Antitumor compounds from Streptomyces sp. KML-2, isolated from Khewra salt mines, Pakistan

BACKGROUND: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds. RESULTS: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemiasalina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus. CONCLUSIONS: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.

Purification and Structure Elucidation of the Polyether Antibiotic Alborixin from Streptomyces pulcher CRF17.

Aims: To screen Streptomyces sp. from saline soil in Pakistan, for its antimicrobial activity and to purify and identify the active metabolites produced by mass spectrometry and NMR spectroscopy. Strain identification by morphological, biochemical, physiological characterization and by 16S rRNA gene sequencing. Study Design: Cultivation in lab fermenter, solvent extraction and purification of the compounds by column chromatography, identification of the compounds by mass spectrometry and NMR spectroscopy, determination of the antimicrobial activity and cytotoxicity. Place and Duration of Study: Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan and Institute of Organic and Bio molecular Chemistry, University of Göttingen, Germany, between February, 2007 and April, 2009. Methodology: The strain was cultivated in a 20 liter fermenter (working volume 10 liters) and the culture broth was extracted with ethyl acetate, acetone and methanol. The resultant crude extract was fractionated on silica gel and the components were purified by column chromatography (silica gel, sephadex column and preparative TLC). The pure component was identified by mass spectrometry (ESI and HRESI-MS), NMR analysis (1H and 13C NMR) and by comparison with reference data, the antimicrobial activity and cytotoxicity was determined by disc diffusion assay and by micro well cytotoxicity assay against Artimia salina. Results: The morphological, biochemical and physiological characterization suggested that isolate CRF17 belongs to the genus Streptomyces. Further NCBI BLAST of the partial 16S rDNA gene sequence 1420 bp (gene bank accession number: EU294134) from the isolate CRF17 showed 99% identity and 98% query coverage towards Streptomyces pulcher. The scale up fermentation of the isolate CRF17 yielded active compound and was identified as alborixin (1). Conclusion: The isolate Streptomyces pulcher CRF17 is a potent producer of the antibiotic alborexin and can be exploited for its commercial production.

Identification, isolation and optimization of antifungal metabolites from the Streptomyces Malachitofuscus ctf9

An indigenous Streptomyces isolate CTF9, exhibiting promising antifungal activity against Mucor miehei and Candida albicans in pre-screening studies, was investigated by cultivation in a 50-L fermenter and by subsequent isolation, purification, and structure elucidation of the active metabolites. Based on the morphological, biochemical, and physiological characterization, as well as the 16S rRNA gene sequence, the isolate CTF9 was identified as Streptomyces malachitofuscus. Using a series of chromatographic techniques, two pure compounds were isolated from the obtained extracts after the fermentation of the isolate CTF9. The isolated compounds were identified as phenylacetic acid and indolyl-3-lactic acid by mass spectrometry (MS) and NMR analysis. The culture optimization studies revealed that the isolate CTF9 can use a variety of low-cost carbon and nitrogen sources to generate the maximum quantity of industrially important metabolites at an elevated temperature of 35°C and at a pH 7.8.