Identification and taxonomy of Streptomyces justiciae strain RA-WS2: a novel setomimycin producing actinobacterium.

The taxonomic position of novel bianthraquinone antibiotic producer Streptomyces strain RA-WS2, a soil isolate from Shivalik region of NW Himalayas, India, has been described. The isolate produces Setomimycin as a major secondary metabolite under defined submerged fermentation conditions. 16S rRNA partial gene sequencing of the isolate indicated its closest similarity (99.4%) with Streptomyces cyaneochromogenes, followed by Streptomyces aquilus. However, the morphological characteristics i.e. colony colour, mycelium and spore chain arrangement were found to be close to Streptomyces aquilus. Therefore, a polyphasic approach was used for taxonomic positioning of the isolate. The Whole genome based similarity with 88.4% dDDH value, 98.65% ANI and 96.99% AAI value indicated its closest identity with Streptomyces justiciae. The taxonomic characteristics such as white colony with smooth surface, cylindrical spores arranged in straight chain, diffusible melanin production, high salt tolerance, 16S rRNA gene sequencing and phylogenomic studies, led to the identification of the strain as Streptomyces justiciae RA-WS2. The predicted biosynthetic gene clusters further confirmed the presence of the BGC for setomimycin biosynthesis in Streptomyces justiciae strain RA-WS2. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03459-5.

Isolation and anticancer activity evaluation of rare Bisaryl anthraquinone antibiotics from novel Streptomyces sp. strain of NW Himalayan region.

Biosynthesis of bisaryl preanthraquinone antibiotics by various microorganisms differs in monomeric subunits as well as their dimerization positions leading to different configurations. The present study relates to the production of rare bisaryl anthraquinone antibiotics by a new Streptomyces strain isolated from Shivalik region of NW Himalayas. In vitro anticancer and anti-migratory effects of Setomimycin (9,9' bisanthraquinone antibiotic) was seen with a significant reduction in the expression of both MEK as well as ERK pathways in a dose dependent manner at 6.5 µM & 8 µM concentration in HCT-116 and 5.5 µM & 7 µM concentration in MCF-7 cells. In vivo studies in aggressive orthotopic mouse mammary carcinoma model (4T1) demonstrated about 76% reduction of primary tumor weight and 90.5% reduction in the tumor volume within two weeks. In vivo pharmacokinetics study of setomimycin revealed that it can be rapidly absorbed with an adequate plasma exposure and half-life which can be linked to its in vivo efficacy.

Discovery of a Secalonic Acid Derivative from Aspergillus aculeatus, an Endophyte of Rosa damascena Mill., Triggers Apoptosis in MDA-MB-231 Triple Negative Breast Cancer Cells.

A new secalonic acid derivative, F-7 (1), was isolated from the endophytic Aspergillus aculeatus MBT 102, associated with Rosa damascena. The planar structure of 1 was established on the basis of 1D and 2D NMR and ESI-TOF-MS spectra. The relative configuration of 1 was determined applying a combined quantum mechanical/NMR approach and, afterward, the comparison of calculated and experimental electronic circular dichroism spectra determined the assignment of its absolute configuration. The compound possesses strong cytotoxic activity against triple negative breast cancer (TNBC) cells. It was found to induce apoptosis, as evidenced by scanning electron microscopy and phase contrast microscopy. Furthermore, flow cytometry analyses demonstrated that 1 induced mitochondrial damage and reactive oxygen species mediated apoptosis, arresting the G1 phase of the cells in a dose-dependent manner. Also, the compound causes significant microtubule disruption in TNBC cells. Subsequently, 1 restricted the cell migration leading to the concomitant increase in expression of cleaved caspase and PARP.

Lipovelutibols A-D: Cytotoxic Lipopeptaibols from the Himalayan Cold Habitat Fungus Trichoderma velutinum.

Four novel lipovelutibols A (1), B (2), C (3), and D (4) containing six amino acid residues with leucinol at the C-terminus and a fatty acyl moiety (n-octanoyl) at its N-terminus were isolated from the psychrotrophic fungus Trichoderma velutinum collected from the Himalayan cold habitat. The structures (1-4) were determined by NMR and MS/MS, and the stereochemistry of amino acids by Marfey's method. Lipopeptaibols 2 and 4 were found to contain d-isovaline, a nonproteinogenic amino acid, but lacked α-aminoisobutyric acid, characteristic of peptaibols. Cytotoxic activity of 2 and 4 was observed against HL-60, LS180, MDA-MB-231, and A549 cancer cell lines.

Revelation and cloning of valinomycin synthetase genes in Streptomyces lavendulae ACR-DA1 and their expression analysis under different fermentation and elicitation conditions.

Streptomyces species are amongst the most exploited microorganisms due to their ability to produce a plethora of secondary metabolites with bioactive potential, including several well known drugs. They are endowed with immense unexplored potential and substantial efforts are required for their isolation as well as characterization for their bioactive potential. Unexplored niches and extreme environments are host to diverse microbial species. In this study, we report Streptomyces lavendulae ACR-DA1, isolated from extreme cold deserts of the North Western Himalayas, which produces a macrolactone antibiotic, valinomycin. Valinomycin is a K+ ionophoric non-ribosomal cyclodepsipeptide with a broad range of bioactivities including antibacterial, antifungal, antiviral and cytotoxic/anticancer activities. Production of valinomycin by the strain S. lavendulae ACR-DA1 was studied under different fermentation conditions like fermentation medium, temperature and addition of biosynthetic precursors. Synthetic medium at 10°C in the presence of precursors i.e. valine and pyruvate showed enhanced valinomycin production. In order to assess the impact of various elicitors, expression of the two genes viz. vlm1 and vlm2 that encode components of heterodimeric valinomycin synthetase, was analyzed using RT-PCR and correlated with quantity of valinomycin using LC-MS/MS. Annelid, bacterial and yeast elicitors increased valinomycin production whereas addition of fungal and plant elicitors down regulated the biosynthetic genes and reduced valinomycin production. This study is also the first report of valinomycin biosynthesis by Streptomyces lavendulae.

Optimization of nonribosomal peptides production by a psychrotrophic fungus: Trichoderma velutinum ACR-P1.

Trichoderma is an anamorphic filamentous fungal genus with immense potential for production of small valuable secondary metabolites with indispensable biological activities. Microbial dynamics of a psychrotrophic strain Trichoderma velutinum ACR-P1, isolated from unexplored niches of the Shiwalik region, bestowed with rich biodiversity of microflora, was investigated for production of nonribosomal peptides (NRPs) by metabolite profiling by intact-cell mass spectrometry (ICMS) employing matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer. Being the first report on NRPs production by T. velutinum, studies on optimization of growth conditions by Response Surface Methodology (RSM) for production of NRPs by ACR-P1 was carried out strategically. Multifold enhancement in the yield of NRPs belonging to subfamily SF4 with medium chain of amino acid residues having m/z 1437.9, 1453.9, and 1452.0 at pH 5.9 at 20 °C and of subfamily SF1 with long-chain amino acid residues having m/z 1770.2, 1784.2, 1800.1, 1802.1, and 1815.1 was achieved at pH 7.0 at 25 °C. Complexities of natural mixtures were thus considerably reduced under respective optimized culture conditions accelerating the production of novel microbial natural products by saving time and resources.

Secondary Metabolites from Endophytic Fungus Penicillium pinophilum Induce ROS-Mediated Apoptosis through Mitochondrial Pathway in Pancreatic Cancer Cells.

The endophytic fungus strain MRCJ-326, isolated from Allium schoenoprasum, which is also known as Snow Mountain Garlic or Kashmiri garlic, was identified as Penicillium pinophilum on the basis of morphological characteristics and internal transcribed spacer region nucleotide sequence analysis. The endophytic fungus extract was subjected to 2D-SEPBOX bioactivity-guided fractionation and purification. The anthraquinone class of the bioactive secondary metabolites were isolated and characterized as oxyskyrin (1), skyrin (2), dicatenarin (3), and 1,6,8-trihydroxy-3-hydroxy methylanthraquinone (4) by spectral analysis. Dicatenarin and skyrin showed marked growth inhibition against the NCI60/ATCC panel of human cancer cell lines with least IC50 values of 12 µg/mL and 27 µg/mL, respectively, against the human pancreatic cancer (MIA PaCa-2) cell line. The phenolic hydroxyl group in anthraquinones plays a crucial role in the oxidative process and bioactivity. Mechanistically, these compounds, i.e., dicatenarin and skyrin, significantly induce apoptosis and transmit the apoptotic signal via intracellular reactive oxygen species generation, thereby inducing a change in the mitochondrial transmembrane potential and induction of the mitochondrial-mediated apoptotic pathway. Our data indicated that dicatenarin and skyrin induce reactive oxygen species-mediated mitochondrial permeability transition and resulted in an increased induction of caspase-3 apoptotic proteins in human pancreatic cancer (MIA PaCa-2) cells. Dicatenarin showed a more pronounced cytotoxic/proapopotic effect than skyrin due to the presence of an additional phenolic hydroxyl group at C-4, which increases oxidative reactive oxygen species generation. This is the first report from P. pinophilum secreating these cytotoxic/proapoptotic secondary metabolites.

Application of electrochemically prepared polypyrrole-polyvinyl sulphonate films to DNA biosensor.

Double stranded calf thymus deoxyribonucleic acid (DNA) was physisorbed onto polypyrrole-polyvinyl sulphonate (PPY-PVS) films electrochemically deposited onto indium-tin-oxide (ITO) coated glass plates. These DNA immobilized PPY-PVS films optimized for various conditions, such as polymerization potential, pH of buffer, DNA concentration and scan rate were characterized using Fourier-transform infrared (FT-IR) spectroscopy, atomic force microscopy (AFM) and cyclic voltammetry (CV) techniques, respectively. The amperometric response studies of these DNA/PPY-PVS electrodes were carried out as a function of 2-aminoantharcene (2-AA, 0.01-20 ppm) and o-chlorophenol (OCP, 0.1-30 ppm) concentration, respectively at 25 degrees C. The observed amperometric current arising due to oxidation of guanine in the DNA/PPY-PVS films decreased linearly with the increase in the concentration of 2-AA and OCP. It has been revealed that 10 ppm of 2-AA is sufficient to reduce the observed guanine oxidation peak current by approximately -95+/-10% as compared to the reported values. A 25 ppm of OCP was capable enough to reduce the guanine oxidation current to zero. These DNA/PPY-PVS electrodes were found to have a shelf life of about 4 months when stored at 25 degrees C.