mpCRISTAR: Multiple Plasmid Approach for CRISPR/Cas9 and TAR-Mediated Multiplexed Refactoring of Natural Product Biosynthetic Gene Clusters.

Multiplexed refactoring provides a tool for rapid transcriptional optimization of biosynthetic gene clusters (BGCs) through simultaneous replacement of multiple native promoters with synthetic counterparts. Here, we present the mpCRISTAR, a multiple plasmid-based CRISPR/Cas9 and TAR (transformation-associated recombination), that enables a rapid and highly efficient, multiplexed refactoring of natural product BGCs in yeast. A series of CRISPR plasmids with different auxotrophic markers that could be stably maintained in yeast cells were constructed to express multiple gRNAs simultaneously. We demonstrated the multiplexing capacity of mpCRISTAR using the actinorhodin biosynthetic gene cluster as a model cluster. mpCRISTAR1, in which each CRISPR plasmid expresses one gRNA, allows for simultaneous replacement of up to four promoter sites with nearly 100% efficiency. By expressing two gRNAs from one CRISPR plasmid, termed mpCRISTAR2, we simultaneously replaced a total of six and eight promoter sites with 68% and 32% efficiency, respectively. The mpCRISTAR could be performed iteratively using two different auxotrophic markers, allowing for refactoring of any type of BGC regardless of their operon complexities. The mpCRISTAR platform we report here would become a useful tool for the discovery of new natural products from transcriptionally silent biosynthetic gene clusters present in microbial genomes.

Library of Synthetic Streptomyces Regulatory Sequences for Use in Promoter Engineering of Natural Product Biosynthetic Gene Clusters.

Promoter engineering has emerged as a powerful tool to activate transcriptionally silent natural product biosynthetic gene clusters found in bacterial genomes. Since biosynthetic gene clusters are composed of multiple operons, their promoter engineering requires the use of a set of regulatory sequences with a similar level of activities. Although several successful examples of promoter engineering have been reported, its widespread use has been limited due to the lack of a library of regulatory sequences suitable for use in promoter engineering of large, multiple operon-containing biosynthetic gene clusters. Here, we present the construction of a library of constitutively active, synthetic Streptomyces regulatory sequences. The promoter assay system has been developed using a single-module nonribosomal peptide synthetase that produces the peptide blue pigment indigoidine, allowing for the rapid screening of a large pool of regulatory sequences. The highly randomized regulatory sequences in both promoter and ribosome binding site regions were screened for their ability to produce the blue pigment, and they are classified into the strong, medium, and weak regulatory sequences based on the strength of a blue color. We demonstrated the utility of our synthetic regulatory sequences for promoter engineering of natural product biosynthetic gene clusters using the actinorhodin gene cluster as a model cluster. We believe that the set of Streptomyces regulatory sequences we report here will facilitate the discovery of new natural products from silent, cryptic biosynthetic gene clusters found in sequenced Streptomyces genomes.

Lepistatins A-C, chlorinated sesquiterpenes from the cultured basidiomycete Lepista sordida.

Three new chlorinated sesquiterpenes, named lepistatins A-C, were isolated from the culture broth of Basidiomycete Lepista sordida. The structures were determined by the analysis of spectroscopic data including HREIMS and 1D and 2D NMR. The absolute configuration of lepistatin B was determined by comparing the specific rotation and circular dichroism spectrum with those of known structurally related compounds bearing the same chiral carbon. The structures of lepistatins A-C feature the indanone core structure, but differ from other indanone-containing sesquiterpenes of fungal origin by the alkyl substitution pattern. This indicates that lepistatins A-C probably possess a new sesquiterpene scaffold derived from the common precursor, trans-humulyl cation, by an alternative cyclization.

Ostalactones A-C, ß- and ε-Lactones with Lipase Inhibitory Activity from the Cultured Basidiomycete Stereum ostrea.

Ostalactones A-C (1-3), three new ß- and ε-lactone natural products, were isolated from the culture broth of the basidiomycete Stereum ostrea. The structures were elucidated by interpretation of HRFABMS and 1D and 2D NMR data. The structures of 1 and 2 are characterized by the presence of a ß-lactone containing a fused 4/5 bicyclic core structure. Compound 3 possesses a 2-oxepinone ring system, which is likely to be a biosynthetic precursor of compounds 1 and 2. Ostalactones A (1) and B (2) displayed potent inhibitory activity against human pancreatic lipase.

Antioxidant activities of Vaccinium uliginosum L. extract and its active components.

Vaccinium uliginosum L. (also known as bog bilberry) is a low-growing deciduous shrub classified in the Ericaceae family of plants, which includes numerous Vaccinium berries, blueberries, and cranberries. Berries of the Ericaceae family are known to contain organic acids, vitamins, glycosides, and anthocyanins and have been reported to have antioxidant activity. In order to identify the antioxidative principles of V. uliginosum, we separated water extracts into polyphenol, anthocyanin-rich (pigment), and sugar/acid fractions by using ethyl acetate, acidic methanol (MeOH), and 0.01 N HCl. Antioxidant activities were assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide radical, and hydroxyl radical assays. The crude extract and fractions containing polyphenol and pigment exhibited the greatest antioxidant activities with 50% inhibitory concentration (IC(50)) values of 85.8 microg/mL, 33.2 microg/mL, and 16.7 microg/mL, respectively, for the DPPH assay and 48.1 microg/mL, 83.8 microg/mL, and 51.9 microg/mL for the nonenzymatic superoxide radical assay. The fractions containing polyphenol, pigment, and sugar/acid significantly inhibited xanthine oxidase. To investigate the functional compounds from the active fractions, we purified the polyphenol fraction and separated the compounds by using chromatographic techniques. The crude extract was dissolved in MeOH and further purified by reversed-phase high-performance liquid chromatography (HPLC) using MeOH-water (35:65 vol/vol) (with 0.04% trifluoroacetic acid) to obtain VU-EA-1 (16.6 mg), VU-EA-2 (8.5 mg), VU-EA-3 (19.8 mg), VU-EA-4 (12.8 mg), VU-EA-5 (6.5 mg), and VU-EA-6 (23.5 mg). The MeOH-washed fraction from the HPLC was concentrated and purified by reversed-phase HPLC using MeOH-water (50:50 vol/vol) to give VU-EA-10 (12.4 mg). Antioxidant activity was assessed by DPPH, superoxide radical, and hydroxyl radical assays. The isolated compounds exhibited dose-dependent antioxidant activity with IC(50) values of 7.6 microg/mL (VU-EA-10) for the DPPH assay, 67.8 microg/mL (VU-EA-4) for the nonenzymatic superoxide radical assay, and 3.7 microg/mL (VU-EA-10) and 7.6 microg/ml (VU-EA-6) for the enzymatic superoxide radical assay and 30% inhibitory concentration values of 0.58 microg/mL (VU-EA-1), 0.57 microg/mL (VU-EA-5), and 0.70 microg/mL (VU-EA-6) for the hydroxyl radical assay. In conclusion, V. uliginosum had potent antioxidative activity, and flavonoids were isolated as the main active principles.

Two new stereoisomers of neolignan and lignan from the flower buds of Magnolia fargesii.

A new stereoisomer of 8-O-4' system neolignan, (7R,8S)-1-(3,4-dimethoxyphenyl)-2-[4-(3-hydroxy-1-propenyl)-2-methoxyphenoxy]-propane-1,3-diol (1) and a new stereoisomer of tetrahydrofuranoid lignan, (7R,8S,7'S,8'R)-(3,4,5,3',4')-pentamethoxy-9,7'-dihydroxy-8.8',7.O.9'-lignan (2) along with seven known lignans and neolignans (3-9) were isolated from the methanol extracts of the flower buds of Magnolia fargesii. The structures of these compounds (1-9) were elucidated by spectroscopic methods including 1D- and 2D-NMR as well as by comparison with reported values. Absolute configurations of new stereoisomers 1 and 2 were determined by circular dichroism (CD) spectra. The absolute configuration of (7S,8S,10S)-[tetrahydro-4-hydroxy-2-(3,4,5-trimethoxyphenyl)furan-3-yl]methyl 3,4-dimethoxy benzoate (3) was determined by Mosher's esterification method for the first time in this study. Three lignans, tanegool (4), (+)-dehydrodiconiferyl alcohol (5), and epieudes-min (6), were isolated from this plant for the first time. Superoxide radical scavenging activities of the isolates (1-9) were measured by irradiated riboflavin/ethylenediaminetetraacetic acid (EDTA)/Nitroblue tetrazolium (NBT) system, and their in vitro rat lens aldose reductase (RLAR) inhibitory activities were also evaluated.

Cyathuscavins A, B, and C, new free radical scavengers with DNA protection activity from the Basidiomycete Cyathus stercoreus.

Three new polyketides, cyathuscavins A (1), B (2), and C (3) were isolated from the mycelium culture of Cyathus stercoreus. The structures of the compounds were elucidated on the basis of NMR and mass spectroscopic data. Antioxidant activities of the compounds were evaluated by the scavenging ability against ABTS(+), DPPH, and superoxide anion radicals. Cyathuscavins A-C showed significant antioxidant activity comparable to those of reference antioxidants, BHA and Trolox. Cyathuscavins A-C protected supercoiled plasmid DNA from Fe(2+)/H(2)O(2)-induced breakage.

Identification of chemical structure and free radical scavenging activity of diphlorethohydroxycarmalol isolated from a brown alga, Ishige okamurae.

To obtain a natural antioxidant from a marine biomass, this study investigated the antioxidative activity of methanolic extracts from the marine brown alga, Ishige okamurae collected off Jeju Island. A potent free radical scavenging activity was detected in the ethyl acetate fraction containing polyphenolic compounds, and the potent antioxidant elucidated as a kind of phlorotannin, diphlorethohydroxycarmalol, by NMR and mass spectroscopic data. The free radical scavenging activities of the diphlorethohydroxycarmalol were investigated in relation to 1,1-diphenyl-2-picrylhydrazyl (DPPH), alkyl, and hydroxyl radicals using an electron spin resonance (ESR) system. The diphlorethohydroxycarmalol was found to scavenge DPPH (IC50=3.41 microM) and alkyl (IC50=4.92 microM) radicals more effectively than the commercial antioxidant, ascorbic acid. Therefore, these results present diphlorethohydroxycarmalol as a new phlorotannin with a potent antioxidative activity that could be useful in cosmetics, foods, and pharmaceuticals.

Synthesis and antioxidant activities of 3,5-dialkoxy-4-hydroxycinnamamides.

A series of 3,5-dialkoxy-4-hydroxycinnamamides 6 and 7 was synthesized, and their antioxidant activity was assessed using the thiobarbituric acid reactive substance (TBARS) assay. Interestingly, cinnamamides with longer alkoxy groups on the C-3 and C-5 positions display enhanced inhibition, and most of the compounds in the series tested exhibit excellent lipid peroxidation inhibitory activities. Some cinamamides bearing hexyloxy or 2,6-di-tert-butyl-4-methyl phenol groups have submicromolar inhibitory activities.

Antioxidant and antiatherogenic activity of cis-Hinokiresinol from Trapa pseudoincisa.

cis-Hinokiresinol, also known as (+)-nyasol, was isolated for the first time from an aquatic herbaceous plant, Trapa pseudoincisa NAKAI, via silica gel and octadecyl silica gel column chromatographies. The chemical structure was determined via analyses of the spectroscopic data, including NMR, MS and IR. cis-Hinokiresinol was also found to exhibit antioxidant and antiatherogenic activities. The ICso values for the scavenging activities of cis-hinokiresinol on ABTS cation and superoxide anion radicals were 45.6 and 40.5 microM, respectively. The IC50 values for the inhibitory effects on Lp-PLA2, hACAT1, hACAT2 and LDL-oxidation were 284.7, 280.6, 398.9 and 5.6 microM, respectively.

Cyathusals A, B, and C, antioxidants from the fermented mushroom Cyathus stercoreus.

Three new polyketide-type antioxidative compounds, cyathusals A (1), B (2), and C (3), and the known pulvinatal (4) were obtained from the fermented mushroom Cyathus stercoreus. The structures of the compounds were characterized on the basis of NMR and mass spectroscopic data. Cyathusals A, B, and C and pulvinatal showed free radical scavenging activities on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical with EC50 values of 41.6, 46.0, 26.6, and 28.6 microM, respectively, and on the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) cation radical with EC50 values of 7.9, 11.1, 9.1, and 8.4 microM, respectively.

Two new stereoisomers of Tetrahydrofuranoid lignans from the flower buds of Magnolia fargesii.

Two new stereoisomers of tetrahydrofuranoid lignans, 7S,8R,7'S,8'R- (1) and 7R,8S,7'S,8'R-3,4,3',4'-tetramethoxy-9,7'-dihydroxy-8.8',7.O.9'-lignan (2) along with nine known lignans including tetrahydrofuranoids (3, 4) and tetrahydrofurofuranoids (5-11) were isolated from a CHCl(3)-soluble fraction of the flower buds of Magnolia fargesii. Two tetrahydrofuranoids, magnostellin A (3) and lariciresinol dimethyl ether (4) were isolated from this species for the first time. The structures of these compounds (1-11) were identified by spectroscopic methods as well as by comparison with published values. Absolute configurations of new stereoisomers (1, 2) were determined by the Mosher's esterification method and Circular Dichroism (CD) studies. All the isolates (1-11) were evaluated for their antioxidant activities using modified superoxide radical-scavenging assay. Compounds 5-8 showed the potent superoxide radical-scavenging activities with the ED(50) values of 19.2, 19.2, 16.5, and 27.7 microM, respectively, as compared with standard antioxidants (BHA: 22.8 microM; Trolox: 940 microM).

Methoxylaricinolic acid, a new sesquiterpene from the fruiting bodies of Stereum ostrea.

Methoxylaricinolic acid (1), a new sesquiterpene with drimane skeleton was isolated from the fruiting bodies of Stereum ostrea, together with the known compound laricinolic acid (2). The structure of 1 was determined as 12-methoxy-7-oxo-11-drimanoic acid on the basis of spectroscopic analysis.

Prevention of UV radiation-induced premature skin aging in hairless mice by the novel compound Melanocin A.

Repetitive exposure of the skin to UV radiation induces various harmful changes, such as thickening, wrinkle formation, inflammation and carcinogenesis. A variety of natural compounds and synthetic compounds have been studied to determine whether they can prevent UV-induced harmful effects. In this study, we investigated the effect of a novel compound, Melanocin A, which was isolated from Eupenicillium shearii F80695, on UV-induced premature skin aging. First, we studied the effect of Melanocin A on UV-induced matrix metalloproteinase (MMP)-9 expression in an immortalized human keratinocyte cell line, HaCaT, in vitro. Acute UV irradiation induced MMP-9 expression at both the mRNA and protein levels and Melanocin A suppressed this expression in a dose-dependent manner. We then investigated the effect of Melanocin A on UV-induced skin changes in hairless mice in vivo. Chronic exposure of hairless mouse dorsal skin to UV increased skin thickness and induced wrinkle formation and the gelatinase activities of MMP-2 and MMP-9. Moreover, Melanocin A significantly suppressed UV-induced morphologic skin changes and MMP-2 and MMP-9 expression. Taken together, these results show that Melanocin A can prevent the harmful effects of UV that lead to skin aging. Therefore, we suggest that Melanocin A should be viewed as a potential therapeutic agent for preventing and/or treating premature skin aging.

Hirsutenols D, E and F, new sesquiterpenes from the culture broth of Stereum hirsutum.

New sesquiterpenes, hirsutenols D-F, were isolated from the fermentation broth of Stereum hirsutum, and their structures were determined on the basis of various spectroscopic analyses. Hirsutenols E and F showed significant scavenging activity against superoxide anion radicals with EC50 values of 1.62 and 0.39 mM, respectively.

Glyscavins A, B and C, new phenolic glycoside antioxidants produced by a fungus Mycelia sterilia F020054.

Three new phenolic glycosides designated glyscavins A (1), B (2), and C (3) were isolated from the culture broth of a fungal strain Mycelia sterilia F020054. Structural elucidation of the compounds was based on the NMR and MS spectroscopic analyses. Glyscavins A, B and C exhibited higher free radical scavenging activity on superoxide and 2,2'-azinobis(3-ethylbenzothialozinesulfonic acid) cation radical (ABTS*+) than butylated hydroxyanisole (BHA).

1,12-substituted tetracyclines as antioxidant agents.

Novel hydroxypyrazoline derivatives of tetracycline and minocycline have been synthesized through the reaction of these tetracyclines with hydrazine. The formation of a new chiral center at C12 is stereospecific to give 12S-12-hydroxy-1,12-pyrazolinotetracycline. A reaction mechanism for the formation of these novel tetracycline derivatives has been proposed. Hydroxypyrazolinotetracyclines exhibit no binding to Mg2+ and Zn2+, features that are required for antibiotic activity and matrix metalloproteinase (MMP) inhibitions, respectively. The modification toward their hydroxypyrazolino derivatives significantly improved the antioxidant activities of tetracycline and minocycline, as shown by three commonly used assays (DPPH, ABTS+, and superoxide scavenging). 12S-Hydroxy-1,12-pyrazolinominocycline is a promising tetracycline-based antioxidant devoid of antibiotic properties and MMP inhibitory activity, which could be beneficial in the treatment of complications related to oxidative stress.

Deoxyverrucosidin, a novel GRP78/BiP down-regulator, produced by Penicillium sp.

Glucose-regulated protein 78 (GRP78) resides in endoplasmic reticulum (ER) and plays a role in protecting tumor cells against the toxic effects of anticancer agents. During the search for down-regulators of GRP78 using a reporter gene (luciferase) assay system, we isolated a novel compound designated as deoxyverrucosidin (1), a congener of verrucosidin (2), from Penicillium sp. and identified it as a down-regulator of the grp78 gene. The structure of 1 was determined by mainly ESI-mass and two-dimensional NMR spectra. 1 dose-dependently inhibited the expression of GRP78 promoter with an IC50 of 30 nM.

Boletunones A and B, highly functionalized novel sesquiterpenes from Boletus calopus.

Highly functionalized novel sesquiterpenes, named boletunones A and B, were isolated from the methanolic extract of the fruiting body of the mushroom Boletus calopus. Their structures and relative stereochemistry were established by various spectral analyses. [structure: see text]

Neuroprotective activity of p-terphenyl leucomentins from the mushroom Paxillus panuoides.

The neuroprotective mechanism of p-terphenyl leucomentins from the mushroom Paxillus panuoides was studied. Leucomentins showed potent inhibition of lipid peroxidation and H2O2 neurotoxicity, but free from any role as reactive oxygen species (ROS) scavengers. Iron-mediated oxidative damage has been implicated in these processes, as a provider of ROS via iron. Leucomentins can chelate iron when DNA is present with iron and H2O2, and so inhibiting DNA single strand breakage. These results suggest that the neuroprotective action of leucomentins is dependent on their ability to chelate iron.