New phenylspirodrimane metabolites MBJ-0030, MBJ-0031, and MBJ-0032 isolated from the soil fungal strain Stachybotrys sp. f23793.

Chemical screening of culture medium from the soil fungus Stachybotrys sp. resulted in the isolation of the three new phenylspirodrimanes MBJ-0030 (1), MBJ-0031 (2) and MBJ-0032 (3). Their structures were determined by detailed analysis of spectroscopic data. The absolute configurations of 1-3 were determined by modified Mosher's and Marfey's methods. In addition, cytotoxic and antimicrobial evaluations of the compounds were conducted.

Isolation and Structure Elucidation of Cytotoxic Saccharothriolides D to F from a Rare Actinomycete Saccharothrix sp. and Their Structure-Activity Relationship.

Three new 10-membered macrolides, saccharothriolides D-F (1-3), were isolated from a rare actinomycete, Saccharothrix sp. A1506. The planar structures were determined from analysis of extensive NMR and HR-ESI-MS data, and the absolute configurations were established by ECD spectroscopy analysis. Saccharothriolides D (1) and E (2) were determined to be C-2 epimers of saccharothriolides A (4) and B (5), respectively. Saccharothriolide F (3) was identified to be a demethylated congener of saccharothriolides D (1) and A (4) at the C-2 position. The availability of compounds 1-6 enabled a structure-activity relationship study that revealed the importance of the phenolic hydroxy group at C-2″ and the stereochemistry of C-2 for the inhibition of human fibrosarcoma HT1080 cell growth.

Synthesis of vitamin D3 derivatives with nitrogen-linked substituents at A-ring C-2 and evaluation of their vitamin D receptor-mediated transcriptional activity.

Binding of a series of novel 1α,25-dihydroxyvitamin D(3) (1,25-VD(3)) derivatives, having a nitrogen-linked substituent at the 2α- or 2ß-position of the A-ring (2-N-substituted compounds), with the vitamin D receptor (VDR) was investigated by means of computational docking studies. Selected compounds were synthesized by coupling A-ring synthons and/or with CD-ring-bearing bromomethylene under Trost's conditions. The 2α- and 2ß-stereoisomers of the A-ring synthons were synthesized from l-serine () as a single chiral source by installing vinyl and propargyl groups at opposite ends of the molecule. The activity of the obtained compounds was evaluated by means of a luciferase-based VDR transcriptional activity assay in NIH3T3 cells. Relatively small substituents incorporating a hydrogen-bonding donor, i.e., NHAc and NHMs, were effective for eliciting VDR transcriptional activity, and 2ß-NHMs-1,25-VD(3) () showed the highest activity, being more potent than 1,25-VD(3). Derivatives with bulky substituents were inactive. These new insights into the structure-activity relationships of 1,25-VD(3) derivatives may be helpful in separating the various biological activities of 1,25-VD(3) and in generating novel therapeutic drug candidates.

Branchiibius hedensis gen. nov., sp. nov., an actinobacterium isolated from a Japanese codling (Physiculus japonicus).

A novel, Gram-stain-positive bacterial strain, Mer 29717(T), was isolated from the branchia (gills) of a Japanese codling, Physiculus japonicus, collected from bottom waters of Suruga Bay in Shizuoka, Japan. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain represents a distinct lineage within the family Dermacoccaceae and was related most closely to members of the genera Demetria and Yimella. It shared highest 16S rRNA gene sequence similarity (95.1 %) with Yimella lutea YIM 45900(T). Strain Mer 29717(T) contained MK-8(H(2)) and MK-8(H(4)) as menaquinones, and iso-C(16 : 0), C(16 : 0), C(17 : 1) cis-9, C(17 : 0), C(18 : 1) cis-9 and C(19 : 1) cis-10 were the major cellular fatty acids. The cell-wall peptidoglycan of strain Mer 29717(T) was composed of l-Lys, d-Ser, l-Ser, Gly, d-Glu and d-Ala. Polar lipids were phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and one unidentified phospholipid. Mycolic acids were not detected. The G+C content of the DNA of strain Mer 29717(T) was 68 mol%. On the basis of differential chemotaxonomic, physiological and biochemical data, strain Mer 29717(T) is considered to represent a novel species of a new genus, for which the name Branchiibius hedensis gen. nov., sp. nov. is proposed. The type strain of Branchiibius hedensis is Mer 29717(T) ( = NBRC 106121(T)  = DSM 22951(T)).

An efficient method to screen for the soil bacteria producing therapeutically effective antibiotics.

The discovery of novel therapeutic antimicrobials has become an urgent issue in response to the global crisis of the spread of multi-drug-resistant bacteria. In this report, we propose an efficient screening method for antimicrobial agents with therapeutic potential from soil bacteria. With this method, colonies of the soil bacteria were formed first on agar plates containing only an extract of soil, followed by an overlay of soft agar containing the pathogens, an antibiotic target. Then, we selected the colonies that formed the inhibitory zones on soft agar and evaluated the therapeutic efficacy of their culture supernatants using a silkworm bacterial infection model. Using Staphylococcus aureus as an indicator strain to obtain bacteria that produce therapeutically effective antimicrobials, we succeeded in reducing the screening size by 20-fold compared to the conventional method. An analysis of 86 antibiotics producers identified in this study indicated that the majority belonged to Streptomyces sp. and Lysobacter sp., well-known producers of secondary metabolites. Besides, the presence of eight genera and 37 species among the identified species indicated the diversity of antibiotic producers. Based on the finding of our study, we propose this method as an efficient way to discover novel antimicrobial agents that are therapeutically effective.

Organization of the biosynthetic gene cluster for the polyketide antitumor macrolide, pladienolide, in Streptomyces platensis Mer-11107.

Pladienolides are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107. They show strong antitumor activity and are a potential lead in the search for novel antitumor agents. We sequenced the 65-kb region covering the biosynthetic gene cluster, and found four polyketide synthase genes (pldAI-pldAIV) composed of 11 modules, three genes involved in post-modifications (pldB-D), and a luxR-family regulatory gene (pldR). The thioesterase domain of pldAIV was more dissimilar to that of polyketide synthase systems synthesizing 12/14-membered macrolide polyketides than to that of systems synthesizing other cyclic polyketides. The pldB gene was identified as a 6-hydroxylase belonging to a cytochrome P450 of the CYP107 family. This was clarified by a disruption experiment on pldB, in which the disruptant produced 6-dehydroxy pladienolide B. Two genes located downstream of pldB, designated pldC and pldD, are thought to be a probable genes for 7-O-acetylase and 18, 19-epoxydase respectively.

Production of human metabolites of cyclosporin A, AM1, AM 4 N and AM 9, by microbial conversion.

In humans, cyclosporin A (CyA) is primarily metabolized to two hydroxylated (AM1 and AM 9) and one N-demethylated (AM 4 N) derivative. To produce these derivatives, 1237 actinomycetes were screened for their ability to convert CyA. Among them, 89 strains (7.2%) produced these derivatives from CyA. Finally, Dactylosporangium variesporum IFO 14104, Actinoplanes sp. ATCC 53771 and Streptosporangium sp. AF 935 were selected for the production of AM1, AM 4 N, and AM 9, respectively. Composition of the production medium and the incubation period with CyA were important for obtaining a high yield of CyA derivatives. Large-scale microbial conversion of CyA (1.5 g) using a 30-l jar fermentor yielded 288 mg of AM1 (19.2%), 147 mg of AM 4 N (9.8%) and 115 mg of AM 9 (7.7%).

Saccharothriolides A-C, novel phenyl-substituted 10-membered macrolides isolated from a rare actinomycete Saccharothrix sp.

Three new 10-membered macrolides, saccharothriolides A-C (1-3), were discovered from a rare actinomycete Saccharothrix sp. A1506. All of the sp(3) carbons in the 10-membered ring had chirality, which was determined by extensive spectroscopic analysis and TDDFT-calculation of ECD spectra. Saccharothriolide B (2) exhibited cytotoxicity against human tumor cell lines HeLa and HT1080.

One-pot fermentation of pladienolide D by Streptomyces platensis expressing a heterologous cytochrome P450 gene.

Pladienolide D is a 16-hydroxylated derivative of pladienolide B, produced by Streptomyces platensis. To facilitate the production of pladienolide D, the gene encoding a pladienolide B 16-hydroxylase from S. bungoensis was introduced into S. platensis. The recombinant produced pladienolide D at a production level comparable to that of pladienolide B.

Increase in pladienolide D production rate using a Streptomyces strain overexpressing a cytochrome P450 gene.

Pladienolide B and its 16-hydroxylated derivative (pladienolide D) are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107 showing strong in vitro and in vivo antitumor activity. While pladienolide B is mainly produced by this strain, pladienolide D is produced to a lesser extent. To facilitate the production of pladienolide D by biotransformation, we found that Streptomyces bungoensis A-1544 was able to hydroxylate pladienolide B at 16-position. We identified psmA from S. bungoensis A-1544, which encoded a pladienolide B 16-hydroxylase PsmA belonging to the CYP105 family of cytochrome P450. To increase the efficiency of pladienolide D production, we constructed recombinant S. bungoensis A-1544 overexpressing psmA and performed biotransformation of pladienolide B to pladienolide D. This biotransformation achieved a production level 15-fold higher than that using the control strain S. bungoensis A-1544/pIJ702.