedral metal carbonyl clusters have been utilised to develop a systematic approach for enumerating the structural alternatives for metal cluster compounds which are isoelectronic. The more usual cluster degradation and aggregation processes are represented in a matrix form which relates the change in electron count to the number of new bonds formed (or broken). The matrices for cluster degradation and aggregation are represented in a complementary fashion so that the search for isoelectronic clusters greatly simplified. The utility of the approach is illustrated by reference to some recent examples from metal carbonyl cluster chemistry.     

Applications and Restrictions of Integrated Genomic and Metabolomic Screening: An Accelerator for Drug Discovery from Actinomycetes?

Since the golden age of antibiotics in the 1950s and 1960s actinomycetes have been the most prolific source for bioactive natural products. However, the number of discoveries of new bioactive compounds decreases since decades. New procedures (e.g., activating strategies or innovative fermentation techniques) were developed to enhance the productivity of actinomycetes. Nevertheless, compound identification remains challenging among others due to high rediscovery rates. Rapid and cheap genome sequencing as well as the advent of bioinformatical analysis tools for biosynthetic gene cluster identification in combination with mass spectrometry-based molecular networking facilitated the tedious process of dereplication. In recent years several studies have been dedicated to accessing the biosynthetic potential of Actinomyces species, especially streptomycetes, by using integrated genomic and metabolomic screening in order to boost the discovery rate of new antibiotics. This review aims to present the various possible applications of this approach as well as the newly discovered molecules, covering studies between 2014 and 2021. Finally, the effectiveness of this approach with regard to find new bioactive agents from actinomycetes will be evaluated.     

SYN-View: A Phylogeny-Based Synteny Exploration Tool for the Identification of Gene Clusters Linked to Antibiotic Resistance

The development of new antibacterial drugs has become one of the most important tasks of the century in order to overcome the posing threat of drug resistance in pathogenic bacteria. Many antibiotics originate from natural products produced by various microorganisms. Over the last decades, bioinformatical approaches have facilitated the discovery and characterization of these small compounds using genome mining methodologies. A key part of this process is the identification of the most promising biosynthetic gene clusters (BGCs), which encode novel natural products. In 2017, the Antibiotic Resistant Target Seeker (ARTS) was developed in order to enable an automated target-directed genome mining approach. ARTS identifies possible resistant target genes within antibiotic gene clusters, in order to detect promising BGCs encoding antibiotics with novel modes of action. Although ARTS can predict promising targets based on multiple criteria, it provides little information about the cluster structures of possible resistant genes. Here, we present SYN-view. Based on a phylogenetic approach, SYN-view allows for easy comparison of gene clusters of interest and distinguishing genes with regular housekeeping functions from genes functioning as antibiotic resistant targets. Our aim is to implement our proposed method into the ARTS web-server, further improving the target-directed genome mining strategy of the ARTS pipeline.     

Malleilactone, a Polyketide Synthase-Derived Virulence Factor Encoded by the Cryptic Secondary Metabolome of Burkholderia pseudomallei Group Pathogens

Sequenced bacterial genomes are routinely found to contain gene clusters that are predicted to encode metabolites not seen in fermentation-based studies. Pseudomallei group Burkholderia are emerging pathogens whose genomes are particularly rich in cryptic natural product biosynthetic gene clusters. We systematically probed the influence of the cryptic secondary metabolome on the virulence of these bacteria and found that disruption of the MAL gene cluster, which is natively silent in laboratory fermentation experiments and conserved across this group of pathogens, attenuates virulence in animal models. Using a promoter exchange strategy to activate the MAL cluster, we identified malleilactone, a polyketide synthase-derived cytotoxic siderophore encoded by this gene cluster. Small molecules targeting malleilactone biosynthesis either alone or in conjunction with antibiotics could prove useful as therapeutics to combat melioidosis and glanders.     

Toblerols: Cyclopropanol‐Containing Polyketide Modulators of Antibiosis in Methylobacteria

Trans‐AT polyketide synthases (PKSs) are a family of biosynthetically versatile modular type I PKSs that generate bioactive polyketides of impressive structural diversity. In this study, we detected, in the genome of several bacteria a cryptic, architecturally unusual trans‐AT PKS gene cluster which eluded automated PKS prediction. Genomic mining of one of these strains, the model methylotroph Methylobacterium extorquens AM1, revealed unique epoxide‐ and cyclopropanol‐containing polyketides named toblerols. Relative and absolute stereochemistry were determined by NMR experiments, chemical derivatization, and the comparison of CD data between the derivatized natural product and a synthesized model compound. Biosynthetic data suggest that the cyclopropanol moiety is generated by carbon–carbon shortening of a more extended precursor. Surprisingly, a knock‐out strain impaired in polyketide production showed strong inhibitory activity against other methylobacteria in contrast to the wild‐type producer. The activity was inhibited by complementation with toblerols, thus suggesting that these compounds modulate an as‐yet unknown methylobacterial antibiotic.     

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.     

Cebulantin,a Cryptic Lanthipeptide Antibiotic Uncovered Using Bioactivity‐Coupled HiTES

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.     

Cebulantin,a Cryptic Lanthipeptide Antibiotic Uncovered Using Bioactivity‐Coupled HiTES

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.     

Catalytic Asymmetric Synthesis of Isoindolinones

As important reactive species, isoindolinones represent a cluster of valuable building blocks for the construction of natural‐product‐like compounds. In particular, chiral isoindolinones are the key structural feature of naturally occurring bioactive molecules. During the past decade, great advances have been made in the asymmetric synthesis of isoindolinone skeleton compounds. Hence, recent progress in catalytic asymmetric synthesis of isoindolinones is reviewed here, with emphasis on chiral organocatalysis and transition metal complexes enabled transformations. Different organocatalysts (chiral phosphoric acids, phase transfer catalysts, chiral thioureas) and chiral transition‐metal complexes (Rh, Pd, Ir, Cu, Mg, and Ni) are included in this transformation.     

Cluster chemical ionization for improved confidence level in sample identification by gas chromatography/mass spectrometry

Upon the supersonic expansion of helium mixed with vapor from an organic solvent (e.g. methanol), various clusters of the solvent with the sample molecules can be formed. As a result of 70 eV electron ionization of these clusters, cluster chemical ionization (cluster CI) mass spectra are obtained. These spectra are characterized by the combination of EI mass spectra of vibrationally cold molecules in the supersonic molecular beam (cold EI) with CI-like appearance of abundant protonated molecules, together with satellite peaks corresponding to protonated or non-protonated clusters of sample compounds with 1-3 solvent molecules. Like CI, cluster CI preferably occurs for polar compounds with high proton affinity. However, in contrast to conventional CI, for non-polar compounds or those with reduced proton affinity the cluster CI mass spectrum converges to that of cold EI. The appearance of a protonated molecule and its solvent cluster peaks, plus the lack of protonation and cluster satellites for prominent EI fragments, enable the unambiguous identification of the molecular ion. In turn, the insertion of the proper molecular ion into the NIST library search of the cold EI mass spectra eliminates those candidates with incorrect molecular mass and thus significantly increases the confidence level in sample identification. Furthermore, molecular mass identification is of prime importance for the analysis of unknown compounds that are absent in the library. Examples are given with emphasis on the cluster CI analysis of carbamate pesticides, high explosives and unknown samples, to demonstrate the usefulness of Supersonic GC/MS (GC/MS with supersonic molecular beam) in the analysis of these thermally labile compounds. Cluster CI is shown to be a practical ionization method, due to its ease-of-use and fast instrumental conversion between EI and cluster CI, which involves the opening of only one valve located at the make-up gas path. The ease-of-use of cluster CI is analogous to that of liquid CI in ion traps with internal ionization, and is in marked contrast to that of CI with most other standard GC/MS systems that require a change of the ion source.     

Heterologous expression of a myxobacterial natural products assembly line in pseudomonads via red/ET recombineering

Natural products of microbial origin are widely used as pharmaceuticals and in agrochemistry. These compounds are often biosynthesized by multifunctional megasynthetases whose genetic engineering and heterologous expression offer considerable promise, especially if the natural hosts are genetically difficult to handle, slow growing, unculturable, or even unknown. We describe a straightforward strategy that combines the power of advanced DNA engineering (recombiogenic cloning) in Escherichia coli with the utility of pseudomonads as the heterologous host for the analysis and mutagenesis of known and unknown secondary metabolite pathways. The myxochromide S biosynthetic gene cluster from Stigmatella aurantiaca was rebuilt and engineered in E. coli to contain the elements required for expression in pseudomonads. The successful production in Pseudomonas putida, at unprecedented levels, demonstrates the feasibility of the new approach to the analysis and mutagenesis of these important pathways.     

The neomycin biosynthetic gene cluster of Streptomyces fradiae NCIMB 8233: characterisation of an aminotransferase involved in the formation of 2-deoxystreptamine

The biosynthetic gene cluster of the 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotic neomycin has been cloned for the first time by screening of a cosmid library of Streptomyces fradiae NCIMB 8233. Sequence analysis has identified 21 putative open reading frames (ORFs) in the neomycin gene cluster (neo) with significant protein sequence similarity to gene products involved in the biosynthesis of other DOS-containing aminoglycosides, namely butirosin (btr), gentamycin (gnt), tobramycin (tbm) and kanamycin (kan). Located at the 5'-end of the neo gene cluster is the previously-characterised neomycin phosphotransferase gene (apH). Three genes unique to the neo and btr clusters have been revealed by comparison of the neo cluster to btr, gnt, tbm and kan clusters. This suggests that these three genes may be involved in the transfer of a ribose moiety to the DOS ring during the antibiotic biosynthesis. The product of the neo-6 gene is characterised here as the L-glutamine : 2-deoxy-scyllo-inosose aminotransferase responsible for the first transamination in DOS biosynthesis, which supports the assignment of the gene cluster.     

Mixing and Matching Siderophore Clusters: Structure and Biosynthesis of Serratiochelins from Serratia sp. V4

Interrogation of the evolutionary history underlying the remarkable structures and biological activities of natural products has been complicated by not knowing the functions they have evolved to fulfill. Siderophores-soluble, low molecular weight compounds-have an easily understood and measured function: acquiring iron from the environment. Bacteria engage in a fierce competition to acquire iron, which rewards the production of siderophores that bind iron tightly and cannot be used or pirated by competitors. The structures and biosyntheses of "odd" siderophores can reveal the evolutionary strategy that led to their creation. We report a new Serratia strain that produces serratiochelin and an analog of serratiochelin. A genetic approach located the serratiochelin gene cluster, and targeted mutations in several genes implicated in serratiochelin biosynthesis were generated. Bioinformatic analyses and mutagenesis results demonstrate that genes from two well-known siderophore clusters, the Escherichia coli enterobactin cluster and the Vibrio cholera vibriobactin cluster, were shuffled to produce a new siderophore biosynthetic pathway. These results highlight how modular siderophore gene clusters can be mixed and matched during evolution to generate structural diversity in siderophores.     

LC‐MS analysis for the components captured by ECV304 cell from extract of Aconitum szechenyianum Gay.

A novel method of cell affinity screening (CAS), cell affinity capture coupled with LC‐MS analysis, was developed for screening the bioactive compounds related to cardiovascular diseases from the natural product libraries. One of the major characteristics lies in its function in affinity‐capturing and separating the bioactive components from the natural product libraries in vitro. Another characteristic is its use in analyzing and identifying the target compounds, by employing high‐performance liquid chromatography and mass spectrometry. CAS was used for screening the bioactive components from the alkaloid extract derived from Aconitum szechenyianum Gay. Of the five components found to be bound to the oxidative‐damaged endothelial cells, the two compounds identified, mesaconitine and aconitine, were recognized in the literature as being related to cardiovascular diseases. Copyright © 2008 John Wiley & Sons, Ltd.