Discovery of Clostrubin,an Exceptional Polyphenolic Polyketide Antibiotic from a Strictly Anaerobic Bacterium

Genome mining of the strictly anaerobic bacterium Clostridium beijerinckii, an industrial producer of solvents, revealed the presence of several cryptic gene clusters for secondary metabolite biosynthesis. To unearth its metabolic potential, a C. beijerinckii strain was cultured under various conditions, which led to the discovery of a deep purple pigment. This novel metabolite, named clostrubin ( 1 ), was isolated and its structure was fully elucidated. The pentacyclic polyphenol features a benzo[a]tetraphene ring topology that is unprecedented for natural products. Stable‐isotope labeling experiments showed that 1 is an aromatic polyketide that folds in a noncanonical manner to form the unusual perifused ring system. In addition to being the first reported polyketide from an anaerobic bacterium, 1 is a potent antibiotic with pronounced activity against various pathogenic bacteria, such as MRSA, VRE, and mycobacteria, with minimum inhibitory concentrations (MIC) of 0.12–0.97 μM .     

Hyperthermostable,Ca<Superscript>2+</Superscript>-Independent,and High Maltose-Forming α-Amylase Production by an Extreme Thermophile <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis>: Whole Cell Immobilization

The synthesis of extracellular α-amylase in Geobacillus thermoleovorans was constitutive. The enzyme was secreted in metabolizable carbon sources as well as non-metabolizable synthetic analogues of glucose, but the titers were higher in the former than that in the latter. G. thermoleovorans is a fast-growing facultatively anaerobic bacterium that grows under both aerobic and anaerobic conditions and produces an extracellular amylolytic enzyme α-amylase with the by-product of lactic acid. G. thermoleovorans is a rich source of various novel thermostable biocatalysts for different industrial applications. α-Amylase synthesis was subject to catabolite repression in the presence of high concentrations of glucose. The addition of cAMP to the medium containing glucose did not result in the repression of α-amylase synthesis. The addition of maltose (1%) to the starch arginine medium resulted in a twofold enhancement in enzyme titers. Polyurethane foam (PUF)-immobilized cells secreted α-amylase, which was higher than that with the free cells. PUF appeared to be a better matrix for immobilization of the thermophilic bacterium than the other commonly used matrices. The repeated use of PUF-immobilized cells was possible over 15 cycles with a sustained α-amylase secretion. The use of this enzyme in starch saccharification eliminates the addition of Ca²⁺ in starch liquefaction and its subsequent removal by ion exchangers from the product streams.     

Characterization of the cellulose-degrading bacterium NCIMB 10462

The gram-negative cellulase-producing bacterium NCIMB 10462 has been previously namedPseudomonas fluorescens subsp. or var.cellulosa. Because of renewed interest in cellulose-degrading bacteria for use in the bioconversion of cellulose to chemical feed stocks and fuels, we re-examined the characteristics of this microorganism to determine its true metabolic potential. Metabolic and physical characterization of NCIMB 10462 revealed that this is an alkalophilic, nonfermentative, gram-negative, oxidase-positive, motile, cellulose-degrading bacterium. The aerobic substrate utilization profile of this bacterium has few characteristics consistent with a classification ofP. fluorescens and a very low probability match with the genusSphingomonas. However, total lipid analysis did not reveal that any sphingo-lipid bases are produced by this bacterium. NCIMB 10462 grows best aerobically, but also grows well in complex media under reducing conditions. NCIMB 10462 grows slowly under anaerobic conditions on complex media, but growth on cellulosic media occurred only under aerobic conditions. Total fatty acid analysis (MIDI) of NCIMB 10462 failed to group this bacterium with a known pseudomonas species. However, fatty acid analysis of the bacteria when grown at temperatures below 37°C suggest that the organism is a pseudomonad. Since a predominant characteristic of this bacterium is its ability to degrade cellulose, we suggest that it be calledPseudomonas cellulosa.

     

Characterization of rat liver microsomal and hepatocytal metabolites of brevetoxins by liquid chromatography–electrospray tandem mass spectrometry

Brevetoxins are natural neurotoxins that are produced by “red tide” algae. This class of compounds can cause neurotoxic shellfish poisoning and other health problems. Brevetoxin-2 is the most abundant among the nine brevetoxins that have been characterized, whereas brevetoxin-1 is the most toxic. In this study, brevetoxin-1 and brevetoxin-2 were incubated with rat liver hepatocytes and rat liver microsomes, respectively. After clean-up steps were taken to remove the proteins, samples were analyzed by liquid chromatography (LC) coupled with electrospray mass spectrometry (LC-MS). After incubation of brevetoxin-1, two metabolites were found: brevetoxin-1-M1 (molecular weight = 900 Da), and brevetoxin-1-M2 (molecular weight = 884 Da). The increase in molecular weight combined with evidence from tandem mass spectrometry showing an increased tendency for loss of water molecules, along with considerations of established precedents for chemical transformations led to the conclusion that brevetoxin-1-M1 was formed by converting one double bond in the E or F ring of brevetoxin-1 into a diol. The second metabolite (brevetoxin-1-M2) is proposed to be a hydrolysis product of brevetoxin-1 involving opening of the lactone ring with the addition of a water molecule. The incubation study of the other starting compound, brevetoxin-2, found two metabolites in the LC-ES-MS selected ion chromatogram. Brevetoxin-2-M1 (molecular weight = 912 Da) gave a large [M−H]⁻ peak at m/z 911, and its product ion mass spectrum allowed the deduction that this metabolite was the hydrolysis product of brevetoxin-2 involving conversion of the lactone to a carboxylic acid and an alcohol. The second metabolite (brevetoxin-2-M2, molecular weight = 896 Da) was deduced to have the same structure as that of brevetoxin-3 based on identical chromatographic retention times and similar mass spectra as those obtained for a brevetoxin-3 standard.     

Anaerobic Origin of Ergothioneine

Ergothioneine is a sulfur metabolite that occurs in microorganisms, fungi, plants, and animals. The physiological function of ergothioneine is not clear. In recent years broad scientific consensus has formed around the idea that cellular ergothioneine primarily protects against reactive oxygen species. Herein we provide evidence that this focus on oxygen chemistry may be too narrow. We describe two enzymes from the strictly anaerobic green sulfur bacterium Chlorobium limicola that mediate oxygen‐independent biosynthesis of ergothioneine. This anoxic origin suggests that ergothioneine is also important for oxygen‐independent life. Furthermore, one of the discovered ergothioneine biosynthetic enzymes provides the first example of a rhodanese‐like enzyme that transfers sulfur to non‐activated carbon.     

Capillary zone electrophoresis with field enhanced sample stacking as a tool for targeted metabolome analysis of adenine nucleotides and coenzymes in Paracoccus denitrificans

The main aim of this work was to demonstrate the applicability of capillary zone electrophoresis in combination with field enhanced sample stacking in targeted metabolome analyses of adenine nucleotides – AMP, ADP, ATP, coenzymes NAD⁺, NADP⁺ and their reduced forms in Paracoccus denitrificans. Sodium carbonate/hydrogencarbonate buffer (100 mM, pH 9.6) with the addition of β‐CD at a concentration of 10 mM was found to be an effective BGE for their separation within 20 min. Besides this, special attention was paid to the development of the procedure for the extraction of specific metabolites from the bacterium P. denitrificans. This procedure was not only optimised to achieve the highest metabolite yields but also to obtain a sample that was fully compatible with the online preconcetration strategy used. The developed methodology was finally applied in a study of the bacterium P. denitrificans at various stages of the active respiratory chain.     

In-Vitro Stability,Metabolism, and Transport of Dental Monomers Made from Bisphenol A and Bisphenol F

The stability and bioavailability of the biomaterial monomers, bisglycidyl methacrylate (BISGMA), bisphenol F diglycidyl ether (BFDGE), and bisphenol A dimethacrylate (BPADM) were investigated using in-vitro techniques. A reverse-phase high-pressure liquid chromatographic/mass spectrometric (HPLC/MS) system was developed to quantitate each monomer and its primary metabolite. Each monomer (10 × 10⁻⁶ M ) was incubated at 37 °C under various conditions. Aliquots (N = 3) were removed at various time intervals and quantitated from a standard curve. The in-vitro transport of each parent monomer and its tetrahydroxy metabolite was measured in a Caco-2 system. BISGMA and BPADM were stable in aqueous solution at pH 1. However, BFDGE, was unstable. Plasma esterase of the rat rapidly hydrolyzed the ester compounds, but human esterase did not have a hydrolytic effect on BISGMA or BPADM. BFDGE disappeared in both rat and human plasma, but no tetrahydroxy metabolite was observed. All three parent compounds were unstable in human- and rat-hepatic fractions producing either tetrahydroxy metabolites or bisphenol A (BPA). The tetrahydroxy metabolites, however, were relatively stable under identical conditions, but BPA disappeared when incubated in hepatic-microsomal fractions. While BPADM metabolism produced BPA, an estrogen disrupter, BISGMA and BFDGE did not appear to produce BPA. These results suggest that the potential toxicity of leached dental monomers is more likely to be a result of the metabolite rather than the parent monomer. From Caco-2 transport studies, BFDGE and its tetrahydroxy metabolite both crossed the Caco-2 membrane at a low rate of transport in 2 h (approximately 3 and 5.2%, respectively). The BISGMA metabolite crossed at approximately 8%, indicative of a moderate rate of transport, and BPA crossed at approximately 10% in 1 h (high rate of transport). The transport of BPADM and BISGMA was unable to be determined due to nonspecific absorption to the acrylic vertical transport well. The transport of BFDGE tetrahydroxy metabolite is of particular interest as BFDGE is likely to be chemically hydrolyzed in stomach acid. It is well known that epoxies react with acids resulting in ring opening, so it is not surprising that BFDGE decomposes at pH 1. As a result, it is necessary to identify the decomposition (hydrolysis) products and test their bioavailability.

Mean (SD) stability of BISGMA (10 × 10⁻⁶ M ) and bisphenol A tetrahydroxy metabolite in human- and rat-S9-hepatic fractions at 37 °C for 1 h.     

Synthesis of Bioactive Side‐Chain Analogues of TAN‐2483B

The fungal metabolite TAN‐2483B has a 2,6‐trans‐relationship across the pyran ring of its furo[3,4‐b]pyran‐5‐one core, which has thwarted previous attempts at its synthesis. We have now developed a chiral pool approach to this core and prepared side‐chain analogues of TAN‐2483B. The synthesis relies on ring expansion of a reactive furan ring‐fused dibromocyclopropane and alkynylation of the resulting pyran. The furan ring is constructed by palladium‐catalysed carbonylative lactonisation. Various side‐chains are appended through Wittig‐type chemistry. The prepared analogues showed micromolar activity towards cancer cell lines HL‐60, 1A9 and MCF‐7 and certain human disease‐relevant kinases, including Bruton's tyrosine kinase (Btk).     

Conformation and Relative Configuration of a Very Potent Glycosylphosphatidylinositol-Anchoring Inhibitor with an Unusual Tricarbocyclic Sesterterpenoid δ-Lactone Skeleton from the Fungus Paecilomyces inflatus

In the course of our screening for glycosylphosphatidylinositol (GPI) inhibitors, we found a fungal strain, Paecilomyces inflatus, which inhibited GPI anchoring in yeast. Bioassay-guided fractionation with gel filtration, MPLC on normal phase and prep. HPLC on reversed-phase yielded a minor secondary metabolite, the substituted hexadecahydroindeno[5′,6′:4,5]cycloocta[1,2-c]pyranyl heptanoate 1 , that inhibited GPI synthesis in vitro by yeast microsomes with a MIC of 3.4 nm. Ester 1 specifically inhibited GPI synthesis in eukaryotic, including mammalian cells, but had no significant activity in protozoa. Based on spectroscopic evidence, including UV, FT-IR, FAB-MS, ESI-HR-MS, ¹H-NMR, ¹³C-NMR, DQ-COSY, ROESY, HSQC, and HMBC data, the metabolite 1 was shown to have an unusual tricarbocyclic sesterterpenoid δ-lactone skeleton. Its solution conformation and relative configuration was elucidated with ¹H,¹H coupling constants and detailed analysis of its ROESY data, coupled with inspection of Dreiding models. In the preliminary investigations on structure-activity relationships, the three derivatives 2 – 4 of ester 1 were prepared by acetylation, catalytic hydrogenation, and intramolecular alkene addition of a hydroxy group, respectively. The test results revealed that the modifications of substituents at the δ-lactone ring or of the C=C bonds of 1 caused a loss of activity by a factor of ca. 500 to 5000. Therefore, it seems that the δ-lactone ring and the C=C bonds in 1 are essential for the potent GPI-inhibitory activity.     

Conversion of arsenobetaine to dimethylarsinic acid by arsenobetaine-decomposing bacteria isolated from coastal sediment

Arsenobetain [(CH₃)₃As⁺CH₂COO^-]-containing growth media (1/5 ZoBell 2216E and solution of inorganic salts) were inoculated with two bacterial strains, which were isolated from a coastal sediment and identified as members of the Vibro-Aeromonas group, and incubated under aerobic and anaerobic conditions. Arsenobetaine was converted to a metabolite only under aerobic conditions. This arsenic metabolite was identified as dimethylarsinic acid [(CH₃)₂AsOOH] by hydride generation/cold trap/GC MS/SIM analysis and high-performance liquid-chromatographic behaviour. The conversion pattern shown by these arsenobetaine-decomposing bacteria (that is, arsenobetaine → dimethylarsinic acid) was fairly different from that shown by the addition of sediment itself as the source of arsenobetaine-decomposing micro-organisms (that is, arsenobetaine → trimethylarsine oxide → inorganic arsenic). This result suggests to us that various micro-organisms, including the arsenobetaine-decomposing bacteria isolated in this study, participate in the degradation of arsenobetaine in marine environments.     

Stereochemistry of the Conversion of 2‐Phenoxyethanol into Phenol and Acetaldehyde by Acetobacterium sp.

The conversion of 2‐phenoxyethanol to phenol and acetate by the anaerobic bacterium Acetobacterium sp. strain LuPhet1 proceeds through acetaldehyde with concomitant migration of a H‐atom from C(1) to C(2) of the glycolic moiety. Separate feeding experiments with (R)‐ and (S)‐2‐phenoxy(1‐²H)ethanol, prepared via chemoenzymatic syntheses, indicate that the H‐atom involved in the 1,2‐shift is the pro‐S one of the enantiotopic couple of the alcohol function.     

Computational studies of the 3-Dimensional structure of cyclopenta polycyclic aromatic hydrocarbons containing a gulf region

Recently, some cyclopenta-fused polyaromatic hydrocarbons, an environmentally relevant subclass of chemicals, have been shown to have carcinogenic activity in animals. It has been suggested that benz[l] aceanthrylene ( I ), an active member of this subclass with a gulf region, has a trans dihydrodiol metabolite that is nonplanar and has two distinct spatial configurations. We have used MMP 2(85) and AM 1 to investigate the three-dimensional structure of this dihydrodiol and other similar derivatives of ( I ) and have found that although ( I ) is somewhat nonplanar the relevant derivatives are all nearly planar. Further, we have computed potential functions for the bending of the angular ring in the gulf region using MMP 2(85), AM 1, and ab initio computed energies for AM 1 spatial configurations and find that these molecules all have only a single potential minimum. We have performed the same calculations for benzo[c]phenanthren and its 1,12 dimethyl derivative, molecules with a similar gulf region for which crystallographic data exists. In agreement with that data, we find that two distinct spatial configurations exist separated by significant barries. The differences between the results generated by the three different methods of computation will be discussed.     

Evidence for a novel mechanism of microbial cellulose degradation

There are two well studied mechanisms that are used by cellulolytic microorganisms to degrade the cellulose present in plant cell walls and a third less well studied oxidative mechanism used by brown rot fungi. The well studied mechanisms use cellulases to hydrolyze the β-1,4 linkages present in cellulose, however the way in which cellulases are presented to the environment are quite different for each mechanism. Most aerobic microorganisms secrete a set of cellulases outside the cell (free cellulase mechanism) while most anaerobic microorganisms produce large multi enzyme complexes on their outer surface (cellulosomal mechanism). Their genomic sequences suggest that the aerobic bacterium, Cytophaga hutchinsonii and the anaerobic bacterium, Fibrobacter succinogenes, do not use either of these mechanisms for degrading cellulose, as these organisms only code for normal endocellulases not for processive cellulases like exocellulases and processive endocellulases which are used in both of the well studied mechanisms.     

Taxane Diterpenoids from Taiwanese Yew Taxus sumatrana

Thirty seven taxanes were characterized from the leaves and twigs of Taiwanese yew (Taxus sumatrana, Taxaceae). Four of these metabolites are new and designated as sumataxins A–D ( 1 – 4 ). Compound 1 possesses an 11(15→1),11(10→9)‐di‐abeo‐taxane skeleton with an unusual spiro‐connected 2,2‐dimethyl‐1,3‐dioxolane ring at C(4), whereas compound 2 has a rare β‐OH orientation at C(13) of taxane diterpene ester. In addition, sumataxin C ( 3 ) is formulated as an 11(15→1)‐abeo‐taxane with a 4,5‐acetonide ring skeleton. Compound 4 is the first metabolite with a 4,20‐epoxy‐taxane structure. The structures of all the taxanes were established by spectroscopic methods. All compounds were evaluated for anti‐HSV‐1 and PBMC activities. Compound 9 exhibited significant enhancement of cell proliferation on peripheral blood mononuclear cells.     

A new antibiotic produced by the cyanobacterium-symbiotic fungus Simplicillium lanosoniveum

The culture broth of the cyanobacterium-symbiotic fungus Simplicillium lanosoniveum var. Tianjinienss Q. L. Dong exhibited unanticipated antibacterial activities against the Gram-positive bacteria, particularly the pathogenic bacterium Staphylococcus aureus, indicating the secretion of antibiotic-like metabolite, for which the modified Sabouraud medium was the suitable medium. The antibiotic-like metabolite was separated with macroporous resins CT-12 (absorption) and 95% ethanol (desorption), purified by ion-exchange resins D301T and displayed a characteristic absorption peak at 228 nm, suggesting the presence of nitrogen. The negative biuret and ninhydrin tests confirmed the absence of –NH₂ and –COOH groups. Further, HPLC and mass spectrometry analyses showed that the retention time and molecular weight of the antibiotic-like metabolite were 4.1031 min and 163.0182 (Δ ± 2.3 ppm), respectively. Taking together, we speculated that the antibiotic-like metabolite was a new antibiotic structurally similar to alkaloid, which was the first one isolated from the species of Simplicillium genus.     

Biosynthesis of the proteasome inhibitor syringolin A: the ureido group joining two amino acids originates from bicarbonate

Background  

Syringolin A, an important virulence factor in the interaction of the phytopathogenic bacterium Pseudomonas syringaepv. syringaeB728a with its host plant Phaseolus vulgaris(bean), was recently shown to irreversibly inhibit eukaryotic proteasomes by a novel mechanism. Syringolin A is synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase and consists of a tripeptide part including a twelve-membered ring with an N-terminal valine that is joined to a second valine via a very unusual ureido group. Analysis of sequence and architecture of the syringolin A synthetase gene cluster with the five open reading frames sylA-sylEallowed to formulate a biosynthesis model that explained all structural features of the tripeptide part of syringolin A but left the biosynthesis of the unusual ureido group unaccounted for.     

Structure Elucidation and Activity of Kolossin A,the D‐/L‐Pentadecapeptide Product of a Giant Nonribosomal Peptide Synthetase

The largest continuous bacterial nonribosomal peptide synthetase discovered so far is described. It consists of 15 consecutive modules arising from an uninterrupted, fully functional gene in the entomopathogenic bacterium Photorhabdus luminescens. The identification of its cryptic biosynthesis product was achieved by using a combination of genome analysis, promoter exchange, isotopic labeling experiments, and total synthesis of a focused collection of peptide candidates. Although it belongs to the growing class of D ‐/ L ‐peptide natural products, the encoded metabolite kolossin A was found to be largely devoid of antibiotic activity and is likely involved in interspecies communication. A stereoisomer of this peculiar natural product displayed high activity against Trypanosoma brucei rhodesiense, a recalcitrant parasite that causes the deadly disease African sleeping sickness.     

Research on heterocyclic compounds. XX. Synthesis of trifluoromethyl derivatives of fused imidazole systems

A series of heterocyclic compounds having an imidazole ring fused with another ring with a bridgehead nitrogen atom and bearing a trifluoromethyl moiety was synthesized by reaction of 3-bromo-1,1,1-trifluoroacetone with various heteroarylamines. In some cases, an intermediate compound obtained together with the required product was isolated and its structure was elucidated: e.g., starting from 2-aminopyridine we have obtained 2-trifluoromethylimidazo[1,2-a]pyridine and 2-hydroxy-2-trifluoromethyl-2,3-dihydro-1H-imidazo [1,2-a]pyridinium bromide. Such results confirmed the mechanism previously proposed for this reaction.     

A Convergent Synthesis of a Twelve‐Membered Macrolide Natural Product, (6R,12S)‐6‐Hydroxy‐12‐methyl‐1‐oxacyclododecane‐2,5‐dione

A new polyketide metabolite, the twelve‐membered macrolide 1 , isolated from the endophytic fungal strain Cladosporium tenuissimum LR 463 of Maytenus hookeri, whose structure had been determined as (6R,12S)‐6‐hydroxy‐12‐methyl‐1‐oxacyclododecane‐2,5‐dione, was synthesized for the first time by a convergent strategy via Yamaguchi esterification of 2 with 3 and ring‐closing metathesis (RCM) to afford the cyclic ester 1 that was eventually transformed to the target molecule. However, the total synthesis revealed that the assigned structure of the natural product is not correct.     

Stereoselective Total Synthesis of Stagonolide C

A convergent and efficient total synthesis of stagonolide C ( 1 ), a phytotoxic metabolite, was achieved (Schemes 2 and 3) The synthesis exploited the high configuration control in the Prins cyclization along with alkene rearrangement and ring‐closing metathesis as key steps.