In-silico and in-vitro evaluation of antifungal bioactive compounds from Streptomyces sp. strain 130 against Aspergillus flavus.

Streptomyces spp. are considered excellent reservoirs of natural bioactive compounds. The study evaluated the bioactive potential of secondary metabolites from Streptomyces sp. strain 130 through PKS-I and NRPS gene-clusters screening. GC-MS analysis was done for metabolic profiling of bioactive compounds from strain 130 in the next set of experiments. Identified antifungal compounds underwent ADMET analyses to screen their toxicity. All compounds' molecular docking was done with the structural gene products of the aflatoxin biosynthetic pathway of Aspergillus flavus. MD simulations were utilized to evaluate the stability of protein-ligand complexes under physiological conditions. Based on the in-silico studies, compound 2,4-di-tert butyl-phenol (DTBP) was selected for in-vitro studies against Aspergillus flavus. Simultaneously, bioactive compounds were extracted from strain 130 in two different solvents (ethyl-acetate and methanol) and used for similar assays. The MIC value of DTBP was found to be 314 µg/mL, whereas in ethyl-acetate extract and methanol-extract, it was 250 and 350 µg/mL, respectively. A mycelium growth assay was done to analyze the effect of compounds/extracts on the mycelium formation of Aspergillus flavus. In agar diffusion assay, zone of inhibitions in DTBP, ethyl-acetate extract, and methanol extract were observed with diameters of 11.3, 13.3, and 7.6 mm, respectively. In the growth curve assay, treated samples have delayed the growth of fungi, which signified that the compounds have a fungistatic nature. Spot assay has determined the fungal sensitivity to a sub-minimum inhibitory concentration of antifungal compounds. The study's results suggested that DTBP can be exploited for antifungal-drug development.Communicated by Ramaswamy H. Sarma.

Natural products from Streptomyces spp. as potential inhibitors of the major factors (holoRdRp and nsp13) for SARS-CoV-2 replication: an in silico approach.

The COVID-19 pandemic caused unprecedented damage to humanity, and while vaccines have been developed, they are not fully effective against the SARS-CoV-2 virus. Limited targeted drugs, such as Remdesivir and Paxlovid, are available against the virus. Hence, there is an urgent need to explore and develop new drugs to combat COVID-19. This study focuses on exploring microbial natural products from soil-isolated bacteria Streptomyces sp. strain 196 and RI.24 as a potential source of new targeted drugs against SARS-CoV-2. Molecular docking studies were performed on holoRdRp and nsp13, two key factors responsible for virus replication factor. Our in silico studies, K-252-C aglycone indolocarbazole alkaloid (K252C) and daunorubicin were found to have better binding affinities than the respective control drugs, with K252C exhibiting binding energy of - 9.1 kcal/mol with holoRdRp and - 9.2 kcal/mol with nsp13, and daunorubicin showing binding energy at - 8.1 kcal/mol with holoRdRp and - 9.3 kcal/mol with nsp13. ADMET analysis, MD simulation, and MM/GBSA studies indicated that K252C and daunorubicin have the potential to be developed as targeted drugs against SARS-CoV-2. The study concludes that K252C and daunorubicin are potential lead compounds that might suppress the inhibition of SARS-CoV-2 replication among the tested microbial compounds and could be developed as targeted drugs against COVID-19. In the future, further in vitro studies are required to validate these findings.

Heat and water flux modeling in an earth dam.

This study aims to identify the water flux in an earth dam using heat flux due to convection. Sixteen earth dam models were constructed in a hydraulic flume by varying geometrical and flow input parameters to identify heat and water flux. Homogeneous as well as earth dams with clay cores were built in a hydraulic flume. Temperature measurements were done to calculate heat flux in the experimental model. A finite element model of the earth dam using Seep/w was developed to obtain water flux, while temp/w was used to obtain heat flux. These results were used as input in Temp/w and Seep/w in Geostudio 2020. Significant reduction of the heat and water flux was seen while comparing the homogeneous models with central impervious core models. An increase in the heat and water flux was observed on increasing the downstream filter's length, longitudinal slope, and vice versa with the upstream slope and the thickness of the clay core. Comparing fluxes in a homogeneous dam model (model 1) with the clay core model (model 9) with top width 2.4 m and bottom width 18 m in model 9, both water flux and heat flux were reduced by 78.46%. While comparing it with model 10, with bottom core width of 18 m and top core width of 1.9 m, both water flux and heat flux reduced by 77.72%. Heat flux measurements were found to be a valuable alternative to detecting water flux and seepage in an earth dam at a reduced cost.

In vitro and in silico anticancer potential analysis of Streptomyces sp. extract against human lung cancer cell line, A549.

During our previous investigation, bioactive compounds present in the extract of Streptomyces sp. strain 196 were characterized using LC-MS/MS and 1H NMR studies. These compounds were K-252-C aglycone indolocarbazole alkaloid, decoyinine, and cycloheximide; the study of these natural drugs against lung carcinoma is still limited. Focus of the current investigation was to study the anticancer effect of strain 196 extract on lung cancer cells (A549). During in vitro studies, anti-proliferative effect of extract was studied using MTT assay in A549 cells. Effect of extract on cell survival was further evaluated using colony assay. Cell death was qualitatively assessed using apoptosis assay. The aftereffect of extract treatment on metastatic potential of cancerous cells was studied using wound closure assay. Effect of extract on the morphology and cytoskeletal arrangement of A549 cells was studied using phalloidin staining. The extract demonstrated concentration and time-dependent cytotoxicity with IC50 value at 0.5 mg/ml (6 h) and 0.15 mg/ml (24 h). The proliferation and metastatic potential of cells, as characterized by MTT and migration assay, decreased over time in a concentration-dependent manner. Discrete changes in cellular morphology were noted as a result of the induced cytotoxicity. Apoptosis assay demonstrated 98.7% cell death at highest concentration of extract (1 mg/ml). During in silico studies, molecular docking revealed that strain 196 compounds are efficiently binding to mutant EGFR form (T790M/L858R) with release of binding energy (∆G) between - 5 and - 6.9 kcal/Mol. In conclusion, strain 196 extract could be a source of therapeutic drugs to treat lung carcinoma.

Protection of surplus food from fungal spoilage using Streptomyces spp.: a green approach.

Consortia of Streptomyces spp. (colonies 169, 194, 165 and 130) used in this study are an efficient producer of secondary metabolites like chitinases and antifungal compounds, which may help in the protection of surplus food from spoilage. Qualitative screening for chitinase production and taxonomy of these colonies were undertaken in our previous studies. In the current study, GC-MS analysis of extract produced from the consortia of Streptomyces strains was done for the identification of antifungal compounds. Treatment of surplus food with activated consortia of Streptomyces spp. has protected powdered food for a month, whereas fresh food (unpowdered) was preserved for two days. A control sample of surplus food (untreated) was kept to check the contamination, which resulted in the growth of three fungi (FP-1, FG-1, and FB-1). Taxonomic characterization of fungi and identification of toxic compounds produced from them were done by ITS amplification and GC-MS analysis, respectively. The study shows that the secondary metabolites from Streptomyces spp. have the potential to protect the food from mycotoxin contamination. Based on literature reports, this is for the first time that bioactive compounds and chitinases produced from Streptomyces are being used for the protection and management of surplus food.

Draft genome and secondary metabolite biosynthetic gene clusters of Streptomyces sp. strain 196.

Emergence of MDR 'superbugs' inflamed a severe sense of urgency amongst scientists aiming at the discovery of novel potential drug molecules. Bacteria of the genus Streptomyces are really worth investigating for their immense potential to produce natural compounds of pharmaceutical importance. In the present study, the genome of Streptomyces sp. strain 196 was sequenced, studied and secondary metabolite biosynthetic gene clusters (smBGCs) were detected. FAME analysis was used for taxonomic validation of strain 196. Genome of strain 196 was sequenced using the Illumina NextSeq system which has resulted in a draft genome of 7.4 Mb. Rapid annotation using subsystem technology (RAST) results revealed the presence of 6682 CDS, 64 tRNA genes and 7 rRNA genes. Comparative studies revealed that strain 196 have 93.5% nucleotide and 96% protein level similarities with Streptomyces rhizosphaericola 1AS2c. Genome mining using antiSMASH predicted the presence of BGCs responsible for diverse bioactive compound production. The detected gene clusters were two PKS-III, one PKS-I, five NRPS, two hybrid PKS-I/NRPS, one thiopeptide/LAP, and one bacteriocin types. Furthermore, many other types BGCs such as three ectoine, two siderophore, one arylpolyene, two butyrolactone, one lassopeptide, one lanthipeptide and one melanin were also found. The results of this study provides information about genome and BGCs of strain 196, this information is valuable for researchers who are interested in isolation of bioactive compounds and working on heterologous expression of cryptic BGCs for novel bioactive compounds production.

Potential applications of extracellular enzymes from Streptomyces spp. in various industries.

Extracellular enzymes produced from Streptomyces have the potential to replace toxic chemicals that are being used in various industries. The endorsement of this replacement has not received a better platform in developing countries. In this review, we have discussed the impact of chemicals and conventional practices on environmental health, and the role of extracellular enzymes to replace these practices. Burning of fossil fuels and agriculture residue is a global issue, but the production of biofuel using extracellular enzymes may be the single key to solve all these issues. We have discussed the replacement of hazardous chemicals with the use of xylanase, cellulase, and pectinase in food industries. In paper industries, delignification was done by the chemical treatment, but xylanase and laccase have the efficient potential to remove the lignin from pulp. In textile industries, the conventional method includes the chemicals which affect the nervous system and other organs. The use of xylanase, cellulase, and pectinase in different processes can give a safe and environment-friendly option to textile industries. Hazardous chemical pesticides can be replaced by the use of chitinase as an insecticide and fungicide in agricultural practices.

Exploitation of potential bioactive compounds from two soil derived actinomycetes, Streptomyces sp. strain 196 and RI.24.

Due to emergence of drug resistant pathogens, nearly all available medicines are becoming ineffective against these life threatening pathogens so there is dire need for the discovery of compounds having unique modes of action. During our previous studies, actinomycetes designated as 196 and RI.24 were isolated, screened for bioactive compounds production and characterized using 16S rRNA gene sequencing. Colony 196 was identified as strain of Streptomyces albolongus (100% sequence similarity) and RI.24 as strain of Streptomyces enissocaesilis (100% sequence similarity). In current study, potential bioactive compounds produced by these strains were characterized. Cold extraction method was applied for taking out of bioactive compounds from actinomycetes. Minimum inhibitory concentration (MIC) determination of compounds from these strains showed activity nearly in the range of commercial antibiotics (strain 196 0.0075 mg/ml, RI.24 0.25 mg/ml and chloramphenicol 0.0075 mg/ml, ampicillin 0.025 mg/ml). Structural elucidation of these compounds was carried out using spectroscopic techniques of LC-MS/MS and 1H NMR. Compounds K-252-C-Aglycone, indolocarbazole alkaloid, decoyinine, cycloheximide were detected from strain 196 whereas daunorubicin, hygromycin B, agecorynin F, indinavir-N-glucuronide and minocycline were identified from strain RI.24.Current study reports these compounds for the first time from strains of Streptomyces albolongus and Streptomyces enissocaesilis. Present investigation also suggests that strains 196 and RI.24 contain polyketide synthase-I (PKS-I) and non-ribosomal peptide synthetase (NRPS) gene clusters which are responsible for the production of bioactive compounds. The results of this study can be used by the scientific world or pharmaceutical industries for the development of new drugs/formulations by applying more advanced techniques.

Draft genome of Streptomyces sp. strain 130 and functional analysis of extracellular enzyme producing genes.

Streptomyces sp. strain 130 possesses multiple uncharacterized extracellular enzyme producing genes. Enzymes from these genes may fulfil the intense demand of stable and effective extracellular enzymes in various industries. Taxonomy of Streptomyces sp. strain 130 was validated by FAME analysis. Strain 130 was screened for the presence of chitinase producing genes of family 18 and 19 using SC1F/SC2R and F19F2/F19R primer sets respectively. Whole genome sequencing was done using Illumina Next Seq 500 system. In the analysis of draft genome of Streptomyces sp. strain 130, the genome size was found to be 7.1 Mb. Blastn and NCBI-conserved domain search tool were used to find similarity percentage with genes in existing database and enzyme family respectively. Ten chitinase, six xylanase and one cellulase producing genes were present in draft genome. Among the ten chitinase producing genes, two were belonging to GH19 family and other eight to GH18 family chitinase. Six out of ten chitinase producing genes were uncharacterized and one belonged to family GH18_PF-ChiA (PF-ChiA is a chitinase found in the hyperthermophilic archaea, prokaryotes). In case of xylanase, four out of six (GH9, 43, 10 and 11 enzyme family) were not showing nucleotide based similarity with any characterized gene. The study of reported genome sequence will help us to identify gene sequence of characterized and uncharacterized extracellular enzyme producing genes. Cloning of each gene and enzyme activity assay of their products will reveal the activity and stability at different variables; and resulting products may have huge applications at industrial scale.