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Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.
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Antraciclinas , Policetídeo Sintases , Streptomyces coelicolor , Policetídeo Sintases/metabolismo , Policetídeo Sintases/genética , Antraciclinas/metabolismo , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/genética , Streptomyces/metabolismo , Streptomyces/genética , Vias Biossintéticas/genética , Hidroxilação , Antibacterianos/biossíntese , Antibacterianos/metabolismo , Antibacterianos/químicaRESUMO
Structural and functional studies of the carminomycin 4-O-methyltransferase DnrK are described, with an emphasis on interrogating the acceptor substrate scope of DnrK. Specifically, the evaluation of 100 structurally and functionally diverse natural products and natural product mimetics revealed an array of pharmacophores as productive DnrK substrates. Representative newly identified DnrK substrates from this study included anthracyclines, angucyclines, anthraquinone-fused enediynes, flavonoids, pyranonaphthoquinones, and polyketides. The ligand-bound structure of DnrK bound to a non-native fluorescent hydroxycoumarin acceptor, 4-methylumbelliferone, along with corresponding DnrK kinetic parameters for 4-methylumbelliferone and native acceptor carminomycin are also reported for the first time. The demonstrated unique permissivity of DnrK highlights the potential for DnrK as a new tool in future biocatalytic and/or strain engineering applications. In addition, the comparative bioactivity assessment (cancer cell line cytotoxicity, 4E-BP1 phosphorylation, and axolotl embryo tail regeneration) of a select set of DnrK substrates/products highlights the ability of anthracycline 4-O-methylation to dictate diverse functional outcomes.
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Metiltransferases , Metiltransferases/metabolismo , Metiltransferases/química , Estrutura Molecular , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Humanos , Antraciclinas/química , Antraciclinas/farmacologia , Especificidade por SubstratoRESUMO
Background: Plant-derived endophytic actinobacteria are the center of attention due to their capacity to produce diverse antimicrobial and anticancer compounds and their metabolites influence plant growth. Methods: In this study, 40 endophytic actinobacteria strains were isolated from the roots of eight medicinal plants used as folk medicine in South Asian region. The isolates were characterized morphologically, biochemically and physiologically and the genus level identification of the selected strains was done by 16SrRNA gene sequencing. In small scale cultivation (50ml broth), the isolates were grown in A-medium to prepare the crude extracts. These crude extracts were subsequently evaluated for their antimicrobial, anticancer and antioxidant activity and the metabolomics profile of each of the extract was determined by TLC and HPLC-UV/MS. Results: The taxonomic studies showed that the isolates belong to the group actinobacteria based on their morphological and physiological characteristics and the 16SrRNA gene sequencing of the selected strains identified the genera including Streptomyces, Micromonospora and Nocardia. Cumulatively,53% of extracts exhibited anti-Gram-(+) activity,47% exhibited anti-Gram-(-) activity,32% exhibited antifungal activity and 30% were cytotoxic to PC3 and A549 cancer cell lines and most of the extracts have shown antioxidant activity greater than 50%. The metabolomics analysis predicted the presence of an array of low molecular weight metabolites and indicated the promising isolates in collection for further studies for novel bioactive metabolite isolation and structure elucidation. Conclusion: Overall the study provides an overview of the endophytic actinobacteria residing in the roots of the selected medicinal plants prevalent in south Asian region and their potential to produce the medicinally and biotechnologically useful compounds.
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Tetracenomycins and elloramycins are polyketide natural products produced by several actinomycetes that exhibit antibacterial and anticancer activities. They inhibit ribosomal translation by binding in the polypeptide exit channel of the large ribosomal subunit. The tetracenomycins and elloramycins are typified by a shared oxidatively modified linear decaketide core, yet they are distinguished by the extent of O-methylation and the presence of a 2',3',4'-tri-O-methyl-α-l-rhamnose appended at the 8-position of elloramycin. The transfer of the TDP-l-rhamnose donor to the 8-demethyl-tetracenomycin C aglycone acceptor is catalyzed by the promiscuous glycosyltransferase ElmGT. ElmGT exhibits remarkable flexibility toward transfer of many TDP-deoxysugar substrates to 8-demethyltetracenomycin C, including TDP-2,6-dideoxysugars, TDP-2,3,6-trideoxysugars, and methyl-branched deoxysugars in both d- and l-configurations. Previously, we developed an improved host, Streptomyces coelicolor M1146::cos16F4iE, which is a stable integrant harboring the required genes for 8-demethyltetracenomycin C biosynthesis and expression of ElmGT. In this work, we developed BioBricks gene cassettes for the metabolic engineering of deoxysugar biosynthesis in Streptomyces spp. As a proof of concept, we used the BioBricks expression platform to engineer biosynthesis for d-configured TDP-deoxysugars, including known compounds 8-O-d-glucosyl-tetracenomycin C, 8-O-d-olivosyl-tetracenomycin C, 8-O-d-mycarosyl-tetracenomycin C, and 8-O-d-digitoxosyl-tetracenomycin C. In addition, we generated four new tetracenomycins including one modified with a ketosugar, 8-O-4'-keto-d-digitoxosyl-tetracenomycin C, and three modified with 6-deoxysugars, including 8-O-d-fucosyl-tetracenomycin C, 8-O-d-allosyl-tetracenomycin C, and 8-O-d-quinovosyl-tetracenomycin C. Our work demonstrates the feasibility of BioBricks cloning, with the ability to recycle intermediate constructs, for the rapid assembly of diverse carbohydrate pathways and glycodiversification of a variety of natural products.
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BACKGROUND: Bioprospecting of actinobacteria isolated from Kubuqi desert, China for antibacterial, antifungal and cytotoxic metabolites production and their structure elucidation. RESULTS: A total of 100 actinobacteria strains were selectively isolated from Kubuqi desert, Inner Mongolia, China. The taxonomic characterization revealed Streptomyces as the predominant genus comprising 37 different species, along with the rare actinobacterial genus Lentzea. The methanolic extracts of 60.8% of strains exhibited potent antimicrobial activities against Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, Salmonella enterica, Saccharomyces cerevisiae and high to mild in vitro cytotoxicity against PC3 (prostate cancer) and A549 (lung carcinoma) cell lines. The metabolomics analysis by TLC, HPLC-UV/vis, HPLC-MS and NMR showed the presence of compounds with molecular weights ranging from 100 to 1000 Da. The scale-up fermentation of the prioritized anti-Gram-negative strain PU-KB10-4 (Streptomyces griseoviridis), yielded three pure compounds including; griseoviridin (1; 42.0 mgL- 1) with 20 fold increased production as compared to previous reports and its crystal structure as monohydrate form is herein reported for the first time, mitomycin C (2; 0.3 mgL- 1) and a new bacterial metabolite 4-hydroxycinnamide (3; 0.59 mgL- 1). CONCLUSIONS: This is the first report of the bioprospecting and exploration of actinobacteria from Kubuqi desert and the metabolite 4-hydroxycinnamide (3) is first time isolated from a bacterial source. This study demonstrated that actinobacteria from Kubuqi desert are a potential source of novel bioactive natural products. Underexplored harsh environments like the Kubuqi desert may harbor a wider diversity of actinobacteria, particularly Streptomyces, which produce unique metabolites and are an intriguing source to develop medicinally valuable natural products.
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Actinobacteria , Produtos Biológicos , Streptomyces , Mitomicina/metabolismo , Bioprospecção , Filogenia , Antibacterianos/química , Produtos Biológicos/farmacologiaRESUMO
Chemical investigation of the endophyte Pseudofusicoccum stromaticum CMRP4328 isolated from the medicinal plant Stryphnodendron adstringens yielded ten compounds, including two new dihydrochromones, paecilins Q (1: ) and R (2: ). The antifungal activity of the isolated metabolites was assessed against an important citrus pathogen, Phyllosticta citricarpa. Cytochalasin H (6: ) (78.3%), phomoxanthone A (3: ) (70.2%), phomoxanthone B (4: ) (63.1%), and paecilin Q (1: ) (50.5%) decreased in vitro the number of pycnidia produced by P. citricarpa, which are responsible for the disease dissemination in orchards. In addition, compounds 3: and 6: inhibited the development of citrus black spot symptoms in citrus fruits. Cytochalasin H (6: ) and one of the new compounds, paecilin Q (1: ), appear particularly promising, as they showed strong activity against this citrus pathogen, and low or no cytotoxic activity. The strain CMRP4328 of P. stromaticum and its metabolites deserve further investigation for the control of citrus black spot disease.
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Antifúngicos , Citrus , Antifúngicos/farmacologia , Doenças das Plantas/prevenção & controle , Doenças das Plantas/microbiologia , EndófitosRESUMO
The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.
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Produtos Biológicos , Escherichia coli , Escherichia coli/genética , Antraquinonas/química , Policetídeo Sintases/genética , Policetídeo Sintases/química , Enedi-Inos/química , Antibióticos AntineoplásicosRESUMO
Actinomycetes produce a variety of clinically indispensable molecules, such as antineoplastic anthracyclines. However, the actinomycetes are hindered in their further development as genetically engineered hosts for the synthesis of new anthracycline analogues due to their slow growth kinetics associated with their mycelial life cycle and the lack of a comprehensive genetic toolbox for combinatorial biosynthesis. In this report, we tackled both issues via the development of the BIOPOLYMER (BIOBricks POLYketide Metabolic EngineeRing) toolbox: a comprehensive synthetic biology toolbox consisting of engineered strains, promoters, vectors, and biosynthetic genes for the synthesis of anthracyclinones. An improved derivative of the production host Streptomyces coelicolor M1152 was created by deleting the matAB gene cluster that specifies extracellular poly-ß-1,6-N-acetylglucosamine (PNAG). This resulted in a loss of mycelial aggregation, with improved biomass accumulation and anthracyclinone production. We then leveraged BIOPOLYMER to engineer four distinct anthracyclinone pathways, identifying optimal combinations of promoters, genes, and vectors to produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone at titers between 15-20 mg/L. Optimization of nogalamycinone production strains resulted in titers of 103 mg/L. We structurally characterized six anthracyclinone products from fermentations, including new compounds 9,10-seco-7-deoxy-nogalamycinone and 4-O-ß-d-glucosyl-nogalamycinone. Lastly, we tested the antiproliferative activity of the anthracyclinones in a mammalian cancer cell viability assay, in which nogalamycinone, auramycinone, and aklavinone exhibited moderate cytotoxicity against several cancer cell lines. We envision that BIOPOLYMER will serve as a foundational platform technology for the synthesis of designer anthracycline analogues.
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Policetídeos , Streptomyces coelicolor , Streptomyces , Animais , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Engenharia Metabólica , Streptomyces/genética , Antraciclinas/metabolismo , Antibióticos Antineoplásicos/metabolismo , Policetídeos/metabolismo , Família Multigênica , Mamíferos/genéticaRESUMO
We report the isolation and characterization of five polyketides [alternariol (1), alternariol-9-methyl ether (2), altertoxin I (3), altertoxin II (4) and tenuazonic acid (5)] from the marine endophytic Alternaria sp. LV52 derived from Cystoseira tamariscifolia, collected from the Red Sea at Nabq-Bay, Egypt. The chemical structures of compounds 1-5 were identified by extensive 1D, 2D NMR, and HR mass measurements. Isolation and phenotypic and genotypic characterization of the producing fungus is reported. The antimicrobial activity of the produced extract and derived compounds was examined against a panel of test organisms. In addition, an in vitro cytotoxic activity of 1-5 was performed against diverse cancer cell lines: HEPG2, HELA, A549 and PC3, revealing that compounds 2 and 4 are potentially cytotoxic against A549 and PC3 with EC50 of 0.73 µg/ml (2.69 µM) and 0.17 µg/ml (0.64 µM) for 2, and 0.40 µg/ml (1.15⯵M) and 0.12 µg/ml (0.33 µM) for 4, respectively.
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Dynemicin is an enediyne natural product from Micromonospora chersina ATCC53710. Access to the biosynthetic gene cluster of dynemicin has enabled the in vitro study of gene products within the cluster to decipher their roles in assembling this unique molecule. This paper reports the crystal structure of DynF, the gene product of one of the genes within the biosynthetic gene cluster of dynemicin. DynF is revealed to be a dimeric eight-stranded ß-barrel structure with palmitic acid bound within a cavity. The presence of palmitic acid suggests that DynF may be involved in binding the precursor polyene heptaene, which is central to the synthesis of the ten-membered ring of the enediyne core.
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Enedi-Inos , Micromonospora , Cristalografia por Raios X , Enedi-Inos/química , Enedi-Inos/metabolismo , Micromonospora/genética , Micromonospora/metabolismo , Família MultigênicaRESUMO
Purpose: Mineralogical and geochemical features of mining and processing wastes collected in Owen County, part of the Central Kentucky Lead-Zinc district, were investigated. The Gratz mine, abandoned in the 1940s, is on a dairy farm. Aside from discerning the nature of mining refuse at the site, the investigation was part of the University of Kentucky College of Pharmacy's mission to explore unusual environments in the search for unique microbiological communities. Materials and methods: Four samples of a soil-plus-spoils mix were collected from spoil piles and two samples, the sluice and coarse samples, were closely associated with the site of the ore processing. Optical petrology (polarized reflected-light, oil-immersion optics at a final magnification of 500 ×), X-ray diffraction, X-ray fluorescence, inductively coupled plasma mass spectrometry, field emission scanning electron microscopy (FE-SEM), and high-resolution transmission electron microscope (HR-TEM) with selected area electron diffraction (SAED) and/or microbeam diffraction (MBD), scanning transmission electron microscopy (STEM), and energy-dispersive X-ray spectrometer (EDS) analyses were employed to characterize the samples. Results and discussion: Calcite is the main mineral in most samples, followed by near equal amounts of quartz and dolomite. Sphalerite and galena are the principal sulfides and barite is the dominant sulfate. Geochemistry of major elements reflected the mineralogy, whereas trace elements showed different groupings between the minerals. Scandium, Cu, Ga, Ge, Cd, and Sb were found predominantly associated with Zn and Pb and sulfide minerals; Bi, Hf, In, Sn, and Zr with heavy mineral fraction; while the remaining trace elements, including the rare earths, were mostly distributed among other present phases, i.e., oxyhalides, oxides, silicates, and carbonaceous material. The data were used to illustrate the processes and conditions that control the sulfide-mineral oxidation and its potential for the environmental release of associated reaction products. Conclusions: The wastes represent a potential source of environmentally disruptive concentrations of Zn, Pb, and other sulfide-associated elements. The high share of carbonates suggests near-neutral conditions in deposited wastes, restricting sulfide weathering and further limiting the oxidant activity of Fe. The low-Fe content and its predominant presence in highly resistant hematite also constrain sulfide weathering. Consequently, the spoils have a low potential for generation of acidity and release of heavy metal(loid)s in the surrounding environment.
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Muraymycins are peptidyl nucleoside antibiotics that contain two Cß-modified amino acids, (2S,3S)-capreomycidine and (2S,3S)-ß-OH-Leu. The former is also a component of chymostatins, which are aldehyde-containing peptidic protease inhibitors thatâlike muraymycinâare derived from nonribosomal peptide synthetases (NRPSs). Using feeding experiments and in vitro characterization of 12 recombinant proteins, the biosynthetic mechanism for both nonproteinogenic amino acids is now defined. The formation of (2S,3S)-capreomycidine is shown to involve an FAD-dependent dehydrogenase:cyclase that requires an NRPS-bound pathway intermediate as a substrate. This cryptic dehydrogenation strategy is both temporally and mechanistically distinct in comparison to the biosynthesis of other capreomycidine diastereomers, which has previously been shown to proceed by Cß-hydroxylation of free l-Arg catalyzed by a member of the nonheme Fe2+- and α-ketoglutarate (αKG)-dependent dioxygenase family and (eventually) a dehydration-mediated cyclization process catalyzed by a distinct enzyme(s). Contrary to our initial expectation, the sole nonheme Fe2+- and αKG-dependent dioxygenase candidate Mur15 encoded within the muraymycin gene cluster is instead demonstrated to catalyze specific Cß hydroxylation of the Leu residue to generate (2S,3S)-ß-OH-Leu that is found in most muraymycin congeners. Importantly, and in contrast to known l-Arg-Cß-hydroxylases, the Mur15-catalyzed reaction occurs after the NRPS-mediated assembly of the peptide scaffold. This late-stage functionalization affords the opportunity to exploit Mur15 as a biocatalyst, proof of concept of which is provided.
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Arginina/metabolismo , Produtos Biológicos/metabolismo , Leucina/metabolismo , Peptídeo Sintases/metabolismo , Peptídeos/metabolismo , Arginina/química , Produtos Biológicos/química , Leucina/química , Estrutura Molecular , Peptídeo Sintases/química , Peptídeos/químicaRESUMO
We report the identification of the ter gene cluster responsible for the formation of the p-terphenyl derivatives terfestatins B and C and echoside B from the Appalachian Streptomyces strain RM-5-8. We characterize the function of TerB/C, catalysts that work together as a dual enzyme system in the biosynthesis of natural terphenyls. TerB acts as a reductase and TerC as a dehydratase to enable the conversion of polyporic acid to a terphenyl triol intermediate. X-ray crystallography of the apo and substrate-bound forms for both enzymes provides additional mechanistic insights. Validation of the TerC structural model via mutagenesis highlights a critical role of arginine 143 and aspartate 173 in catalysis. Cumulatively, this work highlights a set of enzymes acting in harmony to control and direct reactive intermediates and advances fundamental understanding of the previously unresolved early steps in terphenyl biosynthesis.
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Hidroliases/metabolismo , Oxirredutases/metabolismo , Compostos de Terfenil/química , Sequência de Aminoácidos , Arginina/química , Ácido Aspártico/química , Vias Biossintéticas , Catálise , Cristalografia por Raios X , Escherichia coli/metabolismo , Modelos Moleculares , Ligação Proteica , Streptomyces/metabolismo , Relação Estrutura-AtividadeRESUMO
The 1.5â Å resolution crystal structure of DynU16, a protein identified in the dynemicin-biosynthetic gene cluster, is reported. The structure adopts a di-domain helix-grip fold with a uniquely positioned open cavity connecting the domains. The elongated dimensions of the cavity appear to be compatible with the geometry of a linear polyene, suggesting the involvement of DynU16 in the upstream steps of dynemicin biosynthesis.
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Antraquinonas/metabolismo , Antibacterianos/biossíntese , Enedi-Inos/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Família Multigênica , Conformação ProteicaRESUMO
Plant diseases caused by phytopathogens are responsible for significant crop losses worldwide. Resistance induction and biological control have been exploited in agriculture due to their enormous potential. In this study, we investigated the antimicrobial potential of endophytic fungi of leaves and petioles of medicinal plants Vochysia divergens and Stryphnodendron adstringens located in two regions of high diversity in Brazil, Pantanal, and Cerrado, respectively. We recovered 1,304 fungal isolates and based on the characteristics of the culture, were assigned to 159 phenotypes. One isolate was selected as representative of each phenotype and studied for antimicrobial activity against phytopathogens. Isolates with better biological activities were identified based on DNA sequences and phylogenetic analyzes. Among the 159 representative isolates, extracts from 12 endophytes that inhibited the mycelial growth (IG) of Colletotrichum abscissum (≥40%) were selected to expand the antimicrobial analysis. The minimum inhibitory concentrations (MIC) of the extracts were determined against citrus pathogens, C. abscissum, Phyllosticta citricarpa and Xanthomonas citri subsp. citri and the maize pathogen Fusarium graminearum. The highest activity against C. abscissum were from extracts of Pseudofusicoccum stromaticum CMRP4328 (IG: 83% and MIC: 40 µg/mL) and Diaporthe vochysiae CMRP4322 (IG: 75% and MIC: 1 µg/mL), both extracts also inhibited the development of post-bloom fruit drop symptoms in citrus flowers. The extracts were promising in inhibiting the mycelial growth of P. citricarpa and reducing the production of pycnidia in citrus leaves. Among the isolates that showed activity, the genus Diaporthe was the most common, including the new species D. cerradensis described in this study. In addition, high performance liquid chromatography, UV detection, and mass spectrometry and thin layer chromatography analyzes of extracts produced by endophytes that showed high activity, indicated D. vochysiae CMRP4322 and P. stromaticum CMRP4328 as promising strains that produce new bioactive natural products. We report here the capacity of endophytic fungi of medicinal plants to produce secondary metabolites with biological activities against phytopathogenic fungi and bacteria. The description of the new species D. cerradensis, reinforces the ability of medicinal plants found in Brazil to host a diverse group of fungi with biotechnological potential.
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Himalaquinones A-G, seven new anthraquinone-derived metabolites, were obtained from the Himalayan-based Streptomyces sp. PU-MM59. The chemical structures of the new compounds were identified based on cumulative analyses of HRESIMS and NMR spectra. Himalaquinones A-F were determined to be unique anthraquinones that contained unusual C-4a 3-methylbut-3-enoic acid aromatic substitutions, while himalaquinone G was identified as a new 5,6-dihydrodiol-bearing angucyclinone. Comparative bioactivity assessment (antimicrobial, cancer cell line cytotoxicity, impact on 4E-BP1 phosphorylation, and effect on axolotl embryo tail regeneration) revealed cytotoxic landomycin and saquayamycin analogues to inhibit 4E-BP1p and inhibit regeneration. In contrast, himalaquinone G, while also cytotoxic and a regeneration inhibitor, did not affect 4E-BP1p status at the doses tested. As such, this work implicates a unique mechanism for himalaquinone G and possibly other 5,6-dihydrodiol-bearing angucyclinones.
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Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Streptomyces/química , Ambystoma mexicanum , Aminoglicosídeos/isolamento & purificação , Aminoglicosídeos/farmacologia , Animais , Antraquinonas/isolamento & purificação , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Paquistão , Microbiologia do SoloRESUMO
Actinobacteria have proven themselves as the major producers of bioactive compounds with wide applications. In this study, 35 actinobacteria strains were isolated from soil samples collected from the Himalayan mountains region in Pakistan. The isolated strains were identified by polyphasic taxonomy and were prioritized based on biological and chemical screening to identify the strains with ability to produce inimitable metabolites. The biological screening included antimicrobial activity against Staphylococcus aureus, Micrococcus luteus, Salmonella enterica, Escherichia coli, Mycobacterium aurum, and Bacillus subtilis and anticancer activity using human cancer cell lines PC3 and A549. For chemical screening, methanolic extracts were investigated using TLC, HPLC-UV/MS. The actinobacteria strain PU-MM93 was selected for scale-up fermentation based on its unique chemical profile and cytotoxicity (50-60% growth inhibition) against PC3 and A549 cell lines. The scale-up fermentation of PU-MM93, followed by purification and structure elucidation of compounds revealed this strain as a promising producer of the cytotoxic anthracycline aranciamycin and aglycone SM-173-B along with the potent neuroprotective carboxamide oxachelin C. Other interesting metabolites produced include taurocholic acid as first report herein from microbial origin, pactamycate and cyclo(L-Pro-L-Leu). The study suggested exploring more bioactive microorganisms from the untapped Himalayan region in Pakistan, which can produce commercially significant compounds.
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Actinobacteria , Antibacterianos/farmacologia , Humanos , Metabolômica , Testes de Sensibilidade Microbiana , Mycobacteriaceae , PaquistãoRESUMO
Transferases have emerged as among the best catalysts for enzyme-mediated bioorthogonal functional group installation to advance innovative in vitro, cell-based and in vivo chemical biology applications. This review introduces the key considerations for selecting enzyme catalysts and chemoselective reactions most amenable to bioorthogonal platform development and highlights relevant key technology development and applications for one ubiquitous transferase subclass - methyltransferases (MTs). Within this context, recent advances in MT-enabled bioorthogonal labeling/conjugation relevant to DNA, RNA, protein, and natural products (i.e. complex small molecule metabolites) are highlighted.
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Metiltransferases , Proteínas , Catálise , RNARESUMO
BACKGROUND: Histone deacetylases (HDACs) regulate transcriptional responses to injury stimuli that are critical for successful tissue regeneration. Previously we showed that HDAC inhibitor romidepsin potently inhibits axolotl tail regeneration when applied for only 1-minute postamputation (MPA). RESULTS: Here we tested CoCl2, a chemical that induces hypoxia and cellular stress, for potential to reverse romidepsin inhibition of tail regeneration. Partial rescue of regeneration was observed among embryos co-treated with romidepsin and CoCl2 for 1 MPA, however, extending the CoCl2 dosage window either inhibited regeneration (CoCl2 :0 to 30 MPA) or was lethal (CoCl2 :0 to 24 hours postamputation; HPA). CoCl2 :0 to 30 MPA caused tissue damage, tissue loss, and cell death at the distal tail tip, while CoCl2 treatment of non-amputated embryos or CoCl2 :60 to 90 MPA treatment after re-epithelialization did not inhibit tail regeneration. CoCl2 -romidepsin:1 MPA treatment partially restored expression of transcription factors that are typical of appendage regeneration, while CoCl2 :0 to 30 MPA significantly increased expression of genes associated with cell stress and inflammation. Additional experiments showed that CoCl2 :0 to 1 MPA and CoCl2 :0 to 30 MPA significantly increased levels of glutathione and reactive oxygen species, respectively. CONCLUSION: Our study identifies a temporal window from tail amputation to re-epithelialization, within which injury activated cells are highly sensitive to CoCl2 perturbation of redox homeostasis.
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Ambystoma mexicanum/fisiologia , Cobalto/farmacologia , Regeneração/efeitos dos fármacos , Cauda/fisiologia , Amputação Cirúrgica , Animais , Morte Celular/efeitos dos fármacos , Depsipeptídeos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Cauda/efeitos dos fármacos , Fatores de TempoRESUMO
New patterns of gene expression are enacted and regulated during tissue regeneration. Histone deacetylases (HDACs) regulate gene expression by removing acetylated lysine residues from histones and proteins that function directly or indirectly in transcriptional regulation. Previously we showed that romidepsin, an FDA-approved HDAC inhibitor, potently blocks axolotl embryo tail regeneration by altering initial transcriptional responses to injury. Here, we report on the concentration-dependent effect of romidepsin on transcription and regeneration outcome, introducing an experimental and conceptual framework for investigating small molecule mechanisms of action. A range of romidepsin concentrations (0-10 µM) were administered from 0 to 6 or 0 to 12 h post amputation (HPA) and distal tail tip tissue was collected for gene expression analysis. Above a threshold concentration, romidepsin potently inhibited regeneration. Sigmoidal and biphasic transcription response curve modeling identified genes with inflection points aligning to the threshold concentration defining regenerative failure verses success. Regeneration inhibitory concentrations of romidepsin increased and decreased the expression of key genes. Genes that associate with oxidative stress, negative regulation of cell signaling, negative regulation of cell cycle progression, and cellular differentiation were increased, while genes that are typically up-regulated during appendage regeneration were decreased, including genes expressed by fibroblast-like progenitor cells. Using single-nuclei RNA-Seq at 6 HPA, we found that key genes were altered by romidepin in the same direction across multiple cell types. Our results implicate HDAC activity as a transcriptional mechanism that operates across cell types to regulate the alternative expression of genes that associate with regenerative success versus failure outcomes.