Identification of (+)-phyllocladene, (--)-sandaracopimaradiene, and (+)-kaurene as new fungal metabolites from fusicoccin-producing Phomopsis amygdali F6

A chemical analysis of the diterpene hydrocarbons produced by fusicoccin-producing fungus Phomopsis amygdali F6 identified five phyllocladene-related tri- and tetracyclic diterpene hydrocarbons. The presence of (+)-phyllocladene, (--)-sandaracopimaradiene, (+)-isopimara-8,15-diene, and (+)-pimara-8(14),15-diene in the fungus was demonstrated by GC-MS, 1H-NMR, and [alpha]D measurements. (+)-Kaurene was also identified by GC-MS and chiral capillary GC. The possible biosynthetic relationship of these metabolites is discussed.     

Novel Fusicoccins R and S,and the Fusicoccin S Aglycon (Phomopsiol) from Phomopsis amygdali Niigata 2-A,and Their Seed Germination-stimulating Activity in the Presence of Abscisic Acid

Our search for new 3-hydroxyfusicoccins structurally related to cotylenin A from a culture of Phomopsis amygdali Niigata 2-A resulted in the isolation of novel 3-hydroxy fusicoccins, called fusicoccins R and S, and the fusicoccin S aglycon, called phomopsiol, together with known 3α-hydroxyfusicoccin J. The structure of phomopsiol was identified as that of O-demethyl-3-epicotylenol based on spectroscopic evidence. The structures of fusicoccins R and S were also determined to be those of 3′-deacetyl-3α-hydroxyfusicoccin A and 3β-hydroxy-3-epifusicoccin H. The lettuce seed germination-stimulating activity of fusicoccins R and S, phomopsiol and 3α-hydroxyfusicoccin J was examined in the presence of ABA; fusicoccin R and 3α-hydroxyfusicoccin J were highly active, while fusicoccin S and phomopsiol were inactive. The possible biosynthetic relationships among these novel fusicoccins having a 3α- or 3β-hydroxy group in their diterpene moiety are briefly discussed.     

A new 5,6-dihydro-2-pyrone derivative from Phomopsis amygdali,an endophytic fungus isolated from hazelnut (Corylus avellana)

Aims of this study were to isolate endophytes from different parts of hazelnut – Corylus avellana L. to obtain bioactive secondary metabolites and search for the presence of gene region of taxadiene synthase (Ts), a key enzyme in taxol biosynthesis, on selected fungi. Fourteen fungal species were isolated and cultured for the screening studies. The cell-free fermentation broths were extracted with chloroform. The chloroform extracts were tested for cytotoxic activity by MTT method. Based on the activity results and chemical profiles, the isolate identified as Phomopsis amygdali by internal transcribed spaces (ITS) sequence analysis using ITS1 primer was selected for further studies. After large-scale fermentation and purification studies, two major compounds, one of which turned out to be a new secondary metabolite, were isolated and characterized. Structure of the new metabolite was elucidated as (S)-4-butoxy-6-((S)-1-hydroxypentyl)-5,6-dihydro-2H-pyran-2-one by the extensive use of 1D and 2D NMR, and HR-MS, whereas the known compound was identified as (−)pestalotin. Additionally, to evaluate taxol-producing potential of the selected isolate, a PCR amplification study followed by gel electrophoresis analysis was carried out revealing no Ts gene region.     

Biodegradation of melamine and its hydroxy derivatives by a bacterial consortium containing a novel Nocardioides species

Melamine has recently been recognized as a food contaminant with adverse human health effects. Melamine contamination in some crops arises from soil and water pollution from various causes. To remove melamine from the polluted environment, a novel bacterium, Nocardioides sp. strain ATD6, capable of degrading melamine was enriched and isolated from a paddy soil sample. The enrichment culture was performed by the soil-charcoal perfusion method in the presence of triazine-degrading bacteria previously obtained. Strain ATD6 degraded melamine and accumulated cyanuric acid and ammonium, via the intermediates ammeline and ammelide. No gene known to encode for triazine-degrading enzymes was detected in strain ATD6. A mixed culture of strain ATD6 and a simazine-degrading Methyloversatilis sp. strain CDB21 completely degraded melamine, but the degradation rate of cyanuric acid was slow. The degradation of melamine and its catabolites by the mixed culture was greatly enhanced by including Bradyrhizobium japonicum strain CSB1 in the inoculum and adding ethanol to the culture medium. The melamine-degrading consortium consisting of strains ATD6, CDB21, and CSB1 appears to be potentially safer than other known melamine-degrading bacteria for the bioremediation of farmland and other contaminated sites, as no known pathogens were included in the consortium.     

Antimalarial activities of (+)-deoxoartemisitene and its novel C-11, 13 derivatives

(+)-Deoxoartemisitene and its C-11, 13 derivatives were synthesized from artemisinic acid via a short and regiospecific process and several derivatives show 10-20 times more in vitro antimalarial activities against Plasmodium falciparum than artemisinin.     

Fusicocca-3(16),10(14)-diene, and beta- and delta-araneosenes, new fusicoccin biosynthesis-related diterpene hydrocarbons from Phomopsis amygdali

Further isolation and examination of fusicoccane hydrocarbons biosynthetically related to fusicoccin from Phomopsis amygdali allowed us to identify new fungal diterpene hydrocarbons of fusicoccadiene and araneosene. These were assigned as (+)-fusicocca-3(16),10(14)-diene, and (+)-beta- and (+)-delta-araneosenes. These findings led to the experimental clarification of the structures of the biosynthetic hydrocarbon intermediates presumed earlier.     

Ascopyrone P,a novel antibacterial derived from fungi

AIMS: To assess the antimicrobial efficacy of ascopyrone P (APP), a secondary metabolite formed by the fungi Anthracobia melaloma, Plicaria anthracina, Plic. leiocarpa and Peziza petersi belonging to the order Pezizales. METHODS AND RESULTS: In vitro testing using a well diffusion procedure showed that APP at a high concentration (approximately 5%) inhibited the growth of Gram-positive and Gram-negative bacteria. Using an automated microbiology reader, growth curve analysis showed that 2000-4000 mg l(-1) APP caused total or significant bacterial inhibition after incubation for 24 h at 30 degrees C. Against certain yeast strains, 1000- 2000 mg l(-1) APP enhanced growth, although at higher concentrations inhibition of some yeasts was observed. Clostridium and fungal strains were not sensitive to 2000 mg l(-1) APP. No significant cidal effect was observed after 2 h against Listeria monocytogenes or Escherichia coli. Results were identical whether the APP samples tested had been produced enzymatically or chemically. CONCLUSIONS: At a level of 2000 mg l(-1), APP demonstrated growth inhibitory activity against a broad range of bacteria, but not yeasts or moulds. SIGNIFICANCE AND IMPACT OF THE STUDY: A possible application for this novel natural antimicrobial is in food preservation, to control the growth of Gram-negative and Gram-positive bacteria in raw and cooked foods. Effective dosage levels would be 500-4000 mg kg(-1), depending on food type. The efficacy, organoleptic and safety aspects of this compound in food still need to be assessed.     

Novel Fusicoccins R and S, and the fusicoccin S aglycon (phomopsiol) from Phomopsis amygdali niigata 2-A, and their seed germination-stimulating activity in the presence of abscisic acid

Our search for new 3-hydroxyfusicoccins structurally related to cotylenin A from a culture of Phomopsis amygdali Niigata 2-A resulted in the isolation of novel 3-hydroxy fusicoccins, called fusicoccins R and S, and the fusicoccin S aglycon, called phomopsiol, together with known 3alpha-hydroxyfusicoccin J. The structure of phomopsiol was identified as that of O-demethyl-3-epicotylenol based on spectroscopic evidence. The structures of fusicoccins R and S were also determined to be those of 3'-deacetyl-3alpha-hydroxyfusicoccin A and 3beta-hydroxy-3-epifusicoccin H. The lettuce seed germination-stimulating activity of fusicoccins R and S, phomopsiol and 3alpha-hydroxyfusicoccin J was examined in the presence of ABA; fusicoccin R and 3alpha-hydroxyfusicoccin J were highly active, while fusicoccin S and phomopsiol were inactive. The possible biosynthetic relationships among these novel fusicoccins having a 3alpha- or 3beta-hydroxy group in their diterpene moiety are briefly discussed.     

Lithocarols A-F,six tenellone derivatives from the deep-sea derived fungus Phomopsis lithocarpus FS508

Lithocarols A-F (1–6) possessing novel highly-oxygenated isobenzofuran core, together with a related known compound isoprenylisobenzofuran A (7) were isolated from the marine-derived fungus Phomopsis lithocarpus FS508. Among them, lithocarols A-E (1–5) represent the first examples of poly-ketal derivatives in tenellone family. The structures for all these compounds were fully elucidated by spectroscopic analysis, X-ray diffraction, and electronic circular dichroism calculations. Their cytotoxic assay disclosed that compounds 1–4 displayed moderate growth inhibitory effect against four human tumor cell lines with the IC₅₀ values ranging from 10.5 to 38.7 μM.     

Emeriamine, an antidiabetic beta-aminobetaine derived from a novel fungal metabolite

Emeriamine [(R)-3-amino-4-trimethylaminobutyric acid], derived from a novel fungal metabolite "emericedin" [(R)-3-acetylamino-4-trimethylaminobutyric acid], was proved to be a strong and specific inhibitor of carnitine-dependent oxidation of long chain fatty acid (IC50; 3.2 X 10(-6)M) and its main inhibition site was shown to be carnitine palmitoyltransferase I located on the outer-surface of the mitochondrial inner membrane. Emeriamine also showed hypoglycemic and antiketogenic activities in a dose-dependent manner (1 - 10 mg/kg) when administered orally to fasted normal and diabetic animals.     

Synthesis of the antibiotic cortalcerone from D-glucose using pyranose 2-oxidase and a novel fungal enzyme, aldos-2-ulose dehydratase.

Using two enzymes purified from the white-rot fungus, Polyporus obtusus, 5% solutions of D-glucose have been quantitatively converted in vitro into D-arabino-hexos-2-ulose (D-glucosone) and subsequently into a compound having antimicrobial activity. The antibiotic has been shown by nuclear magnetic resonance and mass spectroscopy to be chemically identical to a previously described fungal metabolite known as cortalcerone. Based on kinetic analysis of the synthetic process, a pathway for the biosynthesis of cortalcerone is proposed, involving both chemical rearrangement and enzymically catalyzed steps. Two enzymes, pyranose 2-oxidase and a previously uncharacterized D-arabino-hexos-2-ulose-utilizing enzyme, may be sufficient for the biosynthesis of cortalcerone from glucose in vivo. The D-arabino-hexos-2-ulose-utilizing enzyme dehydrates certain aldosuloses and has been named aldos-2-ulose dehydratase. The enzyme, which appears to be a dimer of 95-kDa subunits, has been purified 450-fold. Additional properties of aldos-2-ulose dehydratase are described, including its apparent ability to catalyze two different steps in the proposed biosynthetic pathway for cortalcerone.     

Protein F2, a novel fibronectin-binding protein from Streptococcus pyogenes, possesses two binding domains

Binding of the group A streptococcus (GAS) to respiratory epithelium is mediated by the fibronectin (Fn)-binding adhesin, protein F1. Previous studies have suggested that certain GAS strains express Fn-binding proteins that are different from protein F1. In this study, we have cloned, sequenced, and characterized a gene ( prtF2 ) from GAS strain 100076 encoding a novel Fn-binding protein, termed protein F2. Insertional inactivation of prtF2 in strain 100076 abolishes its high-affinity Fn binding. prtF2 -related genes exist in most GAS strains that lack prtF1 (encoding protein F1) but bind Fn with high affinity. These observations suggest that protein F2 is a major Fn-binding protein in GAS. Protein F2 is highly homologous to Fn-binding proteins from Streptococcus dysgalactiae and Strep-tococcus equisimilis , particularly in its carboxy-terminal portion. Two domains are responsible for Fn binding by protein F2. One domain (FBRD) consists of three consecutive repeats, whereas the other domain (UFBD) resides on a non-repeated stretch of approximately 100 amino acids and is located 100 amino acids amino-terminal of FBRD. Each of these domains is capable of binding Fn when expressed as a separate protein. In strain 100076, protein F2 activity is regulated in response to alterations in the concentration of atmospheric oxygen.     

A novel Bacillus thuringiensis strain LLB6, isolated from bryophytes, and its new cry2Ac-type gene

AIMS: To isolate and characterize the novel Bacillus thuringiensis strains from bryophytes collected from Wuyi Mountain, Fujian Province of China, and identify new B. thuringiensis strains and toxins active against mosquitoes. METHODS AND RESULTS: Twelve novel B. thuringiensis strains were isolated from 76 bryophyte samples. According to the results of this preliminary screening, LLB6 was the most toxic to Aedes albopictus. Then phase-contrast as well as scanning electron microscopy, bioassays, cloning, sequencing and expression were performed to characterize the novel isolate LLB6 and its new gene cry2Ac5. CONCLUSIONS: Bacillus thuringiensis occurred naturally on bryophytes. LLB6 isolated from Physcomitrium japonicum was toxic to A. albopictus. A new cry2Ac5 gene of LLB6 was detected, cloned and expressed successfully. Bioassays on A. albopictus showed that the expressed Cry2Ac5 was also toxic to the third instar larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of B. thuringiensis strains isolated from bryophytes. It represents a specific source of new B. thuringiensis strains and is of great importance for the knowledge of the ecology of B. thuringiensis. Novel LLB6 harboring the new gene cry2Ac5 and its expressed Cry2Ac5 protein revealed activity against A. albopictus and became a new member of B. thuringiensis toxins.     

Macroscopic fungi from Sierra de Quila, Jalisco, Mexico: diversity and fungal similarity

Diversity and similarity of macrofungi of Sierra de Quila, Jalisco, México were analized in three different kinds of vegetation. Fungal diversity in the area is high. The pine-oak and cloud forests, were more diverse in their community structure than the oak forest. Similarity among the three kinds of vegetation was low, there are few species share among them; pine-oak and cloud forests show higher affinity than oak forests. This pattern of similarity is a general condition for others regions with environmental conditions similar to Sierra de Quila.     

AVP2, a sequence-divergent, K(+)-insensitive H(+)-translocating inorganic pyrophosphatase from Arabidopsis

Plant vacuolar H(+)-translocating inorganic pyrophosphatases (V-PPases; EC 3.6.1.1) have been considered to constitute a family of functionally and structurally monotonous intrinsic membrane proteins. Typified by AVP1 (V. Sarafian, Y. Kim, R.J. Poole, P.A. Rea [1992] Proc Natl Acad Sci USA 89: 1775-1779) from Arabidopsis, all characterized plant V-PPases share greater than 84% sequence identity and catalyze K(+)-stimulated H(+) translocation. Here we describe the molecular and biochemical characterization of AVP2 (accession no. AF182813), a sequence-divergent (36% identical) K(+)-insensitive, Ca(2+)-hypersensitive V-PPase active in both inorganic pyrophosphate hydrolysis and H(+) translocation. The differences between AVP2 and AVP1 provide the first indication that plant V-PPases from the same organism fall into two distinct categories. Phylogenetic analyses of these and other V-PPase sequences extend this principle by showing that AVP2, rather than being an isoform of AVP1, is but one representative of a novel category of AVP2-like (type II) V-PPases that coexist with AVP1-like (type I) V-PPases not only in plants, but also in apicomplexan protists such as the malarial parasite Plasmodium falciparum.     

Genome sequencing of a yeast-like fungal strain P6, a novel species of Aureobasidium spp.: insights into its taxonomy,evolution, and biotechnological potentials

Purpose

This study aimed to look insights into taxonomy, evolution, and biotechnological potentials of a yeast-like fungal strain P6 isolated from a mangrove ecosystem.

Methods

The genome sequencing for the yeast-like fungal strain P6 was conducted on a Hiseq sequencing platform, and the genomic characteristics and annotations were analyzed. The central metabolism and gluconate biosynthesis pathway were studied through the genome sequence data by using the GO, KOG, and KEGG databases. The secondary metabolite potentials were also evaluated.

Results

The whole genome size of the P6 strain was 25.41Mb and the G + C content of its genome was 50.69%. Totally, 6098 protein-coding genes and 264 non-coding RNA genes were predicted. The annotation results showed that the yeast-like fungal strain P6 had complete metabolic pathways of TCA cycle, EMP pathway, pentose phosphate pathway, glyoxylic acid cycle, and other central metabolic pathways. Furthermore, the inulinase activity associated with β-fructofuranosidase and high glucose oxidase activity in this strain have been demonstrated. It was found that this yeast-like fungal strain was located at root of most species of Aureobasidium spp. and at a separate cluster of all the phylogenetic trees. The P6 strain was predicted to contain three NRPS gene clusters, five type-I PKS gene clusters, and one type-I NRPS/PKS gene cluster via analysis at the antiSMASH Website. It may synthesize epichloenin A, fusaric acid, elsinochromes, and fusaridione A.

Conclusions

Based on its unique DNA sequence, taxonomic position in the phylogenetic tree and evolutional position, the yeast-like fungal strain P6 was identified as a novel species Aureobasidium hainanensis sp. nov. P6 isolate and had highly potential applications.

     

Inactivation of glucosamine-6-phosphate synthetase from Salmonella typhimurium LT2 by fumaroyl diaminopropanoic acid derivatives, a novel group of glutamine analogs

A novel group of glutamine analogs, N3-fumaroyl-L-2,3-diaminopropanoic acid (FDP) and its derivatives and analogs including amide (FCDP), methyl ester (FMDP) and its homologue, N4-(4-methoxyfumaroyl)-L-2,4-diaminobutanoic acid, inactivate glucosamine-6-phosphate synthetase (L-glutamine: D-fructose-6-phosphate aminotransferase (hexose-isomerizing), EC 2.6.1.16), isolated from Salmonella typhimurium, by covalent modification. For comparative purposes, selected known glutamine analogs were also examined. Anticapsin, 6-diazo-5-oxo-L-norleucine and, at high concentration, azaserine inactivate the enzyme. The pseudo-first-order rate constants show a hyperbolic dependence on inhibitor concentration for all the above-mentioned inhibitors, suggesting the formation of a reversible complex prior to covalent modification. Dissociation constants for inhibitors were determined and ranged from 10(-4) M for FCDP to 10(-6) M for FMDP. Albizziin, gamma-glutamylhydroxamate and, at low concentration, azaserine inhibit glucosamine synthetase only reversibly. All inhibitors tested are competitive in relation to glutamine. and competitive inhibitors, albizziin and gamma-glutamylhydroxamate protect the enzyme against inactivation. Fructose 6-phosphate accelerates the rate of inactivation. Some analogs of FDP, such as SMDP, CRDP, O-FMSer, MMDP and AADP, are not active against glucosamine-6-phosphate synthetase. The structure-activity relationship of the novel group of glutamine analogs is discussed and structural requirements for the activity of these compounds is established. It is postulated that the compounds examined can be classified as mechanism-based enzyme inactivators.     

Molecular cloning and expression of a novel laccase showing thermo- and acid-stability from the endophytic fungus Phomopsis liquidambari and its potential for growth promotion of plants

A novel laccase (LACB3) from the endophytic fungus, Phomopsis liquidambari, was cloned and its potential to promote peanut growth was evaluated. The full-length cDNA is 1,731 bp, encoding a mature protein of 556 amino acids with a molecular mass of 60.1 kDa. Using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), LACB3 exhibited a K _m and k _cat of 85 μM and 92.7 s^?1, respectively. The enzyme had maximal activity at pH 2.5 and 50 °C and retained 50 % of its activity after 20 h at 50 °C. When LACB3 was applied to soil, the peanut biomass was increased by 12 %, and the content of vanillic acid, coumaric acid and 4-hydroxybenzoic acid in soil were decreased by 21, 27 and 40 %, respectively. These results suggest substantial potential for the use of P. liquidambari or its laccase in agriculture.