Yōko Takahashi

Spoxazomicins A–C, novel antitrypanosomal alkaloids produced by an endophytic actinomycete, Streptosporangium oxazolinicum K07-0460 T

Three novel antitrypanosomal alkaloids, named spoxazomicins A–C, were isolated by silica gel column chromatography and HPLC from the culture broth of a new endophytic actinomycete species, Streptosporangium oxazolinicum K07-0460T. The structures of the spoxazomicins were elucidated by NMR and X-ray crystal analyses and shown to be new types of pyochelin family antibiotic. Spoxazomicin A showed potent and selective antitrypanosomal activity with an IC50 value of 0.11 μg ml−1 in vitro without cytotoxicity against MRC-5 cells (IC50=27.8 μg ml−1).

Phytohabitans suffuscus gen. nov., sp. nov., an actinomycete of the family Micromonosporaceae isolated from plant roots.

An actinomycete strain, K07-0523(T), was isolated from the roots of an orchid collected in Okinawa prefecture, Japan. 16S rRNA gene sequence analysis indicated that the new strain belonged to the family Micromonosporaceae and the similarity values between strain K07-0523(T) and the type species of 24 genera in the family Micromonosporaceae were 93.3-97.7 %. Strain K07-0523(T) contained D-glutamic acid, glycine, D-alanine, meso-diaminopimelic acid, hydroxydiaminopimelic acid and L-lysine in the cell wall. The major menaquinones were MK-9(H(6)), MK-10(H(4)) and MK-10(H(6)). Galactose, glucose, mannose, ribose and xylose were present in the whole-cell sugars. The acyl type of the peptidoglycan was glycolyl. Major fatty acids were anteiso-C(17 : 0), iso-C(17 : 0), iso-C(16 : 0) and iso-C(15 : 0). Phosphatidylethanolamine was detected as the major phospholipid and corresponded to phospholipid type II. The G+C content of the genomic DNA was 73 mol%. On the basis of phylogenetic and chemotaxonomic data, the new strain represents a member of a new genus and novel species, namely Phytohabitans suffuscus gen. nov., sp. nov., in the family Micromonosporaceae. The type strain of the type species is K07-0523(T) (=DSM 45306(T)=NBRC 105367(T)).

Trehangelins A, B and C, novel photo-oxidative hemolysis inhibitors produced by an endophytic actinomycete, Polymorphospora rubra K07-0510

Three new natural products, designated trehangelins A, B and C, were isolated by solvent extraction, silica gel and octadecylsilyl silica gel column chromatographies and subsequent preparative HPLC from the cultured broth of an endophytic actinomycete strain, Polymorphospora rubra K07-0510. The trehangelins consisted of a trehalose moiety and two angelic acid moieties. Trehangelins A (IC50 value, 0.1 mg ml−1) and C (IC50 value, 0.4 mg ml−1), with symmetric structures, showed potent inhibitory activity against hemolysis of red blood cells induced by light-activated pheophorbide a. However, trehangelin B, with an asymmetric structure, displayed only a slight inhibition (IC50 value, 1.0 mg ml−1).

Mangromicins A and B: structure and antitrypanosomal activity of two new cyclopentadecane compounds from Lechevalieria aerocolonigenes K10-0216.

Two new cyclopentadecane antibiotics, named mangromicins A and B, were separated out from the culture broth of Lechevalieria aerocolonigenes K10-0216 by Diaion HP-20, silica gel and ODS column chromatography, and were finally purified by HPLC. The chemical structures of the two novel compounds were elucidated by instrumental analyses, including various NMR, MS and X-ray crystallography. Mangromicins A and B consist of cyclopentadecane skeletons with a tetrahydrofuran unit and a 5,6-dihydro-4-hydroxy-2-pyrone moiety. Mangromicins A and B showed in vitro antitrypanosomal activity with IC50 values of 2.4 and 43.4 μg ml−1, respectively. The IC50 values of both compounds were lower than those of cytotoxicity against MRC-5 human fetal lung fibroblast cells.

Mangromicins, six new anti-oxidative agents isolated from a culture broth of the actinomycete, Lechevalieria aerocolonigenes K10-0216

We have been continually searching for novel chemical compounds from culture broths of various actinomycetes using a physicochemical screening system. During the course of this program, we have previously reported the discovery of two new natural products, designated mangromicins A and B, discovered in a broth of a rare actinomycete strain, Lechevalieria aerocolonigenes K10-0216. Mangromicins have a unique and rare structure, a cyclopentadecane skeleton with a tetrahydrofuran unit and a 5,6-dihydro-4-hydroxy-2-pyrone moiety. New mangromicin analogs were isolated by using an improved production medium. As a consequence, six analogs, together with mangromicins A and B, were isolated from a cultured broth of L. aerocolonigenes K10-0216. We named them mangromicins D, E, F, G, H and I. All mangromicins showed radical scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals and nitric oxide generated from LPS-stimulated RAW264.7 cells, a murine macrophage cell line. Among the analogs, mangromicins A and I showed the most potent DPPH radical scavenging activity and nitric oxide scavenging activity, respectively.

Streptomyces lactacystinicus sp. nov. and Streptomyces cyslabdanicus sp. nov., producing lactacystin and cyslabdan, respectively

Actinomycete strains OM-6519T and K04–0144T produce the bioactive compounds lactacystin and cyslabdan, respectively. Here, the taxonomic positions of these two strains were determined. The morphological and chemical features of strains OM-6519T and K04–0144T indicated that they belonged to the genus Streptomyces. Strain OM-6519T showed the highest 16S rRNA gene sequence similarities with Streptomyces xanthocidicus NBRC 13469T (99.7%), Streptomyces chrysomallus subsp. fumigatus NBRC 15394T (99.6%) and Streptomyces aburaviensis NRRL B-2218T (99.5%). However, the DNA–DNA relatedness values between strain OM-6519T and the three related strains were below 70%. Strain K04–0144T showed the highest 16S rRNA gene sequence similarities with Streptomyces corchorusii NBRC 13032T (99.4%), Streptomyces olivaceoviridis NBRC 15394T (99.4%) and Streptomyces canarius NRRL B-2218T (99.3%). However, the DNA–DNA relatedness values between strain K04–0144T and the three related strains were also below 70%. Based on morphological, cultural and physiological characteristics and DNA–DNA relatedness data, strains OM-6519T and K04–0144T should be classified as new species of the genus Streptomyces, for which the names Streptomyces lactacystinicus sp. nov. and Streptomyces cyslabdanicus sp. nov. are proposed. The type strain of S. lactacystinicus is OM-6519T (=NBRC 110082T, DSM 43136T). The type strain of S. cyslabdanicus is K04–0144T (=NBRC 110081T, DSM 42135T).

Mangromicin C, a new analog of mangromicin.

Mangromicin analogs were discovered in a cultured broth of a rare actinomycete, Lechevalieria aerocolonigenes K10-0216.1,2 The mangromicin analogs showed antitrypanosomal and reactive oxygen species (ROS) scavenging bioactivities. Mangromicin analogs have unique structures, including common partial structures, notably a cyclopentadecane skeleton with a tetrahydrofuran unit and a 5,6-dihydro-4-hydroxy-2-pyrone moiety. In a previous report, we described eight new mangromicin analogs, which possessed anti-oxidative properties. At that time, we discovered mangromicin C (1) but were unable to determine its structure, which was clearly different from all the other analogs. We have now found that 1 has a structure which includes a tetrahydropyran ring (Figure 1). In this paper, we report the fermentation, isolation, structural elucidation and biological activity of 1.

Endophytic actinomycetes: promising source of novel bioactive compounds

Endophytic actinomycetes associated with plant roots are a relatively untapped source of potential new bioactive compounds. This is becoming increasingly important, as the returns from discovery research on soil-dwelling microbes, have been continuously diminishing. We have isolated more than 1000 strains of actinomycetes from plant roots in our search for novel bioactive compounds, identified and assayed their bioactive metabolites, as well as investigated their biosynthetic genes for generating secondary metabolites. This has resulted in the discovery of several interesting compounds. Creation of plant root clone libraries enabled us to confirm that we had, indeed, isolated endophytes. In this paper, we introduce our approach to this promising line of research, incorporating data from other publications, and illustrate the potential that endophytic actinomycetes offer as a new source of novel lead compounds.

Actinomycetes, an Inexhaustible Source of Naturally Occurring Antibiotics

Global public health faces a desperate situation, due to the lack of effective antibiotics. Coordinated steps need to be taken, worldwide, to rectify this situation and protect the advances in modern medicine made over the last 100 years. Work at Japan’s Kitasato Institute has been in the vanguard of many such advances, and work is being proactively tailored to promote the discovery of urgently needed antimicrobials. Efforts are being concentrated on actinomycetes, the proven source of most modern antibiotics. We devised a novel physicochemical screening mechanism, whereby simple physico-chemical properties, in conjunction with related detection methods, such as LC/MS, LC/UV, and polarity, could be used to identify or predict new compounds in a culture broth, simply by comparing results with existing databases. New compounds are isolated, purified, and their structure determined before being tested for any bioactivity. We used lyophilized actinomycete strains from the Kitasato Microbial Library, most more than 35 years old, and found 330 strains were producers of useful bioactive substances. We also tested organisms found in fresh samples collected in the complex environments from around plant roots, as well as from sediments of mangrove forests and oceans, resulting in the discovery of 36 novel compounds from 11 actinomycete strains. A compound, designated iminimycin, containing an iminium ion in the structure was discovered from the culture broth of Streptomyces griseus OS-3601, which had been stored for a long time as a streptomycin-producing strain. This represented the first iminium ion discovery in actinomycetes. Compounds with a cyclopentadecane skeleton containing 5,6-dihydro-4-hydroxyl-2-pyrone ring and tetrahydrofuran ring, designated mangromicins, were isolated from the culture broth of Lechevalieria aerocolonigenes K10-0216 obtained from sediment in a mangrove forest. These structures are extremely unique among natural compounds. From the same culture broth, new steroid compounds, named K10-0216 KA and KB, and other new compounds having a thiazole and a pyridine ring, named pyrizomicin A and B, were discovered. New substances can be found from actinomycetes that have been exhaustively studied. Novel compounds with different skeletons can be found from a single broth of one strain. The sought after new antibiotics will arise from continued exploitation of the actinomycetes, especially rare actinomycetes. Work on new organisms and samples should be augmented by re-examination of known actinomycetes already in storage. New research should also be carried out on the manipulation of culture media, thereby stimulating actinomycete strains to produce novel chemicals. The establishment of wide-ranging international research collaborations will facilitate and expedite the efficient and timely discovery and provision of bioactive compounds to help maintain and promote advances in global public health.

Actinokineospora bangkokensis sp. nov., isolated from rhizospheric soil.

A novel actinomycete, strain 44EHW(T), was isolated from rhizospheric soil under an Elephant ear plant (Colocasia esculenta) in Bangkok, Thailand. Strain 44EHW(T) produced long branching hyphae and abundant aerial mycelia with chains of rod-shaped spores. Whole-cell hydrolysates contained galactose, glucose, arabinose, ribose, mannose and rhamnose as diagnostic sugars. meso-Diaminopimelic acid was the diamino acid and glycine, alanine and glutamic acid were present in the cell-wall peptidoglycan with the acyl type of the peptidoglycan being acetyl. Phospholipids consisted of phosphatidylethanolamine, phosphatidylethanolamine with hydroxy fatty acids and diphosphatidylglycerol, as well as other unknown phospholipids; however, no mycolic acids were detected. The predominant menaquinone observed was MK-9(H4) and major fatty acids were iso-C16 : 0 and 2-OH iso-C16 : 0. The G+C content of genomic DNA was 74 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that this isolate was most similar to Actinokineospora enzanensis NBRC 16517(T). However, DNA-DNA hybridization revealed a low relatedness between this isolate and A. enzanensis NBRC 16517(T), indicating that this isolate represented a novel species in the genus Actinokineospora. On the basis of 16S rRNA gene sequence analysis, phenotypic characteristics and DNA-DNA hybridization data, we propose that strain 44EHW(T) represents a novel species in the genus Actinokineospora, Actinokineospora bangkokensis. The type strain is 44EHW(T) ( = BCC 53155(T) = NBRC 108932(T)).