Naoya Oku

Rakicidin D, an inhibitor of tumor cell invasion from marine-derived Streptomyces sp.

Tumor metastasis is the leading cause of death in cancer patients. It is the process by which a tumor cell leaves the primary tumor, disseminates to a distant site through the circulatory system and establishes a secondary tumor.1 During the metastatic cascade, tumor cells must pass through the extracellular matrix barriers to accomplish the metastasis. Although the genetic basis of tumorigenesis can vary greatly, the steps required for metastasis are similar for all tumor cells. Therefore, interruption of metastasis is a promising approach to the treatment of cancers of various genetic origins. In our continuing search for anti-invasive compounds from microbial secondary metabolites,2–5 rakicidin D (1) was isolated from the culture broth of an actinomycete strain of the genus Streptomyces. Rakicidins are the 15-membered depsipeptides consisting of three amino acids and a 3-hydroxyfatty acid (Figure 1). To date, three congeners, rakicidins A (2) and B (3) from Micromonospora and rakicidin C (4) from Streptomyces, have been reported.6,7 Rakicidins contain a rare unusual amino acid, 4-amino-2,4-pentadienoate, which has been found only in the secondary metabolites from actinomycetes. Except for rakicidins, only two classes of cyclic peptides, BE435478 and vinylamycin,9 are reported to date to contain this unusual amino acid. Herein, we describe the isolation, structure elucidation and biological properties of rakicidin D (1). The producing strain Streptomyces sp. MWW064 was isolated from a marine sediment sample collected in Samut Sakhon province, Thailand. The strain was cultured in our standard medium for actinomycetes and the whole culture broth was extracted with 1-butanol. The extract showed inhibitory activity toward tumor cell invasion into Matrigel, the reconstituted extracellular matrix proteins.10 Bioassay-guided fractionation of the extract led to the isolation of a new compound, rakicidin D (1). Compound 1 was obtained as a colorless amorphous powder. The high-resolution ESITOFMS indicated a molecular formula of C24H38N4O7, which was consistent with the 1H and 13C NMR data. The IR spectrum of 1 indicated the presence of OH or NH (3356 cm 1) and carbonyl (1688 and 1648 cm 1) functionalities. The UV spectrum and the 1H and 13C NMR spectra of 1 showed high similarity to those for rakicidins A and B.7 The 13C NMR and HMQC analysis confirmed the presence of 24 carbons attributable to six deshielded signals including carbonyl carbons, two sp2 methines, one sp2 methylene, five sp3 methines, six sp3 methylenes, four methyl carbons and five exchangeable protons. 2-Amino-2,4-pentadienoate moiety was elucidated by a COSY correlation for H-9/H-10 and a series of HMBC correlations from H-12 to C-10 and C-11, from H-10 to C-8, C-11 and C-12, and from H-9 to C-8 and C-10 (Figure 2). An NOE between H-10 and an exomethylene proton at dH 5.41 indicated that the latter proton and C-10 were located on the same side of the C-11/C-12 double bond. E configuration for the double bond between C-9 and C-10 was confirmed by a large coupling constant for H-9 and H-10 (JHH1⁄415.0 Hz). HMBC correlations from H-7 to C-6 and C-8, and from H-6 to C-5 and C-8 established the connectivity of N-methylglycine and the pentadienoate. COSY correlations for 2-NH/H-2/H-3 and HMBC correlations from H-2 to C-1, H-3 to C-4 and 4-NH2 to C-3 confirmed the presence of a b-hydroxyasparagine moiety. Connection of this amino acid to the glycine moiety was established by an HMBC correlation from 2-NH to C-5. Three additional fragments, H-23/H-14/H-15, H-24/H-16 and H-21/H-22, were recognized from the COSY spectrum. HMBC correlations from H-23 to C-13, C-14 and C-15, and from H-24 to C-15, C-16 and C-17, established the 2,4-dimethyl-3-hydroxyalkanoate substructure. Connectivity of this fragment to the peptide unit was elucidated by HMBC correlations from 11-NH to C-13 and from H-15 to C-1. Finally, the fragment C-22/C-21 containing the triplet methyl group, the methylene fragment C-20 that had an HMBC correlation from H-22 and

Genome Mining Reveals a Minimum Gene Set for the Biosynthesis of 32-Membered Macrocyclic Thiopeptides Lactazoles

Although >100 thiopeptides have been discovered, the number of validated gene clusters involved in their biosynthesis is lagging. We use genome mining to identify a silent thiopeptide biosynthetic gene cluster responsible for biosynthesis of lactazoles. Lactazoles are structurally unique thiopeptides with a 32-membered macrocycle and a 2-oxazolyl-6-thiazolyl pyridine core. We demonstrate that lactazoles originate from the simplest cluster, containing only six unidirectional genes (lazA to lazF). We show that lazC is involved in the macrocyclization process, leading to central pyridine moiety formation. Substitution of the endogenous promoter with a strong promoter results in an approximately 30-fold increase in lactazole A production and mutagenesis of lazC precursor gene in production of two analogs. Lactazoles do not exhibit antimicrobial activity butxa0may modulate signaling cascades triggered by bone morphogenetic protein. Our approach facilitates the production of a more diverse set of thiopeptide structures, increasing the semisynthetic repertoire for use in drug development.

Insights into the biosynthesis of dehydroalanines in goadsporin

Dehydroalanines in goadsporin are proposed to be formed by GodF and GodG, which show slight homology to the N‐terminal glutamylation and C‐terminal elimination domains, respectively, of LanB, a classu2005I lanthipeptide dehydratase. Although similar, separated‐type LanBs are conserved among thiopeptides and indispensable for their biosynthesis and biological activities, these enzymes had not yet been characterized. Here, we identified goadsporinu2005B, which has unmodified Ser4 and Ser14, from both godF and godG disruptants. The godG disruptant also produced goadsporinu2005C, a glutamylated‐Ser4 variant of goadsporinu2005B. These results suggested that dehydroalanines are formed by glutamylation and glutamate elimination. NMR analysis revealed for the first time that the glutamyl group was attached to a serine via an ester bond, by the catalysis of LanB‐type enzymes. Our findings provide insights into the function of separated‐type LanBs involved in the biosynthesis of goadsporin and thiopeptides.

Identification of the n-1 fatty acid as an antibacterial constituent from the edible freshwater cyanobacterium Nostoc verrucosum

The cyanobacterium Nostoc verrucosum occurs in cool, clear streams and its gelatinous colonies, called “ashitsuki,” have been eaten in ancient Japan. Its ethanolic extract was found to inhibit the growth of Gram-positive bacteria and activity-guided fractionation yielded an unusual n-1 fatty acid, (9Z,12Z)-9,12,15-hexadecatrienoic acid (1), as one of the active principles. It inhibited the growth of Staphylococcus aureus at MIC 64u2009μg/mL.

Sacrolide A, a new antimicrobial and cytotoxic oxylipin macrolide from the edible cyanobacterium Aphanothece sacrum.

Summary Macroscopic gelatinous colonies of freshwater cyanobacterium Aphanothece sacrum, a luxury ingredient for Japanese cuisine, were found to contain a new oxylipin-derived macrolide, sacrolide A (1), as an antimicrobial component. The configuration of two chiral centers in 1 was determined by a combination of chiral anisotropy analysis and conformational analysis of different ring-opened derivatives. Compound 1 inhibited the growth of some species of Gram-positive bacteria, yeast Saccharomyces cerevisiae and fungus Penicillium chrysogenum, and was also cytotoxic to 3Y1 rat fibroblasts. Concern about potential food intoxication caused by accidental massive ingestion of A. sacrum was dispelled by the absence of 1 in commercial products. A manual procedure for degrading 1 in raw colonies was also developed, enabling a convenient on-site detoxification at restaurants or for personal consumption.

Wenyingzhuangia gracilariae sp. nov., a novel marine bacterium of the phylum Bacteroidetes isolated from the red alga Gracilaria vermiculophylla.

A Gram-negative, strictly aerobic, beige-pigmented, non-motile, rod-shaped bacterial strain designated N5DB13-4T was isolated from the red alga Gracilaria vermiculophylla (Rhodophyta) collected at Sodegaura Beach, Chiba, Japan. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the novel isolate is affiliated with the family Flavobacteriaceae within the phylum Bacteroidetes and that it showed highest sequence similarity (97.3xa0%) to Wenyingzhuangia heitensis H-MN17T. The hybridization values for DNA–DNA relatedness between the strains N5DB13-4T and W. heitensis H-MN17T were 34.1xa0±xa03.5xa0%, which is below the threshold accepted for the phylogenetic definition of a novel prokaryotic species. The DNA G+C content of strain N5DB13-4T was determined to be 31.8xa0mol%; MK-6 was identified as the major menaquinone; and the presence of iso-C15:0, iso-C15:0 3-OH and iso-C17:0 3-OH as the major (>10xa0%) cellular fatty acids. A complex polar lipid profile was present consisting of phosphatidylethanolamine, two unidentified glycolipids and four unidentified lipids. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel species of the genus Wenyingzhuangia for which the name Wenyingzhuangia gracilariae sp. nov. is proposed. The type strain of W. gracilariae sp. nov. is N5DB13-4T (=KCTC 42246 T=NBRC 110602T).

Linfuranones B and C, Furanone-Containing Polyketides from a Plant-Associated Sphaerimonospora mesophila

Two new furanone-containing polyketides, linfuranones B and C, were isolated from a plant-associated actinomycete of the genus Sphaerimonospora. Their structures were determined by NMR and MS spectroscopic analyses, and the absolute configurations were established by anisotropic methods and chemical degradation approaches. In silico analysis of biosynthetic genes suggested that linfuranone B is generated from linfuranone C by oxidative cleavage of the polyketide chain. Linfuranones B and C induced preadipocyte differentiation into matured adipocytes at 20-40 μM without showing cytotoxicity.

Ascidiimonas aurantiaca gen. nov., sp. nov., a member of Flavobacteriaceae isolated from a sea squirt.

A Gram-stain negative, strictly aerobic, chemoheterotrophic, tangerine orange pigmented, curved-rod shaped bacterium, designated N5DA8-2CT, was isolated from a sea squirt by use of a bait-streaked agar technique. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the novel marine strain is affiliated with the family Flavobacteriaceae of the phylum Bacteroidetes and that it shared high (92.6xa0%) sequence similarity with Frondibacter aureus A5Q-67T. The strain could be differentiated phenotypically from the related members of the family Flavobacteriaceae. The major fatty acids of strain N5DA8-2CT were iso-C15:1 G, iso-C17:0 3-OH, iso-C15:0 and C16:1 ω7c and/or C16:1 ω6c. A polar lipid profile was present consisting of phosphatidylethanolamine and three unidentified amino lipids. The DNA G+C content of the strain was determined to be 41xa0mol% and the major respiratory quinone was identified as menaquinone 6 (MK-6). From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel genus in the family Flavobacteriaceae, for which the name Ascidiimonas aurantiaca gen. nov., sp. nov. is proposed. The type strain of A. aurantiaca gen. nov., sp. nov. is N5DA8-2CT (=xa0KCTC 32992Txa0= NBRC 110020T).

Sarmentosamide, a novel hexadienamide from Thai soil actinomycetes

A new hexadienamide derivative named sarmentosamide (1) was identified from the culture of Streptomyces sp. SBI108 isolated from Thai soil under an herb. The structure was elucidated on the basis of spectroscopic data, and the absolute configuration was determined by chemical degradation.