[show abstract][hide abstract] ABSTRACT: An industrial microorganism, Streptomyces avermitilis, which is a producer of anthelmintic macrocyclic lactones, avermectins, has been constructed as a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis. Twenty of the entire biosynthetic gene clusters for secondary metabolites were successively cloned and introduced into a versatile model host S. avermitilis SUKA17 or 22. Almost all S. avermitilis transformants carrying the entire gene cluster produced metabolites as a result of the expression of biosynthetic gene clusters introduced. A few transformants were unable to produce metabolites, but their production was restored by the expression of biosynthetic genes using an alternative promoter or the expression of a regulatory gene in the gene cluster that controls the expression of biosynthetic genes in the cluster using an alternative promoter. Production of metabolites in some transformants of the versatile host was higher than that of the original producers, and cryptic biosynthetic gene clusters in the original producer were also expressed in a versatile host.
[show abstract][hide abstract] ABSTRACT: Many bioactive natural products are produced as "secondary metabolites" by plants, bacteria, and fungi. During the middle of the 20th century, several secondary metabolites from fungi revolutionized the pharmaceutical industry, for example, penicillin, lovastatin, and cyclosporine. They are generally biosynthesized by enzymes encoded by clusters of coordinately regulated genes, and several motif-based methods have been developed to detect secondary metabolite biosynthetic (SMB) gene clusters using the sequence information of typical SMB core genes such as polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS). However, no detection method exists for SMB gene clusters that are functional and do not include core SMB genes at present. To advance the exploration of SMB gene clusters, especially those without known core genes, we developed MIDDAS-M, a motif-independent de novodetection algorithm for SMB gene clusters. We integrated virtual gene cluster generation in an annotated genome sequence with highly sensitive scoring of the cooperative transcriptional regulation of cluster member genes. MIDDAS-M accurately predicted 38 SMB gene clusters that have been experimentally confirmed and/or predicted by other motif-based methods in 3 fungal strains. MIDDAS-M further identified a new SMB gene cluster for ustiloxin B, which was experimentally validated. Sequence analysis of the cluster genes indicated a novel mechanism for peptide biosynthesis independent of NRPS. Because it is fully computational and independent of empirical knowledge about SMB core genes, MIDDAS-M allows a large-scale, comprehensive analysis of SMB gene clusters, including those with novel biosynthetic mechanisms that do not contain any functionally characterized genes.
PLoS ONE 01/2013; 8(12):e84028. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Three new prenylated flavonoids, namely, solophenols B (1), C (2), and D (3), as well as a new prenylated stilbene, solomonin (4), were isolated from propolis collected from the Solomon Islands. In addition, 17 known compounds were identified. The structures of the new compounds were determined by a combination of methods, including mass spectrometry and NMR. These new compounds and several known compounds were tested for antibacterial activity against Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa. Most of them exhibited potent antibacterial activity. These findings may indicate that propolis from the Solomon Islands has potential applications as an ingredient in food additives or pharmaceuticals.
Journal of Agricultural and Food Chemistry 10/2012; · 2.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: To investigate the biological activity of a novel 24-membered macrolide compound, JBIR-19, isolated from the culture broth of the entomopathogenic fungus Metarhizium sp. fE61, morphological changes in yeast cells were examined using the automated image-processing program CalMorph. Principal components analysis was used to elucidate dynamic changes in the phenotypes, revealing two independent effects of JBIR-19 in yeast cells: bud elongation and increased size of the actin region. Using a fitness assay, we identified the genes required for robust growth in the presence of JBIR-19. Among these were CCW12, YLR111W, and DHH1, which are also involved in abnormal bud morphology. Based on these results and others, we predict intracellular targets of JBIR-19 and its functional interactions.
FEMS Yeast Research 11/2011; 12(3):293-304. · 2.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: Searching for metabolites from Streptomyces sp. RI051-SDHV6 resulted in the discovery of a novel peptide, JBIR-96 (1). The structure of 1 was established as an N-phenylacetylated pentapeptide involving a cysteic acid and a peptide lactone structure by extensive NMR and MS analyses. In addition, the absolute configuration of 1 was established by Marfey's and modified Mosher's methods.
Journal of Natural Products 05/2011; 74(5):1344-7. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Terrestrial actinobacteria have served as a primary source of bioactive compounds; however, a rapid decrease in the discovery of new compounds strongly necessitates new investigational approaches. One approach is the screening of actinobacteria from marine habitats, especially the members of the genus Streptomyces. Presence of this genus in a marine sponge, Haliclona sp., was investigated using culture-dependent and -independent techniques. 16S rRNA gene clone library analysis showed the presence of diverse Streptomyces in the sponge sample. In addition to the dominant genus Streptomyces, members of six different genera were isolated using four different media. Five phylogenetically new strains, each representing a novel species in the genus Streptomyces were also isolated. Polyphasic study suggesting the classification of two of these strains as novel species is presented. Searching the strains for the production of novel compounds and the presence of biosynthetic genes for secondary metabolites revealed seven novel compounds and biosynthetic genes with unique sequences. In these compounds, JBIR-43 exhibited cytotoxic activity against cancer cell lines. JBIR-34 and -35 were particularly interesting because of their unique chemical skeleton. To our knowledge, this is the first comprehensive study detailing the isolation of actinobacteria from a marine sponge and novel secondary metabolites from these strains.
[show abstract][hide abstract] ABSTRACT: Four novel glycosylated derivatives of versipelostatin (1), versipelostatins B-E (2-5), were isolated from the culture broth of Streptomyces versipellis 4083-SVS6. The inhibitory activities of the isolated compounds against the expression of molecular chaperone GRP78 induced by 2-deoxyglucose were evaluated. Of the five versipelostatin family members, 1 and 4 were the more potent with IC(50) values of 3.5 and 4.3 microM. These results suggest that the alpha-L-oleandropyranosyl (1-->4)-beta-D-digitoxopyranosyl residue in the sugar moiety may play an important role in down-regulating GRP78 expression induced by 2-deoxyglucose.
[show abstract][hide abstract] ABSTRACT: In the course of our screening program for active compounds that induce cell morphological changes of Saccharomyces cerevisiae, the culture broth of an entomopathogenic fungus Metarhizium sp. fE61 exhibited a unique morphological phenotype. We conducted an activity-guided isolation from the fermentation broth of Metarhizium sp. fE61 to yield two new macrolide compounds named JBIR-19 (1) and -20 (2) as active substances. Their structures were determined to be 24-membered macrolide analogs containing a 2-aminoethyl phosphate ester on the basis of NMR and other spectroscopic data. Compounds 1 and 2 induced striking elongated morphology of S. cerevisiae at concentrations of 3.1 and 13 μM, but showed weak antiyeast activity at MICs of 200 and >200 μM, respectively.Keywords: cell morphology, JBIR-19, JBIR-20, 24-membered macrolide, Metarhizium sp., Saccharomyces cerevisiae
The Journal of Antibiotics 02/2009; 62(3):159-162. · 2.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ammocidins B, C and D were isolated from the culture broth of Saccharothrix sp. AJ9571, an ammocidin A-producing strain. Their structures were determined by a detailed spectroscopic analysis and by a comparison of their NMR data with those of ammocidin A. Ammocidins A and B showed potent anti-proliferative activities against human cancer cell lines.
The Journal of Antibiotics 02/2009; 62(3):123-7. · 2.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Two pamamycin homologues with different side chain lengths were isolated from Streptomyces sp. HKI-0118. Aerial mycelium-inducing activity decreased by ca. 1/10 per methylene unit in the side chain.
The Journal of Antibiotics 03/2008; 61(2):98-102. · 2.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Viable microbial cells distributed in a 130 microim thick surface layer of cotton fabrics were stained with a fluorescent glucose, 2- [N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino] -2-deoxy-D-glucose (2-NBDG), and automatically mapped with an ultra-deep focusing range microscope (UDF) system. The software of the UDF system was upgraded and the number of Candida albicans cells could be counted at a higher precision than before. Bacterial cells of Pseudomonas fluorescens, Serratia marcescens, and Citrobacter freundii, which were smaller than 1-2 microm, were successfully mapped for the first time. These results indicate the practical importance of the present method in the evaluation of the antibacterial properties of fabrics and the efficacy of washing.
[show abstract][hide abstract] ABSTRACT: The efficacy of microbial cell removal (EMR) from fabrics is a practically important indicator for the evaluation of cleansers and detergents. EMR is expressed quantitatively by the relative number of viable cells remaining on a fabric swatch after the treatment with these reagents. In order to count the viable cells on the swatch directly and rapidly, we have developed a unique microscopic imaging system with an ultra-deep focusing range. Standard swatches of cotton fabric were inoculated with microorganisms such as Pseudomonas fluorescence, Staphylococcus aureus, or Candida albicans. After the incubation on an agar medium, each swatch was treated with a fluorescent glucose, 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxyglucose, to stain only viable cells. The images of every cell distributed within the surface layer with no greater than 130 microm thickness could be integrated into one image. Thus visualized cells could be counted automatically by a novel imaging program. Using a pair of cotton swatches (0.5 x 1.0 cm(2)) inoculated with C. albicans, EMR was evaluated quantitatively. Before washing, the total number of viable cells found on the observation area (3.8 x 10(-4 )cm(2)) was 288 cells. After washing with a test detergent, no cell (<1) was detected. For this case, EMR was given by the formula: log(288/<1)=greater than 2.5. The imaging and cell count of a test fabric could be performed within 1 h.
Journal of Industrial Microbiology and Biotechnology 01/2007; 33(12):995-1002. · 2.32 Impact Factor