Secondary Metabolites of the Genus Amycolatopsis: Structures,Bioactivities and Biosynthesis

Actinomycetes are regarded as important sources for the generation of various bioactive secondary metabolites with rich chemical and bioactive diversities. Amycolatopsis falls under the rare actinomycete genus with the potential to produce antibiotics. In this review, all literatures were searched in the Web of Science, Google Scholar and PubMed up to March 2021. The keywords used in the search strategy were “Amycolatopsis”, “secondary metabolite”, “new or novel compound”, “bioactivity”, “biosynthetic pathway” and “derivatives”. The objective in this review is to summarize the chemical structures and biological activities of secondary metabolites from the genus Amycolatopsis. A total of 159 compounds derived from 8 known and 18 unidentified species are summarized in this paper. These secondary metabolites are mainly categorized into polyphenols, linear polyketides, macrolides, macrolactams, thiazolyl peptides, cyclic peptides, glycopeptides, amide and amino derivatives, glycoside derivatives, enediyne derivatives and sesquiterpenes. Meanwhile, they mainly showed unique antimicrobial, anti-cancer, antioxidant, anti-hyperglycemic, and enzyme inhibition activities. In addition, the biosynthetic pathways of several potent bioactive compounds and derivatives are included and the prospect of the chemical substances obtained from Amycolatopsis is also discussed to provide ideas for their implementation in the field of therapeutics and drug discovery.     

Spatial regulation of a common precursor from two distinct genes generates metabolite diversity

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase genes usually encode proteins that generate various backbone precursors. These precursors are modified by other tailoring enzymes to yield a large variety of different secondary metabolites. The number of core synthesis genes in a given species correlates, therefore, with the number of types of secondary metabolites the organism can produce. In our study, heterologous expression of all the A. terreus NRPS-like genes showed that two NRPS-like proteins, encoded by atmelA and apvA, release the same natural product, aspulvinone E. In hyphae this compound is converted to aspulvinones whereas in conidia it is converted to melanin. The genes are expressed in different tissues and this spatial control is probably regulated by their own specific promoters. Comparative genomics indicates that atmelA and apvA might share a same ancestral gene and the gene apvA is located in a highly conserved region in Aspergillus species that contains genes coding for life-essential proteins. Our data reveal the first case in secondary metabolite biosynthesis in which the tissue specific production of a single compound directs it into two separate pathways, producing distinct compounds with different functions. Our data also reveal that a single trans-prenyltransferase, AbpB, prenylates two substrates, aspulvinones and butyrolactones, revealing that genes outside of contiguous secondary metabolism gene clusters can modify more than one compound thereby expanding metabolite diversity. Our study raises the possibility of incorporation of spatial, cell-type specificity in expression of secondary metabolites of biological interest and provides new insight into designing and reconstituting their biosynthetic pathways.     

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.     

Genomic and Metabolomic Investigation of a Rhizosphere Isolate Streptomyces netropsis WLXQSS-4 Associated with a Traditional Chinese Medicine

Rhizosphere microorganisms play important ecological roles in promoting herb growth and producing abundant secondary metabolites. Studies on the rhizosphere microbes of traditional Chinese medicines (TCMs) are limited, especially on the genomic and metabolic levels. In this study, we reported the isolation and characterization of a Steptomyces netropsis WLXQSS-4 strain from the rhizospheric soil of Clematis manshurica Rupr. Genomic sequencing revealed an impressive total of 40 predicted biosynthetic gene clusters (BGCs), whereas metabolomic profiling revealed 13 secondary metabolites under current laboratory conditions. Particularly, medium screening activated the production of alloaureothin, whereas brominated and chlorinated pimprinine derivatives were identified through precursor-directed feeding. Moreover, antiproliferative activities against Hela and A549 cancer cell lines were observed for five compounds, of which two also elicited potent growth inhibition in Enterococcus faecalis and Staphylococcus aureus, respectively. Our results demonstrated the robust secondary metabolism of S. netropsis WLXQSS-4, which may serve as a biocontrol agent upon further investigation.     

Secondary Metabolites of Purpureocillium lilacinum

Fungi can synthesize a wealth of secondary metabolites, which are widely used in the exploration of lead compounds of pharmaceutical or agricultural importance. Beauveria, Metarhizium, and Cordyceps are the most extensively studied fungi in which a large number of biologically active metabolites have been identified. However, relatively little attention has been paid to Purpureocillium lilacinum. P. lilacinum are soil-habituated fungi that are widely distributed in nature and are very important biocontrol fungi in agriculture, providing good biological control of plant parasitic nematodes and having a significant effect on Aphidoidea, Tetranychus cinnbarinus, and Aleyrodidae. At the same time, it produces secondary metabolites with various biological activities such as anticancer, antimicrobial, and insecticidal. This review attempts to provide a comprehensive overview of the secondary metabolites of P. lilacinum, with emphasis on the chemical diversity and biological activity of these secondary metabolites and the biosynthetic pathways, and gives new insight into the secondary metabolites of medical and entomogenous fungi, which is expected to provide a reference for the development of medicine and agrochemicals in the future.     

Aminoglycoside Antibiotics: New Insights into the Biosynthetic Machinery of Old Drugs

2‐Deoxystreptamine (2DOS) is the unique chemically stable aminocyclitol scaffold of clinically important aminoglycoside antibiotics such as neomycin, kanamycin, and gentamicin, which are produced by Actinomycetes. The 2DOS core can be decorated with various deoxyaminosugars to make structurally diverse pseudo‐oligosaccharides. After the discovery of biosynthetic gene clusters for 2DOS‐containing aminoglycoside antibiotics, the function of each biosynthetic enzyme has been extensively elucidated. The common biosynthetic intermediates 2DOS, paromamine and ribostamycin are constructed by conserved enzymes encoded in the gene clusters. The biosynthetic intermediates are then converted to characteristic architectures by unique enzymes encoded in each biosynthetic gene cluster. In this Personal Account, we summarize both common biosynthetic pathways and the pathways used for structural diversification.

     

Genome Mining,Heterologous Expression,Antibacterial and Antioxidant Activities of Lipoamides and Amicoumacins from Compost-Associated Bacillus subtilis fmb60

Bacillus subtilis fmb60, which has broad-spectrum antimicrobial activities, was isolated from plant straw compost. A hybrid NRPS/PKS cluster was screened from the genome. Sixteen secondary metabolites produced by the gene cluster were isolated and identified using LC-HRMS and NMR. Three lipoamides D–F (1–3) and two amicoumacin derivatives, amicoumacins D, E (4, 5), were identified, and are reported here for the first time. Lipoamides D–F exhibited strong antibacterial activities against harmful foodborne bacteria, with the MIC ranging from 6.25 to 25 µg/mL. Amicoumacin E scavenged 38.8% of ABTS⁺ radicals at 1 mg/mL. Direct cloning and heterologous expression of the NRPS/PKS and ace gene cluster identified its importance for the biosynthesis of amicoumacins. This study demonstrated that there is a high potential for biocontrol utilization of B. subtilis fmb60, and genome mining for clusters of secondary metabolites of B. subtilis fmb60 has revealed a greater biosynthetic potential for the production of novel natural products than previously anticipated.     

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.     

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.     

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.     

Lentzeacins A-E,New Bacterial-Derived 2,5- and 2,6-Disubstituted Pyrazines from a BGC-Rich Soil Bacterium Lentzea sp. GA3-008

Pyrazines (1,4-diazirines) are an important group of natural products that have tremendous monetary value in the food and fragrance industries and can exhibit a wide range of biological effects including antineoplastic, antidiabetic and antibiotic activities. As part of a project investigating the secondary metabolites present in understudied and chemically rich Actinomycetes, we isolated a series of six pyrazines from a soil-derived Lentzea sp. GA3-008, four of which are new. Here we describe the structures of lentzeacins A-E (1, 3, 5 and 6) along with two known analogues (2 and 4) and the porphyrin zincphyrin. The structures were determined by NMR spectroscopy and HR-ESI-MS. The suite of compounds present in Lentzea sp. includes 2,5-disubstituted pyrazines (compounds 2, 4, and 6) together with the new 2,6-disubstituted isomers (compounds 1, 3 and 5), a chemical class that is uncommon. We used long-read Nanopore sequencing to assemble a draft genome sequence of Lentzea sp. which revealed the presence of 40 biosynthetic gene clusters. Analysis of classical di-modular and single module non-ribosomal peptide synthase genes, and cyclic dipeptide synthases narrows down the possibilities for the biosynthesis of the pyrazines present in this strain.     

NMR Characterization of Lignans

Lignans are particularly interesting secondary metabolites belonging to the phenyl-propanoid biosynthetic pathway. From the structural point of view, these molecules could belong to the aryltetralin, arylnaphtalene, or dibenzylbutyrolactone molecular skeleton. Lignans are present in different tissues of plants but are mainly accumulated in seeds. Extracts from plant tissues could be characterized by using the NMR-based approach, which provides a profile of aromatic molecules and detailed structural information for their elucidation. In order to improve the production of these secondary metabolites, elicitors could effectively stimulate lignan production. Several plant species are considered in this review with a particular focus on Linum species, well recognized as the main producer of lignans.     

Characterization of the secondary metabolite biosynthetic gene clusters in archaea

Secondary metabolites are a range of bioactive compounds yielded by bacteria, fungi and plants, etc. The published archaea genomic data provide the opportunity for efficient identification of secondary metabolite biosynthetic gene clusters (BGCs) by genome mining. However, the study of secondary metabolites in archaea is still rare. By using the antiSMASH, we found two main putative secondary metabolite BGCs, bacteriocin and terpene in 203 Archaea genomes. Compared with the genomes of Euryarchaeota that usually lives in less complexity of environment, the genomes of Crenarchaeota usually contained more abundant bacteriocin. In these archaea genomes, we also found the positive correlation between the abundance of bacteriocin and the abundance of CRISPR spacer, suggesting the bacteriocin might be a crucial component of the innate immune system that defense the microbe living in the common environment. The structure analysis of the bacteriocin gene clusters gave a clue that the assisted genes located at the edge of clusters evolved faster than the core biosynthetic genes. To the best of our knowledge, we are the first to systematically explore the distribution of secondary metabolites in archaea, and the investigation of the relationship between BGC and CRISPR spacer expands our understanding of the evolutionary dynamic of these functional molecules.     

Development of Genetic Dereplication Strains in Aspergillus nidulans Results in the Discovery of Aspercryptin

To reduce the secondary metabolite background in Aspergillus nidulans and minimize the rediscovery of compounds and pathway intermediates, we created a “genetic dereplication” strain in which we deleted eight of the most highly expressed secondary metabolite gene clusters (more than 244,000 base pairs deleted in total). This strain allowed us to discover a novel compound that we designate aspercryptin and to propose a biosynthetic pathway for the compound. Interestingly, aspercryptin is formed from compounds produced by two separate gene clusters, one of which makes the well‐known product cichorine. This raises the exciting possibility that fungi use differential regulation of expression of secondary metabolite gene clusters to increase the diversity of metabolites they produce.     

Possibility to Biotransform Anthracyclines by Peroxidases Produced by Bjerkandera adusta CCBAS 930 with Reduction of Geno- and Cytotoxicity and Pro-Oxidative Activity

The aim of this study was to evaluate the bioremoval mechanism of anthracycline antibiotics by the white-rot fungus B. adusta CCBAS 930. The activity of oxidoreductases and levels of phenolic compounds and free radicals were determined during the biotransformation of anthraquinone antibiotics: daunomycin (DNR) and doxorubicin (DOX) by B. adusta strain CCBAS 930. Moreover, phytotoxicity (Lepidium sativum L.), ecotoxicity (Vibrio fischeri), genotoxicity and cytotoxicity of anthraquinone dyes were evaluated before and after biological treatment. More than 80% and 90% of DNR and DOX were removed by biodegradation (decolorization). Initial solutions of DNR and DOX were characterized by eco-, phyto-, geno- and cytotoxicity. Despite efficient decolorization, secondary metabolites, toxic to bacteria, formed during biotransformation of anthracycline antibiotics in B. adusta CCBAS 930 cultures. DNR and DOX metabolites did not increase reactive oxygen species (ROS) production in human fibroblasts and resazurin reduction. DNR metabolites did not change caspase-3 activity.     

Metabolomic Strategy to Characterize the Profile of Secondary Metabolites in Aspergillus aculeatus DL1011 Regulated by Chemical Epigenetic Agents

Chemical epigenetic regulation (CER) is an effective method to activate the silent pathway of fungal secondary metabolite synthesis. However, conventional methods for CER study are laborious and time-consuming. In the meantime, the overall profile of the secondary metabolites in the fungi treated by the CER reagent is not well characterized. In this study, suberohydroxamic acid (SBHA), a histone deacetylase inhibitor, was added to a culture of Aspergillus aculeatus DL1011 and a new strategy based on LC-MS/MS analysis integrated with various metabolomic tools (MetaboAnalyst, MS-DIAL, SIRIUS and GNPS) was developed to characterize the profile of induced metabolites. As a result, 13.6%, 29.5% and 27.2% of metabolites were identified as newly biosynthesized, increasing and decreasing in abundance by CER, respectively. The structures of the 18 newly induced secondary metabolites were further identified by the new strategy to demonstrate that 72.2% of them (1 novel compound and 12 known compounds) were first discovered in A. aculeatus upon SBHA treatment. The accuracy of the new approach was confirmed by purification and NMR data analysis of major newly biosynthesized secondary metabolites. The bioassay showed that the newly biosynthesized compounds, roseopurpurin analogues, showed selective activities against DPPH scavenging, cytotoxicity and SHP1 inhibition. Our research demonstrated that CER was beneficial for changing the secondary metabolic profile of fungi and was an effective means of increasing the diversity of active metabolites. Our work also supplied a metabolomic strategy to characterize the profile changes and determine the newly induced compounds in the secondary metabolites of fungi treated with the chemical epigenetic regulator.     

Metabolomic Profiling and Molecular Networking of Nudibranch-Associated Streptomyces sp. SCSIO 001680

Antibiotic-resistant bacteria are the primary source of one of the growing public health problems that requires global attention, indicating an urgent need for new antibiotics. Marine ecosystems are characterized by high biodiversity and are considered one of the essential sources of bioactive chemical compounds. Bacterial associates of marine invertebrates are commonly a source of active medicinal and natural products and are important sources for drug discovery. Hence, marine invertebrate-associated microbiomes are a fruitful resource for excavating novel genes and bioactive compounds. In a previous study, we isolated Streptomyces sp. SCSIO 001680, coded as strain 63, from the Red Sea nudibranch Chromodoris quadricolor, which exhibited antimicrobial and antitumor activity. In addition, this isolate harbors several natural product biosynthetic gene clusters, suggesting it has the potential to produce bioactive natural products. The present study aimed to investigate the metabolic profile of the isolated Streptomyces sp. SCSIO 001680 (strain 63) and to predict their potential role in the host’s survival. The crude metabolic extracts of strain 63 cultivated in two different media were characterized by ultra-high-performance liquid chromatography and high-resolution mass spectrometry. The metabolomics approach provided us with characteristic chemical fingerprints of the cellular processes and the relative abundance of specific compounds. The Global Products Social Molecular Networking database was used to identify the metabolites. While 434 metabolites were detected in the extracts, only a few compounds were identified based on the standards and the public spectral libraries, including desferrioxamines, marineosin A, and bisucaberin, halichoblelide, alternarin A, pachastrelloside A, streptodepsipeptide P1 1B, didemnaketal F, and alexandrolide. This finding suggests that this strain harbors several novel compounds. In addition, the metabolism of the microbiome of marine invertebrates remains poorly represented. Thus, our data constitute a valuable complement to the study of metabolism in the host microbiome.     

Labdane Diterpenoids from Salvia tingitana Etl. Synergize with Clindamycin against Methicillin-Resistant Staphylococcus aureus

Quorum-sensing (QS) is a regulatory mechanism in bacterial communication, important for pathogenesis control. The search for small molecules active as quorum-sensing inhibitors (QSI) that can synergize with antibiotics is considered a good strategy to counteract the problem of antibiotic resistance. Here the antimicrobial labdane diterpenoids sclareol (1) and manool (2) extracted from Salvia tingitana were considered as potential QSI against methicillin-resistant Staphylococcus aureus. Only sclareol showed synergistic activity with clindamycin. The quantification of these compounds by LC–MS analysis in the organs and in the calli of S. tingitana showed that sclareol is most abundant in the flower spikes and is produced by calli, while manool is the major labdane of the roots, and is abundant also in the leaves. Other metabolites of the roots were abietane diterpenoids, common in Salvia species, and pentacyclic triterpenoids, bearing a γ-lactone moiety, previously undescribed in Salvia. Docking simulations suggested that 1 and 2 bind to key residues, involved in direct interactions with DNA. They may prevent accessory gene regulator A (AgrA) binding to DNA or AgrA activation upon phosphorylation, to suppress virulence factor expression. The antimicrobial activity of these two compounds probably achieves preventing upregulation of the accessory gene regulator (agr)-regulated genes.     

Dielectric properties of water clusters containing CO and NO molecules. Computational experiment

The absorption of CO and NO molecules by (H₂O)₂₀ clusters was studied by the method of molecular dynamics. In general, the clusters containing CO molecules are more stable mechanically, while the clusters with NO molecules are more stable against heating. The mobility of NO molecules in such clusters is higher than that of CO molecules. The total dipole moment, the static dielectric permeability, the number of active electrons in the clusters, and the specific number of hydrogen bonds between water molecules possess peak values when the number of doping molecules i = 6. IR absorption spectra mostly acquire a smooth shape at i > 6. Capture of CO and NO molecules by water cluster operates as anti-greenhouse effect.