A systematic analysis of biosynthetic gene clusters in the human microbiome reveals a common family of antibiotics.

In complex biological systems, small molecules often mediate microbe-microbe and microbe-host interactions. Using a systematic approach, we identified 3,118 small-molecule biosynthetic gene clusters (BGCs) in genomes of human-associated bacteria and studied their representation in 752 metagenomic samples from the NIH Human Microbiome Project. Remarkably, we discovered that BGCs for a class of antibiotics in clinical trials, thiopeptides, are widely distributed in genomes and metagenomes of the human microbiota. We purified and solved the structure of a thiopeptide antibiotic, lactocillin, from a prominent member of the vaginal microbiota. We demonstrate that lactocillin has potent antibacterial activity against a range of Gram-positive vaginal pathogens, and we show that lactocillin and other thiopeptide BGCs are expressed in vivo by analyzing human metatranscriptomic sequencing data. Our findings illustrate the widespread distribution of small-molecule-encoding BGCs in the human microbiome, and they demonstrate the bacterial production of drug-like molecules in humans. PAPERCLIP:

Metabolomic profiling of Dezhou donkey seminal plasma related to freezability.

Donkeys are indispensable livestock in China because they have transport function and medicinal value. With the popularization of artificial insemination on donkeys, semen cryopreservation technology has gradually become a research hotspot. Seminal plasma is a necessary medium for transporting sperm and provides energy and nutrition for sperm. Seminal plasma metabolites play an important role in the process of sperm freezing, and also have an important impact on sperm motility and fertilization rate after freezing and thawing. In this study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to compare the metabolic characteristics of seminal plasma of high freezability (HF) and low freezability (LF) male donkeys. We identified 672 metabolites from donkey seminal plasma, of which 33 metabolites were significantly different between the two groups. Metabolites were identified and categorized according to their major chemical classes, including homogeneous non-metal compounds, nucleosides, nucleotides, and analogues, organosulphur compounds, phenylpropanoids and polyketide, organoheterocyclic compounds, organic oxygen compounds, benzenoids, organic acids and derivatives, lipids and lipid-like molecules, organooxygen compounds, alkaloids and derivatives, organic nitrogen compounds. The results showed that the contents of phosphatidylcholine, piceatannol and enkephalin in donkey semen of HF group were significantly higher than those of LF group (p < .05), while the contents of taurocholic and lysophosphatidic acid were significantly lower than those of LF group (p < .05). The different metabolites were mainly related to sperm biological pathway response and oxidative stress. These metabolites may be considered as candidate biomarkers for different fertility in jacks.

Mother-to-infant transmission of the carcinogenic colibactin-producing bacteria.

BACKGROUND: The Escherichia coli strain that is known to produce the genotoxic secondary metabolite colibactin is linked to colorectal oncogenesis. Therefore, understanding the properties of such colibactin-positive E. coli and the molecular mechanism of oncogenesis by colibactin may provide us with opportunities for early diagnosis or prevention of colorectal oncogenesis. While there have been major advances in the characterization of colibactin-positive E. coli and the toxin it produces, the infection route of the clb + strain remains poorly characterized. RESULTS: We examined infants and their treatments during and post-birth periods to examine potential transmission of colibactin-positive E. coli to infants. Here, analysis of fecal samples of infants over the first month of birth for the presence of a colibactin biosynthetic gene revealed that the bacterium may be transmitted from mother to infant through intimate contacts, such as natural childbirth and breastfeeding, but not through food intake. CONCLUSIONS: Our finding suggests that transmission of colibactin-positive E. coli appears to be occurring at the very early stage of life of the newborn and hints at the possibility of developing early preventive measures against colorectal cancer.

LC-MS/MS Method Development for the Discovery and Identification of Amphidinols Produced by Amphidinium.

Amphidinols are polyketides produced by dinoflagellates suspected of causing fish kills. Here, we demonstrate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification and quantification of amphidinols (AM). Novel AM were detected by neutral loss (NL) scan and then quantified together with known AM by selection reaction monitoring (SRM). With the new method, AM were detected in four of eight analyzed strains with a maximum of 3680 fg toxin content per cell. In total, sixteen novel AM were detected by NL scan and characterized via their fragmentation patterns. Of these, two substances are glycosylated forms. This is the first detection of glycosylated AM.

Structural analysis of small to medium-sized molecules by mass spectrometry after electron-ion fragmentation (ExD) reactions.

Electron capture dissociation (ECD) is a tandem mass spectrometry (MS/MS) method that utilizes the interaction of ions and electrons. Its unique ability to preserve labile bonds distinguishes it from conventional threshold-based MS/MS methods, the most important of which is collision-induced dissociation (CID). During the last decade, ECD has opened up several new venues in protein analyses, for example top-down sequencing, identification of post-translational modifications, and characterization of protein-protein interactions. In recent years, a number of related dissociation techniques, so-called ExD techniques, particularly electron transfer dissociation (ETD), electron detachment dissociation (EDD), electron induced dissociation (EID), and negative electron transfer dissociation (NETD), have emerged and have extended the application range of ion-electron dissociations further. Importantly, ExD techniques have been applied beyond protein analyses, which is the focus of the current paper. This short introduction describes the application of ExD to small and medium-sized molecules and reviews important applications to natural products, biomedical compounds, synthetic molecules, crude oils, and environmental toxins.

Exploration of industrially important pigments from soil fungi.

The worldwide interest of the current era is to increase tendency towards the use of natural substances instead of synthetic ones. So, alternative and effective environment friendly sustainable technologies are highly needed. Due to a broad range of biological activities, fungi are considered as a significant source of pigments. Among the fungal species in the soil, the genera of Aspergillus, Fusarium, Penicillium, Paecilomyces, and Trichoderma are dominant. The pigments commonly produced by fungi belong to aromatic polyketide groups such as melanins, quinones, flavins, ankaflavin, anthraquinone, and naphthoquinone. The use of fungal pigments has benefits which comprise easy and fast growth in the cheap culture medium and different color shades being independent of weather conditions and would be useful in various industrial applications. In relation to the toxic effects of the synthetic dyes, the natural dyes are easily degradable since they cause no detrimental effects. Thus, the study of pigments produced by soil fungi has tremendous use in medical, textile coloring, food coloring, and cosmetics.

Polyketides from mantis-associated fungus Daldinia eschscholzii IFB-TL01.

Three new polyketides, named daldinone F (1), nodulisporin G (2), and dalmanol C (3), together with five known compounds, 4-8, were isolated from cultures of Daldinia eschscholzii. The structures of the new compounds were elucidated by extensive NMR and MS analyses. Compound 1 showed moderate cytotoxic activity against SW480 cancer cells with an IC50 value of 9.59 µM, and its absolute configuration was determined by single crystal X-ray diffraction.

Isolation of polyketides from Prymnesium parvum (Haptophyta) and their detection by liquid chromatography/mass spectrometry metabolic fingerprint analysis.

Prymnesium parvum is a microalga that forms blooms coupled with the presence of potent exotoxins; however, no chemical standards are currently available for the toxins. Streamlined methods are presented for the separation and enrichment of polyketide toxins, prymnesin-1 (prym1) and prymnesin-2 (prym2). Prymnesins were separated by reversed-phase chromatography and detected by positive-mode electrospray ionization MS to generate a unique metabolic fingerprint. More than 10 ions were detected and mass assignments were in agreement with predicted isotopic distributions for the intact compounds and related fragments; ions occurred as multiply protonated species and with common salt adducts. The most prevalent ion was observed at 919.88 m/z, which represents the aglycone [prymagly+2H](2+) backbone structure common to both molecules. Expanded mass spectra for this and related ions were in excellent agreement (<0.5ppm) with empirically derived spectra based on elemental composition and naturally occurring isotopes. These investigations have confirmed the isolation of polyketide prymnesins from whole cells, which heretofore has not been reproduced since their original characterization. Moreover, this study represents the first time these compounds have been verified in aqueous materials. These tools should allow the direct identification and analysis of polyketide prymnesins, which will greatly improve our understanding of these toxins in P. parvum.

Environmental control of the calicheamicin polyketide synthase leads to detection of a programmed octaketide and a proposal for enediyne biosynthesis.

A light in the dark: the fermentation products of the polyketide synthase CalE8 (without its cognate thioesterase) were identified and gave some insight into the elusive early steps of calicheamicin biosynthesis. Fermentation in either the light or dark resulted in different proportions of a new octaketide and led to an updated mechanistic proposal.

Gut microbiota of hepatitis B virus-infected patients in the immune-tolerant and immune-active phases and their implications in metabolite changes.

BACKGROUND: The microbes and metabolomics of microbiota dysbiosis in the gut in the different phases of hepatitis B virus (HBV) infection are not fully understood. AIM: To investigate the specific gut microbiota and metabolites of the immune-tolerant (IT) and immune-active (IA) phases of chronic hepatitis B (CHB). METHODS: Clinical fecal samples from healthy individuals and patients in the IT and IA phases of HBV infection were collected. Next, non-target metabolomics, bioinformatics, and 16S rDNA sequencing analyses were performed. RESULTS: A total of 293 different metabolites in 14 phyla, 22 classes, 29 orders, 51 families, and 190 genera were identified. The four phyla of Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria were the most abundant, accounting for 99.72%, 99.79%, and 99.55% in the healthy controls, IT-phase patients, and IA-phase patients, respectively. We further identified 16 genera with different richness in the IT phase and IA phase of HBV infection. Of the 134 named metabolites, 57 were upregulated and 77 were downregulated. A total of 101 different metabolic functions were predicted in this study, with 6 metabolic pathways having the highest enrichments, namely carbohydrate metabolism (14.85%), amino acid metabolism (12.87%), lipid metabolism (11.88%), metabolism of cofactors and vitamins (11.88%), xenobiotic biodegradation (9.9%), and metabolism of terpenoids and polyketides (7.92%). CONCLUSION: These findings provide observational evidence of compositional alterations of the gut microbiome and some related metabolites in patients with IT-phase or IA-phase HBV infection. Further studies should investigate whether microbiota modulation can facilitate the progression of CHB and the cause-effect relationship between the gut microbiota and CHB.

A Capture Strategy for the Identification of Thio-Templated Metabolites.

Nonribosomal peptide synthetase and polyketide synthase systems are home to complex enzymology and produce compounds of great therapeutic value. Despite this, they have continued to be difficult to characterize due to their substrates remaining enzyme-bound by a thioester bond. Here, we have developed a strategy to directly trap and characterize the thioester-bound enzyme intermediates and applied the strategy to the azinomycin biosynthetic pathway. The approach was initially applied in vitro to evaluate its efficacy and subsequently moved to an in situ system, where a protein of interest was isolated from the native organism to avoid needing to supply substrates. When the nonribosomal peptide synthetase AziA3 was isolated from Streptomyces sahachiroi, the capture strategy revealed AziA3 functions in the late stages of epoxide moiety formation of the azinomycins. The strategy was further validated in vitro with a nonribosomal peptide synthetase involved in colibactin biosynthesis. In the long term, this method will be utilized to characterize thioester-bound metabolites within not only the azinomycin biosynthetic pathway but also other cryptic metabolite pathways.

Secondary metabolites of the genus Penicillium from undisturbed and anthropogenically altered Antarctic habitats.

From undisturbed Antarctic habitats (permafrost sediments 30-150 thousand years of age, water of Radok Lake) and superficial deposits contaminated with petroleum products, we isolated 14 and 9 strains of Penicillium fungi, respectively. Comparison of the fungal complexes showed them to differ by species composition; only two species-P. palitans and P. solitum-were in the species lists of both groups. The identified secondary metabolites in the investigated strains belonged to diketopiperazine (group of roquefortines, rugulosuvin B), benzodiazepine (anacin, cyclopenins), quinoline alkaloids (viridicatins), clavine ergot alkaloids (α-cyclopiazonic acid, festuclavine, fumigaclavines), polycyclic indole alkaloids (communesin B, chaetoglobosin A), amino acid derivatives (N-acetyltryptamine, chrysogins, penicillin G), polyketides (citreoviridin A, mycophenolic acid), and terpenes (andrastins, phomenone). Strains isolated from anthropogenically altered habitats produced a more complete and characteristic profile of exometabolites, as compared with strains isolated from undisturbed habitats. It is only from contaminated soils there were isolated fungi that produced more structurally diverse secondary metabolites pertaining to polycyclic indole alkaloids and terpenoids. The fungi isolated from contaminated samples can be used in biodegradation of oil spills and bioremediation of the environment, and also as producers of promising biologically active compounds.

Application of Optimized and Validated Agar Overlay TLC-Bioautography Assay for Detecting the Antimicrobial Metabolites of Pharmaceutical Interest.

The agar overlay TLC-bioautography is one of the crucial methods for simultaneous in situ detection and separation of antimicrobial metabolites of pharmaceutical interest. The main focus of this research relies on the dereplication of an antimicrobial metabolite coriloxin derived from mycoendophytic Xylaria sp. NBRTSB-20 with a validation of agar overlay TLC-bioautography technique. This polyketide metabolite coriloxin was purified by column chromatography, and its purity was assessed by HPLC, UPLC-ESI-QTOF-MS, FT-IR and NMR spectral analysis. The antimicrobial capability of ethyl acetate extract and the purified compound coriloxin was determined by disc diffusion, minimal inhibitory concentration and agar overlay TLC-bioautography assay. The visible LOD of coriloxin antimicrobial activity was found at 10 µg for Escherichia coli and 20 µg for both Staphylococcus aureus and Fusarium oxysporum. Inter- and intra-day precision was determined as the relative standard deviation is less than 6.56%, which proved that this method was precise. The accuracy was expressed as recovery, and the values were found ranging from 91.18 to 108.73% with RSD values 0.94-2.30%, respectively. The overall findings of this investigation suggest that agar overlay TLC-bioautography assay is a suitable and acceptable method for the in situ determination of antimicrobial pharmaceuticals.

On-line screening and identification of free radical scavenging compounds in Angelica dahurica fermented with Eurotium cristatum using an HPLC-PDA-Triple-TOF-MS/MS-ABTS system.

Eurotium cristatum, a beneficial fungus isolated from Fuzhuan tea, was used to ferment Angelica dahurica for the first time. The antioxidant capacities of the extracts before and after fermentation were compared using ABTS, DPPH and FRAP assays. The results showed that the antioxidant capacities of the extracts acquired using organic solvents were greater after fermentation. Moreover, based on a comparison of the HPLC chromatograms, the chemical composition of Angelica dahurica changed substantially during fermentation. To further understand the changes in its antioxidant constituents, an on-line HPLC-PDA-Triple-TOF-MS/MS-ABTS system was employed. Twelve antioxidants belonging to three different classes were detected and identified, and their antioxidant capacities were preliminarily evaluated. The results indicated that the substances produced during the fermentation of Eurotium cristatum played important roles in enhancing the antioxidant capacity.

Vitis vinifera 'Pinot noir' leaves as a source of bioactive nutraceutical compounds.

Agricultural by-products are often hidden sources of healthy plant ingredients. The investigation of the nutritional values of these by-products is essential towards sustainable agriculture and improved food systems. In the vine industry, grape leaves are a bulky side product which is strategically removed and treated as waste in the process of wine production. In this work we performed an untargeted metabolomic profiling of the methanol extract of the leaves of Vitis vinifera cultivar 'Pinot noir', analysed their fatty acid content, and estimated their antioxidative capacity, with the purpose of investigating its nutritional and medicinal value. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) analysis identified the presence of numerous compounds which are known to possess diverse nutritional and pharmacological properties, particularly polyphenols and phenolic compounds (e.g. caffeic acid, catechin, kaempferol and quercetin), several phytosterols and fatty acids. Fatty acids were the most represented lipids' secondary class, with the essential alpha-linolenic acid being the most abundant in 'Pinot noir' leaves, with a relative content of 42%. Also, we have found that 'Pinot noir' leaves present a high antioxidant capacity, putting grapevine leaves at the top of the list of foods with the highest antioxidative activity. Our findings scientifically confirmed that 'Pinot noir' leaves have a high content and diversity of biologically active phytochemical compounds which make it of exceptional interest for pharmaceutical and food industries.

Mass Spectrometry-Based Integration and Expansion of the Chemical Diversity Harbored Within a Marine Sponge.

Marine sponges and their associated symbionts produce a structurally diverse and complex set of natural products including alkaloids, terpenoids, peptides, lipids, and steroids. A single sponge with its symbionts can produce all of the above-mentioned classes of molecules and their analogs. Most approaches to evaluating sponge chemical diversity have focused on major metabolites that can be isolated and characterized; therefore, a comprehensive evaluation of intra- (within a molecular family; analogs) and inter-chemical diversity within a single sponge remains incomplete. We use a combination of metabolomics tools, including a supervised approach via manual library search and literature search, and an unsupervised approach via molecular networking and MS2LDA analysis to describe the intra and inter-chemical diversity present in Smenospongia aurea. Furthermore, we use imaging mass spectrometry to link this chemical diversity to either the sponge or the associated cyanobacteria. Using these approaches, we identify seven more molecular features that represent analogs of four previously known peptide/polyketide smenamides and assign the biosynthesis of these molecules to the symbiotic cyanobacteria by imaging mass spectrometry. We extend this analysis to a wide diversity of molecular classes including indole alkaloids and meroterpenes.

Unprecedented antioxidative and anti-inflammatory aryl polyketides from the brown seaweed Sargassum wightii.

Previously undescribed aryl polyketide lactones, 4-(8-ethyl-tetrahydro-7-oxo-2H-pyran-5-yl)-propyl-4'-methylbenzoate (compound 1) and methyl-2-(12-oxo-7-phenyl-8-vinyl-1-oxa-4,9-cyclododecadien-3-yl)-acetate (compound 2) were purified from ethyl acetate-methanol fraction of the brown seaweed Sargassum wightii. The structures were proposed based on their NMR and mass spectrometric data. The antioxidative activities of the lactones were significantly greater (P<0.05) (IC50 1,1-diphenyl-2-picrylhydrazyl radical scavenging 0.24-0.32mg/mL) than α-tocopherol (IC50 0.63mg/mL). The title compounds displayed considerably greater 5-lipoxygenase inhibitory activity (IC50 0.56 and 0.29mg/mL, respectively) in conjunction with higher selectivity indices (anti-cycloxygense-1IC50/anti-cycloxygense-2IC50 >1) compared to non-steroidal anti-inflammatory drugs (SIaspirin 0.03, SIibuprofen 0.43). Putative biosynthetic pathway of title polyketide products through polyketide synthase enzyme cascade catalyzed reactions substantiated the structural attributions of the hitherto unreported aryl polyketides. This is the first report of the occurrence and characterization of two rare skeletal types, oxo-2H-pyranyl and oxa-cyclododecadienyl macrolactone featuring the aryl substituent from marine organisms with potential antioxidative and anti-inflammatory activities.

Volatile Compounds from the Different Organs of Houttuynia cordata and Litsea cubeba (L. citriodora).

The volatile compounds obtained from the different organs of Houttuynia cordata (Saururaceae) and Litsea cubeba (Lauraceae) were analyzed by Gas Chromatography/Mass Spectrometry (GC/MS), Headspace Solid Phase Micro Extraction-Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS), and GC/olfactometry (GC/O). The major component of all parts of H. cordata is assigned as 4-tridecanone. Each organ produces myrcene as the major monoterpenoid. The major monoterpene in the rhizomes and roots was ß-pinene instead of myrcene. 1-Decanal which was responsible for the unpleasant odor of this plant, was the predominant polyketide in both leaves and stems. The presence of 1-decanal was very poor in flowers, stem collected in summer, rhizomes, and roots. GC/MS analyses were very simple in case of the crude extracts of flowers. The content of sesquiterpenoids was extremely poor. (8Z)-Heptadecene, geranial, and neral were detected as the major components in Litsea cubeba. Odor-contributing components by GC/O analysis of the ether extract of the fresh flowers of L. cubeba were neral and geranial which played an important role in sweet-lemon fragrance of the flowers. The role of a high content of (8Z)-heptadecene was still unknown but it might play a significant role in the dispersion of the volatile monoterpene hydrocarbons and aldehydes. The flower volatiles of the Japanese L. cubeba were chemically quite different from those of the Chinese same species.

Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly.

Malonyl carba(dethia) N-decanoyl cysteamine methyl esters and novel acetoxymethyl esters were utilised as second-generation probes for polyketide intermediate capture. The use of these tools in vivo led to the characterisation of an almost complete set of biosynthetic intermediates from a modular assembly line, providing a first kinetic overview of intermediate processing leading to complex natural product formation.

A new polyketide, penicillolide from the marine-derived fungus Penicillium sacculum.

A new polyketide, penicillolide (1) was isolated from the fermentation broth of the marine-derived fungus Penicillium sacculum GT-308. Compound 1 is a polyketide with a unique carbon skeleton. The structure of this compound was established via extensive spectroscopic analyses including 1D-, 2D-NMR, and HRESI-MS.