Isolation of C-29 oxygenated oleanane triterpenoids and a (+)-muurolene type sesquiterpenoid from the fruiting bodies of Fuscoporia torulosa and their bioactivities.

Basidiomycetes with a wide variety of skeletons of secondary metabolites can be expected to be the source of new interesting biological compounds. During our research on basidiomycetes, two new C-29 oxygenated oleanane-type triterpenes (1 and 2) and torulosacid (3), a muurolene type sesquiterpenoid with a five-membered ether ring along with nine known compounds (4-12), were isolated from the MeOH extract of the fruiting bodies of Fuscoporia torulosa. The structures of 1-3 were determined by NMR and HREIMS analysis.

An Aromatic Farnesyltransferase Functions in Biosynthesis of the Anti-HIV Meroterpenoid Daurichromenic Acid.

produces daurichromenic acid, an anti-HIV meroterpenoid, via oxidative cyclization of the farnesyl group of grifolic acid. The prenyltransferase (PT) that synthesizes grifolic acid is a farnesyltransferase in plant specialized metabolism. In this study, we demonstrated that the isoprenoid moiety of grifolic acid is derived from the 2--methyl-d-erythritol-4-phosphate pathway that takes place in plastids.

Identification and Characterization of Daurichromenic Acid Synthase Active in Anti-HIV Biosynthesis.

Daurichromenic acid (DCA) synthase catalyzes the oxidative cyclization of grifolic acid to produce DCA, an anti-HIV meroterpenoid isolated from We identified a novel cDNA encoding DCA synthase by transcriptome-based screening from young leaves of The gene coded for a 533-amino acid polypeptide with moderate homologies to flavin adenine dinucleotide oxidases from other plants. The primary structure contained an amino-terminal signal peptide and conserved amino acid residues to form bicovalent linkage to the flavin adenine dinucleotide isoalloxazine ring at histidine-112 and cysteine-175. In addition, the recombinant DCA synthase, purified from the culture supernatant of transgenic , exhibited structural and functional properties as a flavoprotein.

A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from .

L. produces daurichromenic acid, the anti-HIV meroterpenoid consisting of sesquiterpene and orsellinic acid (OSA) moieties. To characterize the enzyme responsible for OSA biosynthesis, a cDNA encoding a novel polyketide synthase (PKS), orcinol synthase (ORS), was cloned from young leaves of .

Comparative analysis of transcriptomes in aerial stems and roots of Ephedra sinica based on high-throughput mRNA sequencing.

Ephedra plants are taxonomically classified as gymnosperms, and are medicinally important as the botanical origin of crude drugs and as bioresources that contain pharmacologically active chemicals. Here we show a comparative analysis of the transcriptomes of aerial stems and roots of Ephedra sinica based on high-throughput mRNA sequencing by RNA-Seq. De novo assembly of short cDNA sequence reads generated 23,358, 13,373, and 28,579 contigs longer than 200 bases from aerial stems, roots, or both aerial stems and roots, respectively.

Cloning and Functional Analysis of Three Chalcone Synthases from the Flowers of Safflowers Carthamus tinctorius.

The flowers of safflowers (Carthamus tinctorius L.) are very important as they are the sole source of their distinct pigments, i.e.

Seed dormancy breaking diterpenoids from the liverwort Plagiochila sciophila and their differentiation inducing activity in human promyelocytic leukemia HL-60 cells.

To obtain the structural diversity of bioactive compounds similar to cotylenins and fusicoccins that modulate 14-3-3 protein-protein interactions in eukaryotes, screening tests were carried out using the lettuce seed dormancy breaking-assay. An acetone extract of the liverwort Plagiochila sciophila exhibited significant activity against the seeds in the presence of the plant hormone abscisic acid. Activity-guided fractionation of the extract afforded the isolation of seven novel fusicoccane-type diterpenoids, named fusicosciophins A-E (1-5), 8-deacetyl (6) and 9-deacetyl fusicosciophin E (7).

Total synthesis of (-)-thallusin: utilization of enzymatic hydrolysis resolution.

(-)-Thallusin, isolated from a marine bacterium, is the only known natural product to act as an algal morphogenesis inducer. Because (-)-thallusin can only be obtained in exceedingly limited amounts from microbial cultivation, a synthetic supply of this compound is highly desirable. Here, we describe a novel synthetic pathway to (±)-thallusin and the first asymmetric synthesis of (-)-thallusin utilizing the enzymatic hydrolysis resolution with the combination of lipase PS-30 and lipase M Amamo-10.

Calyculin biogenesis from a pyrophosphate protoxin produced by a sponge symbiont.

The Japanese marine sponge Discodermia calyx contains a major cytotoxic compound, calyculin A, which exhibits selective inhibition of protein phosphatases 1 and 2A. It has long been used as a chemical tool to evaluate intracellular signal transduction regulated by reversible protein phosphorylation. We describe the identification of the biosynthetic gene cluster of calyculin A by a metagenome mining approach.

Seed dormancy breaking diterpenoids, including novel brassicicenes J and K, from fungus Alternaria brassicicola, and their necrotic/apoptotic activities in HL-60 cells.

To find new metabolites similar to cotylenins and fusicoccins from the fungus Alternaria brassicicola, screening tests were carried out using the lettuce seed dormancy breaking assay. Activity-guided fractionation of the EtOAc extract from the culture using the assay afforded the isolation of two novel fusicoccane diterpenoids named brassicicenes J (1) and K (2), along with three known brassicicenes A (3), B (4), and F (5). Their structures were elucidated from extensive NMR spectral data and by comparison of these with those reported in the literature.

Generation of mast cells from mouse fetus: analysis of differentiation and functionality, and transcriptome profiling using next generation sequencer.

While gene knockout technology can reveal the roles of proteins in cellular functions, including in mast cells, fetal death due to gene manipulation frequently interrupts experimental analysis. We generated mast cells from mouse fetal liver (FLMC), and compared the fundamental functions of FLMC with those of bone marrow-derived mouse mast cells (BMMC). Under electron microscopy, numerous small and electron-dense granules were observed in FLMC.

Activity-guided isolation of cytotoxic bis-bibenzyl constituents from Dumortiera hirsuta.

Activity-guided fractionation of the ether extract of Dumortiera hirsute (Japanese liverwort), using cytotoxicity testing with cultured HL 60 and KB cells, resulted in the isolation of a new cytotoxic bis-bibenzyl compound, along with the two known bis-bibenzyls: isomarchantin C and isoriccardin C. The structural determination of the new bis-bibenzyl through extensive NMR spectral data indicated a derivative of marchantin A, which has been isolated from the liverwort Marchantia polymorpha. The cytotoxicity of the bis-bibenzyls was evaluated by the MTT (3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay using cultured HL 60 and KB cells.

Heterologously expressed β-hydroxyl fatty acids from a metagenomic library of a marine sponge.

Functional screening based on the antibacterial activity of a metagenomic library of the Japanese marine sponge, Discodermia calyx, afforded three β-hydroxyl fatty acids: 3-hydroxypalmitic acid, 3-hydroxylauric acid and 3-hydroxymyristic acid, heterologously expressed in an antibacterial clone, pDC113. 3-Hydroxypalmitic acid showed moderate antibacterial activity against Bacillus cereus and Candida albicans. A sequence analysis of the insert DNA revealed 23 putative ORFs, with most sharing homology to bacterial fatty acid synthase II and lipid A biosynthesis enzymes.

De novo sequencing and transcriptome analysis of the central nervous system of mollusc Lymnaea stagnalis by deep RNA sequencing.

The pond snail Lymnaea stagnalis is among several mollusc species that have been well investigated due to the simplicity of their nervous systems and large identifiable neurons. Nonetheless, despite the continued attention given to the physiological characteristics of its nervous system, the genetic information of the Lymnaea central nervous system (CNS) has not yet been fully explored. The absence of genetic information is a large disadvantage for transcriptome sequencing because it makes transcriptome assembly difficult.

Porphyrins from a metagenomic library of the marine sponge Discodermia calyx.

Marine sponges harbouring uncultured symbiotic bacteria are important sources of biologically active compounds. Since they would be interesting resources to explore unknown functional genes by means of a metagenomic approach, we constructed a metagenomic library of the Japanese marine sponge Discodermia calyx. The functional screening afforded the two clones producing porphyrins as red pigments.

Cloning and characterization of a novel gene that encodes (S)-beta-bisabolene synthase from ginger, Zingiber officinale.

Ginger, Zingiber officinale Roscoe, contains a fragrant oil mainly composed of sesquiterpenes and monoterpenes. We isolated a cDNA that codes for a sesquiterpene synthase from young rhizomes of ginger, Z. officinale Roscoe, Japanese cultivar "Kintoki".

Transannular proton transfer in the cyclization of geranylgeranyl diphosphate to fusicoccadiene, a biosynthetic intermediate of fusicoccins.

Enzymatic cyclization of geranylgeranyl diphosphate to fusicoccadiene involves a transannular proton transfer process. Label distribution in the cyclized products derived from deuterium-labeled GGDPs showed that a proton generated from C-10 migrates to C-6 in the intermediary dolabellane framework prior to the second ring formation. Although a direct 1,5-proton transfer would achieve this process, semiempirical MO calculations suggested an alternative pathway, which involves successive 1,4- and 1,5-proton transfers using C-2 as a springboard.

Identification of diterpene biosynthetic gene clusters and functional analysis of labdane-related diterpene cyclases in Phomopsis amygdali.

Two diterpene biosynthesis gene clusters in the fusicoccin-producing fungus, Phomopsis amygdali, were identified by genome walking from PaGGS1 and PaGGS4 which encode the geranylgeranyl diphosphate (GGDP) synthases. The diterpene cyclase-like genes, PaDC1 and PaDC2, were respectively located proximal to PaGGS1 and PaGGS4. The amino acid sequences of these two enzymes were similar to those of fungal labdane-related diterpene cyclases.

Phaeoside, a novel galactoside of hydroxymanoyl oxide from the gibberellin A1-producing Phaeosphaeria sp. L487.

Isolation and examination of a diterpene glycoside from the culture filtrate of the gibberellin A(1)-producing Phaeosphaeria sp. L487 allowed us to identify a novel fungal galactoside of hydroxymanoyl oxide together with (-)-ent-13-epi-manoyl oxide. It was designated phaeoside and determined to be 1alpha-hydroxy-ent-13-epi-manoyl oxide 1-O-beta-D-galactopyranoside based on its chemical degradation and spectroscopic methods.

Emission of ent-kaurene, a diterpenoid hydrocarbon precursor for gibberellins, into the headspace from plants.

ent-Kaurene is a tetracyclic hydrocarbon precursor for gibberellins (GAs) in plants and fungi. To address whether fungal GA biosynthesis enzymes function in plants, we generated transgenic Arabidopsis plants overexpressing ent-kaurene synthase (GfCPS/KS) from a GA-producing fungus Gibberella fujikuroi. GfCPS/KS catalyzes a two-step reaction corresponding to ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) activities in plants.

Diterpene cyclases responsible for the biosynthesis of phytoalexins, momilactones A, B, and oryzalexins A-F in rice.

Rice (Oryza sativa L.) produces diterpene phytoalexins, such as momilactones, oryzalexins, and phytocassanes. Using rice genome information and in vitro assay with recombinant enzymes, we identified genes (OsKS4 and OsKS10) encoding the type-A diterpene cyclases 9beta-pimara-7,15-diene synthase and ent-sandaracopimaradiene synthase which are involved in the biosynthesis of momilactones A, B and oryzalexins A-F respectively.

Biological functions of ent- and syn-copalyl diphosphate synthases in rice: key enzymes for the branch point of gibberellin and phytoalexin biosynthesis.

Rice (Oryza sativa L.) produces ent-copalyl diphosphate (ent-CDP) and syn-CDP as precursors for several classes of phytoalexins and the phytohormones, gibberellins (GAs). It has recently been shown that a loss-of-function mutation of OsCPS1, a gene encoding a putative ent-CDP synthase, results in a severely GA-deficient dwarf phenotype in rice.

Erinacol (cyatha-3,12-dien-14beta-ol) and 11-O-acetylcyathin A3, new cyathane metabolites from an erinacine Q-producing Hericium erinaceum.

In our search for new cyathane metabolites related to the biosynthesis of erinacine Q in Hericium erinaceum, we isolated a novel cyatha-3,12-dien-14beta-ol named erinacol together with known 11-O-acetylcyathatriol (the erinacine Q aglycon) and new metabolite 11-O-acetylcyathin A(3) from the mycelial extract. The structure of each compound was determined by spectral methods. Possible biosynthetic relationships of these metabolites are discussed from their structural features.