Exploration of Diverse Secondary Metabolites From Streptomyces sp. YINM00001, Using Genome Mining and One Strain Many Compounds Approach.

A talented endophytic bacteria strain YINM00001, which showed strong antimicrobial activity and multiple antibiotic resistances, was isolated from a Chinese medicinal herb Peperomia dindygulensis Miq. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that strain was closely related to Streptomyces anulatus NRRL B-2000T (99.93%). The complete genome of strain YINM00001 was sequenced. The RAxML phylogenomic tree also revealed that strain YINM00001 was steadily clustered on a branch with strain Streptomyces anulatus NRRL B-2000T under the 100 bootstrap values. The complete genome of strain YINM00001 consists of an 8,372,992 bp linear chromosome (71.72 mol% GC content) and a 317,781 bp circular plasmid (69.14 mol% GC content). Genome mining and OSMAC approach were carried out to investigate the biosynthetic potential of producing secondary metabolites. Fifty-two putative biosynthetic gene clusters of secondary metabolites were found, including the putative cycloheximide, dinactin, warkmycin, and anthracimycin biosynthetic gene clusters which consist with the strong antifungal and antibacterial activities exhibited by strain YINM00001. Two new compounds, peperodione (1) and peperophthalene (2), and 17 known compounds were isolated from different fermentation broth. Large amounts and high diversity of antimicrobial and/or anticancer compounds cycloheximide, dinactin, anthracimycin, and their analogs had been found as predicted before, which highlights strain YINM00001 as an ideal candidate for further biosynthetic studies and production improvement of these valuable compounds. Meanwhile, several gene clusters that were highly conserved in several sequenced actinomycetes but significantly different from known gene clusters might be silent under proceeding fermentation conditions. Further studies, such as heterologous expression and genetic modification, are needed to explore more novel compounds from this talented endophytic Streptomyces strain.

Plant-derived bioactive compounds produced by Streptomyces variabilis LCP18 associated with Litsea cubeba (Lour.) Pers as potential target to combat human pathogenic bacteria and human cancer cell lines.

To date, endophytic actinomycetes have been well-documented as great producers of novel antibiotics and important pharmaceutical leads. The present study aimed to evaluate potent bioactivities of metabolites synthesized by the strain LCP18 residing in the Vietnamese medicinal plant Litsea cubeba (Lour.) Pers towards human pathogenic bacteria and human cancer cell lines. Endophytic actinomycete strain LCP18 showed considerable inhibition against seven bacterial pathogens and three human tumor cell lines and was identified as species Streptomyces variabilis. Strain S. variabilis LCP18 was phenotypically resistant to fosfomycin, trimethoprim-sulfamethoxazole, dalacin, cefoxitin, rifampicin, and fusidic acid and harbored the two antibiotic biosynthetic genes such as PKS-II and NRPS. Further purification and structural elucidation of metabolites from the LCP18 extract revealed five plant-derived bioactive compounds including isopcrunetin, genistein, daidzein, syringic acid, and daucosterol. Among those, isoprunetin, genistein, and daidzein exhibited antibacterial activity against Salmonella typhimurium ATCC 14,028 and methicillin-resistant Staphylococcus epidermidis ATCC 35,984 with the MIC values ranging from 16 to 128 µg/ml. These plant-derived compounds also exhibited cytotoxic effects against human lung cancer cell line A549 with IC50 values of less than 46 µM. These findings indicated that endophytic S. variabilis LCP18 can be an alternative producer of plant-derived compounds which significantly show potential applications in combating bacterial infections and inhibition against lung cancer cell lines.

Bactericidal and In-Vitro Cytotoxic Efficacy of Silver Nanoparticles (Ag-NPs) Fabricated by Endophytic Actinomycetes and Their Use as Coating for the Textile Fabrics.

An endophytic strain of Streptomyces antimycoticus L-1 was isolated from healthy medicinal plant leaves of Mentha longifolia L. and used for the green synthesis of silver nanoparticles (Ag-NPs), through the use of secreted enzymes and proteins. UV-vis spectroscopy, Fourier-transform infrared (FT-IR), transmission electron microscopy (TEM), X-ray diffraction (XRD), and dynamic light scattering (DLS) analyses of the Ag-NPs were carried out. The XRD, TEM, and FT-IR analysis results demonstrated the successful biosynthesis of crystalline, spherical Ag-NPs with a particle size of 13-40 nm. Further, the stability of the Ag-NPs was assessed by detecting the surface Plasmon resonance (SPR) at 415 nm for one month or by measuring the NPs surface charge (-19.2 mV) by zeta potential analysis (ζ). The green-synthesized Ag-NPs exhibited broad-spectrum antibacterial activity at different concentrations (6.25-100 ppm) against the pathogens Staphylococcus aureus, Bacillus subtilis Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhimurium with a clear inhibition zone ranging from (9.5 ± 0.4) nm to (21.7 ± 1.0) mm. Furthermore, the green-synthesized Ag-NPs displayed high efficacy against the Caco-2 cancerous cell line (the half maximal inhibitory concentration (IC50) = 5.7 ± 0.2 ppm). With respect to antibacterial and in-vitro cytotoxicity analyses, the Ag-NPs concentration of 100 ppm was selected as a safe dose for loading onto cotton fabrics. The scanning electron microscopy connected with energy-dispersive X-ray spectroscopy (SEM-EDX) for the nano-finished fabrics showed the distribution of Ag-NPs as 2% of the total fabric elements. Moreover, the nano-finished fabrics exhibited more activity against pathogenic Gram-positive and Gram-negative bacteria, even after 10 washing cycles, indicating the stability of the treated fabrics.

Endophytic Streptomyces laurentii Mediated Green Synthesis of Ag-NPs with Antibacterial and Anticancer Properties for Developing Functional Textile Fabric Properties.

Improvement of the medical textile industry has received more attention recently, especially with widespread of microbial and viral infections. Medical textiles with new properties, such as bacterial pathogens self-cleaning, have been explored with nanotechnology. In this study, an endophytic actinomycetes strain of Streptomyces laurentii R-1 was isolated from the roots of the medicinal plant Achillea fragrantissima. This is used as a catalyst for the mediated biosynthesis of silver nanoparticles (Ag-NPs) for applications in the textile industry. The biosynthesized Ag-NPs were characterized using UV-vis spectroscopy, Fourier transform infrared (FT-IR), transmission electron microscopy (TEM), and X-ray Diffraction (XRD), which confirmed the successful formation of crystalline, spherical metal nanoparticles. The biosynthesized Ag-NPs exhibited broad-spectrum antibacterial activity. Our data elucidated that the biosynthesized Ag-NPs had a highly cytotoxic effect against the cancerous caco-2 cell line. The selected safe dose of Ag-NPs for loading on cotton fabrics was 100 ppm, regarding their antibacterial activity and safe cytotoxic efficacy. Interestingly, scanning electron microscope connected with energy dispersive X-ray spectroscopy (SEM-EDX) of loaded cotton fabrics demonstrated the smooth distribution of Ag-NPs on treated fabrics. The obtained results highlighted the broad-spectrum activity of nano-finished fabrics against pathogenic bacteria, even after 5 and 10 washing cycles. This study contributes a suitable guide for the performance of green synthesized NPs for utilization in different biotechnological sectors.

Endophytic actinomycetes associated with Cinnamomum cassia Presl in Hoa Binh province, Vietnam: Distribution, antimicrobial activity and, genetic features.

Endophytic microbes associated with medicinal plants are considered to be potential producers of various bioactive secondary metabolites. The present study investigated the distribution, antimicrobial activity and genetic features of endophytic actinomycetes isolated from the medicinal plant Cinnamomum cassia Presl collected in Hoa Binh province of northern Vietnam. Based on phenotypic characteristics, 111 actinomycetes were isolated from roots, stems and leaves of the host plants by using nine selective media. The isolated actinomycetes were mainly recovered from stems (n = 67; 60.4%), followed by roots (n = 29; 26.1%) and leaves (n = 15; 13.5%). The isolates were accordingly assigned into 5 color categories of aerial mycelium, of which gray is the most dominant (n = 42; 37.8%), followed by white (n = 33; 29.7%), yellow (n = 25; 22,5%), red (n = 8; 7.2%) and green (n = 3; 2.7%). Of the total endophytic actinomycetes tested, 38 strains (occupying 34.2%) showed antimicrobial activity against at least one of nine tested microbes and, among them, 26 actinomycetes (68.4%) revealed anthracycline-like antibiotics production. Analysis of 16S rRNA gene sequences deposited on GenBank (NCBI) of the antibiotic-producing actinomycetes identified 3 distinct genera, including Streptomyces, Microbacterium, and Nocardia, among which Streptomyces genus was the most dominant and represented 25 different species. Further genetic investigation of the antibiotic-producing actinomycetes found that 28 (73.7%) and 11 (28.9%) strains possessed genes encoding polyketide synthase (pks) and nonribosomal peptide synthetase (nrps), respectively. The findings in the present study highlighted endophytic actinomycetes from C. cassia Presl which possessed broad-spectrum bioactivities with the potential for applications in the agricultural and pharmaceutical sectors.

Molecular profiling of endophytic Streptomyces cavourensis MH16 inhabiting Millingtonia hortensis Linn. and influence of different culture media on biosynthesis of antimicrobial metabolites.

Endophytic actinomycetes, a prolific source of natural products, are well known for their diverse metabolic versatility, and their association with medicinal plants and antimicrobial potential are well worth exploring. We isolated and identified the Streptomyces cavourensis strain MH16 inhabiting the tree Millingtonia hortensis Linn. using phylogenetic analysis based on a 16S rRNA molecular approach. We used the disc diffusion method to evaluate the impact of differences in the compositions of the media on the production of secondary metabolites from strain MH16. The production of antimicrobial metabolites was determined by the observation of inhibition zones on intensive bands when using a TLC-bioautography assay. Biosynthesis of secondary metabolites was optimal when the strain MH16 was cultured in ISP-2 medium as depicted by a zone of inhibition. Strain MH16 effectively inhibited methicillin-resistant Staphylococcus aureus, Escherichia coli, Candida albicans, and other multi drug-resistant pathogens. The minimum inhibitory concentration of the antimicrobial metabolites was 25-100 µg mL-1. The study manifests the optimization and utilization of different fermentation media which best suits for increased production of the secondary metabolites from Streptomyces cavourensis. This research suggests that the antimicrobial metabolites of strain MH16 found in M. hortensis has great potential for the biodiscovery of new anti-infective drugs against a wide range of multidrug-resistant pathogens.

Endophytic Actinomycetes as Potential Producers of Hemicellulases and Related Enzymes for Plant Biomass Degradation

Abstract Tailor made enzymatic preparation must be design to hydrolyze efficiently plant biomass, once that each plant biomass possesses a distinct cell wall composition. Most of actinomycetes used for plant cell wall degradation are focused on the cellulases and xylanases production. However, a wide range of enzymes must be produced for an efficient degradation of lignocellulose materials. During the last decade several unusual environments were studied to obtain strains that produce glycohydrolases with innovator characteristics. In this context, the present work concerned the selection of endophytic actinomycetes as producers of hemicellulases and related enzymes with different enzymatic profiles, for use in the deconstruction of lignocellulosic biomass. A total of 45 Brazilian actinomycetes previously isolated from plants (endophytics) and soil were prospected for hemicellulases and β-glucosidase production. Four strains highlighted for hemicellulase production (DR61, DR63, DR69 and DR66) and were selected for cultivation under other inductors substrates (xylan and pectin). All strains belong to Streptomyces genera and have their extracts tested for degradation of several hemicellulolytic substrates. The strains presented different glicohydrolyse enzymes profiles mainly for xylans and glucans that can be used for specific formulations of enzymes applied on the biomass deconstruction, principally on sugar cane bagasse.

Diversity and bioprospecting of actinomycete endophytes from the medicinal plants.

The endophytic actinomycetes constitute one of the fascinating group of microorganisms associated with a wide range of plant species. The diversity of actinomycetes in plants and their tissue parts is a matter of debate as no consensus are derived between individual studies. Nevertheless, their diversity correlates with the occurrence in plant species harboured in unique regions of biologically diverse areas called "hot spots." Recent advances in the isolation techniques have facilitated the isolation of rare taxa from these environments. The biosynthetic ability of the endophytic actinomycetes has proven beyond doubt that these organisms have the potential to synthesize an array of compounds with novelty in structure and bioactivity and as a result are preferred in the natural product screening programs. In the years to come, the scientific world may await to discover many more novel actinomycete taxa with metabolic diversity and applications in therapeutics. SIGNIFICANCE AND IMPACT OF THE STUDY: "Endophytes" - the microbes residing in the living tissues of plants are virtually omnipresent. Actinomycete endophytes are diverse in distribution within plant tissues, especially in the roots as they have a close association with the rhizhosphere. An introspection into diversity studies necessitates careful sampling, analysis, and isolation data from the biodiverse and nonbiodiverse regions represented by unique environments. The key to the recovery of novel species and their bioprospection lies in these regions.

Isolation and identification of an antifungal compound from endophytic Streptomyces sp. CEN26 active against Alternaria brassicicola.

UNLABELLED: 2,5-Bis(hydroxymethyl)furan monoacetate (BHMF-OAc) compound was isolated from the culture filtrate secreted by Streptomyces sp. CEN26, an endophytic actinomycete found in the root nodes of Centella asiatica (L.) Urban. The compound was purified using various chromatographic techniques and bioassay-guided fractionation. The compound was then identified through a structural characterization using (1) H, (13) C-NMR and MS analysis. After the purification and identification process, a series of experiments were conducted in order to test whether the compound affected conidial germination of Alternaria brassicicola. Findings show that the compound inhibited and deformed the conidial germination of A. brassicicola. Its minimal inhibitory concentrations of MIC90 and MIC50 were found to be 100 and 25 ppm respectively. Further experimentation show that the aforementioned concentrations of MIC90 and MIC50 completely suppressed the appressorium formation of the fungal pathogen. Therefore, the BHMF-OAc compound does have antifungal properties that help protect plants from infection by A. brassicicola. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic Streptomyces sp. CEN26 produces 2,5-bis(hydroxymethyl)furan monoacetate (BHMF-OAc), which is a compound that inhibits the infection process of Alternaria brassicicola in cabbage. Such an antifungal property would allow for the use of the BHMF-OAc as an alternative to chemical fungicides.

Antimicrobial biosynthetic potential and genetic diversity of endophytic actinomycetes associated with medicinal plants.

Endophytic actinomycetes are one of the primary groups that share symbiotic relationships with medicinal plants and are key reservoir of biologically active compounds. In this study, six selective medicinal plants were targeted for the first time for endophytic actinomycetes isolation from Gibbon Wild Life Sanctuary, Assam, India, during winter and summer and 76 isolates were obtained. The isolates were found to be prevalent in roots followed by stem and leaves. 16S rRNA gene sequence analysis revealed 16 genera, including rare genera, Verrucosispora, Isoptericola and Kytococcus, which have never been previously reported as endophytic. The genus Streptomyces (66%) was dominant in both seasons. Shannon's diversity index showed that Azadirachta indica (1.49), Rauwolfia serpentina (1.43) and Emblica officinalis (1.24) were relatively good habitat for endophytic actinomycetes. Antimicrobial strains showed prevalence of polyketide synthase (PKS) type-II (85%) followed by PKS type-I (14%) encoded in the genomes. Expression studies showed 12-fold upregulation of PKSII gene in seventh day of incubation for Streptomyces antibioticus (EAAG90). Our results emphasize that the actinomycetes assemblages within plant tissue exhibited biosynthetic systems encoding for important biologically active compounds.

Isolation, abundance and phylogenetic affiliation of endophytic actinomycetes associated with medicinal plants and screening for their in vitro antimicrobial biosynthetic potential.

Microorganisms associated with medicinal plants are of interest as the producers of important bioactive compounds. To date, the diversity of culturable endophytic actinomycetes associated with medicinal plants is in its initial phase of exploration. In this study, 42 endophytic actinomycetes were isolated from different organs of seven selected medicinal plants. The highest number of isolates (n = 22, 52.3%) of actinomycetes was isolated from roots, followed by stems (n = 9, 21.4%), leaves (n = 6, 14.2%), flowers (n = 3, 7.1%), and petioles (n = 2, 4.7%). The genus Streptomyces was the most dominant among the isolates (66.6%) in both the locations (Dampa TRF and Phawngpuii NP, Mizoram, India). From a total of 42 isolates, 22 isolates were selected for further studies based on their ability to inhibit one of the tested human bacterial or fungal pathogen. Selected isolates were identified based on 16S rRNA gene analysis and subsequently the isolates were grouped to four different genera; Streptomyces, Brevibacterium, Microbacterium, and Leifsonia. Antibiotic sensitivity assay was performed to understand the responsible antimicrobials present in the isolates showing the antimicrobial activities and revealed that the isolates were mostly resistant to penicillin G and ampicillin. Further, antimicrobial properties and antibiotic sensitivity assay in combination with the results of amplification of biosynthetic genes polyketide synthase (PKS-I) and non-ribosomal peptide synthetase (NRPS) showed that the endophytic actinomycetes associated with the selected medicinal plants have broad-spectrum antimicrobial activity. This is the first report of the isolation of Brevibacterium sp., Microbacterium sp., and Leifsonia xyli from endophytic environments of medicinal plants, Mirabilis jalapa and Clerodendrum colebrookianum. Our results emphasize that endophytic actinomycetes associated with medicinal plants are an unexplored resource for the discovery of biologically active compounds.

Diversity, bioactivities, and metabolic potentials of endophytic actinomycetes isolated from traditional medicinal plants in Sichuan, China.

The present study was designed to determine the taxonomic diversity and metabolic activity of the actinomycetes community, including 13 traditional medicinal plants collected in Sichuan province, China, using multiple approaches such as morphological and molecular identification methods, bioactivity assays, and PCR screening for genes involved in antibiotics biosynthesis. 119 endophytic actinomycetes were recovered; 80 representative strains were chosen for 16S rRNA gene partial sequence analyses, with 66 of them being affiliated to genus Streptomyces and the remaining 14 strains being rare actinomycetes. Antimicrobial tests showed that 12 (15%) of the 80 endophytic actinomycetes displayed inhibitory effects against at least one indicator pathogens, which were all assigned to the genus Streptomyces. In addition, 87.5% and 58.8% of the isolates showed anticancer and anti-diabetic activities, respectively. Meanwhile, the anticancer activities of the isolates negatively correlated with their anti-diabetic activities. Based on the results of PCR screening, five genes, PKS-I, PKS-II, NRPS, ANSA, and oxyB, were detected in 55.0%, 58.8%, 90.0%, 18.8% and 8.8% of the 80 actinomycetes, respectively. In conclusion, the PCR screening method employed in the present study was conducive for screening and selection of potential actinomycetes and predicting potential secondary metabolites, which could overcome the limitations of traditional activity screening models.

Diversity, bioactivities, and metabolic potentials of endophytic actinomycetes isolated from traditional medicinal plants in Sichuan, China / 中国天然药物

The present study was designed to determine the taxonomic diversity and metabolic activity of the actinomycetes community, including 13 traditional medicinal plants collected in Sichuan province, China, using multiple approaches such as morphological and molecular identification methods, bioactivity assays, and PCR screening for genes involved in antibiotics biosynthesis. 119 endophytic actinomycetes were recovered; 80 representative strains were chosen for 16S rRNA gene partial sequence analyses, with 66 of them being affiliated to genus Streptomyces and the remaining 14 strains being rare actinomycetes. Antimicrobial tests showed that 12 (15%) of the 80 endophytic actinomycetes displayed inhibitory effects against at least one indicator pathogens, which were all assigned to the genus Streptomyces. In addition, 87.5% and 58.8% of the isolates showed anticancer and anti-diabetic activities, respectively. Meanwhile, the anticancer activities of the isolates negatively correlated with their anti-diabetic activities. Based on the results of PCR screening, five genes, PKS-I, PKS-II, NRPS, ANSA, and oxyB, were detected in 55.0%, 58.8%, 90.0%, 18.8% and 8.8% of the 80 actinomycetes, respectively. In conclusion, the PCR screening method employed in the present study was conducive for screening and selection of potential actinomycetes and predicting potential secondary metabolites, which could overcome the limitations of traditional activity screening models.

Streptomycete endophytes from anti-diabetic medicinal plants of the Western Ghats inhibit alpha-amylase and promote glucose uptake.

UNLABELLED: α-amylase inhibitor retards the liberation of glucose from dietary complex carbohydrates and delays the absorption of glucose. The purpose of the study was to isolate and select α-amylase inhibitor-producing endophytic actinomycetes from the leaves and stems of Leucas ciliata and Rauwolfia densiflora, two of the well-known medicinal plants used in the treatment for diabetes. Sterilized plant samples were inoculated on the actinomycete isolation agar medium containing 50 ppm cycloheximide and incubated for 4-8 weeks at room temperature. The actinomycetes were isolated on agar medium and identified on the basis of 16S rRNA sequences, the isolates exhibiting >99% similarities were submitted to NCBI, and gene accession numbers were obtained. They were inoculated to International Streptomyces Project 1 medium (ISP 1) for fermentation. The extracts obtained were tested for the anti-diabetic potential by the inhibition of alpha-amylase by colorimetric assay and glucose uptake in the porcine hemidiaphragm. Streptomyces longisporoflavus (JX965948) isolated from the stem fragments of L. ciliata exhibited alpha-amylase inhibitory activity (IC50 values = 162.3 ± 1.05 µg ml⁻¹) in comparison with the standard Acarbose™ (IC50 value = 73.1 ± 1.12 µg ml⁻¹). Extract of Streptomyces sp. (JQ926174) from R. densiflora indicated glucose uptake in the porcine hemidiaphragm. Results indicate for the first time the potential of endophytic streptomycete extracts with anti-diabetic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic actinomycetes were isolated from two medicinal species of the Western Ghats, a biodiversity 'hotspot' in southern India and screened for the anti-diabetic potential for inhibition of α-amylase and improved glucose uptake in the porcine hemidiaphragm. Results indicate the inhibition of α-amylase by Streptomyces longisporoflavus extract with IC50 values of 162.3 ± 1.05 µg ml⁻¹ in comparison with the standard inhibitor Acarbose™ with IC50 value 73.1 ± 1.12 µg ml⁻¹. Further, extract from Streptomyces sp. showed increased glucose uptake by hemidiaphragm. The present investigation implicates the potential of endophytic actinomycetes as sources of anti-diabetic agents.

Generic and functional diversity in endophytic actinomycetes from wild Compositae plant species at South Sinai - Egypt.

The diversity of culturable endophytic actinomycetes associated with wild Compositae plants is scantily explored. In this study, one hundred and thirty one endophytic actinobacteria were isolated from ten Compositae plant species collected from South Sinai in Egypt. Microscopic and chemotaxonomic investigation of the isolates indicated fourteen genera. Rare genera, such as Microtetraspora, and Intrasporangium, which have never been previously reported to be endophytic, were identified. Each plant species accommodated between three to eight genera of actinobacteria and unidentified strains were recovered from seven plant species. The generic diversity analysis of endophytic assemblages grouped the plant species into three main clusters, representing high, moderate and low endophytic diversity. The endophytes showed high functional diversity, based on forty four catabolic and plant growth promotion traits; providing some evidence that such traits could represent key criteria for successful residence of endophytes in the endosphere. Stress-tolerance traits were more predictive measure of functional diversity differences between the endophyte assemblages (Shannon's index, p = 0.01). The results indicate a potential prominent role of endophytes for their hosts and emphasize the potency of plant endosphere as a habitat for actinobacteria with promising future applications.