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Modeling improved production of the chemotherapeutic polypeptide actinomycin D by a novel Streptomyces sp. strain from a Saharan soil.

ABSTRACT: The novel bioactive actinobacterial strain GSBNT10 obtained from a Saharan soil, was taxonomically characterized using a polyphasic approach. 16S rRNA gene sequence analysis supported the classification of the isolate within the genus Streptomyces indicating it as a novel species. The major metabolite responsible of the bioactivity was purified and structurally characterized as actinomycin D (act-D) by mass spectrometric and nuclear magnetic resonance analyses Plackett-Burman design (PBD) and response surface methodology (RSM) were applied in order to optimize the medium formulation for the production of this bioactive metabolite. By PBD experiments, NaNO3, K2HPO4 and initial pH value were selected as significant variables affecting the metabolite production. Central Composite Design (CCD) showed that adjustment of the fermentative medium at pH 8.25, K2HPO4 at 0.2 gL-1 and NaNO3 at 3.76 gL-1 were the values suiting the production of act-D. Moreover, the results obtained by the statistical approach were confirmed by act-D detection using the HPLC equipped with a diode array detector and coupled online with electrospray-mass spectrometry (ESIMS) technique. act-D production was highly stimulated, obtaining a good yield (656.46 mgL-1) which corresponds to a 58.56% increase compared with the non-optimized conditions and data from LC-ESIMS technique efficiently confirmed the forecast from RSM.

SUBMITTER: Djinni I

PROVIDER: S-EPMC6538969 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Modeling improved production of the chemotherapeutic polypeptide actinomycin D by a novel <i>Streptomyces</i> sp. strain from a Saharan soil.

Djinni Ibtissem I Defant Andrea A Djoudi Warda W Chaabane Chaouch Faouzia F Souagui Samiha S Kecha Mouloud M Mancini Ines I

Heliyon 20190527 5

The novel bioactive actinobacterial strain GSBNT10 obtained from a Saharan soil, was taxonomically characterized using a polyphasic approach. 16S rRNA gene sequence analysis supported the classification of the isolate within the genus <i>Streptomyces</i> indicating it as a novel species. The major metabolite responsible of the bioactivity was purified and structurally characterized as actinomycin D (act-D) by mass spectrometric and nuclear magnetic resonance analyses Plackett-Burman design (PBD) ...[more]

PMID: 31193702

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Project description:The Actinomycetia isolate PBR11 was isolated from the forest rhizosphere soil of Pobitora Wildlife Sanctuary (PWS), Assam, India. The isolate was identified as Streptomyces sp. with 92.91% sequence similarity to their closest type strain, Streptomyces atrovirens NRRL B-16357 DQ026672. The strain demonstrated significant antimicrobial activity against 19 test pathogens, including multidrug-resistant (MDR) clinical isolates and dermatophytes. Phenol, 2,5-bis(1,1-dimethylethyl), is the major chemical compound detected by gas chromatography-mass spectrometry in the ethyl acetate extract of PBR11 (EtAc-PBR11). The presence of the PKS type II gene (type II polyketide synthases) and chitinase gene suggested that it has been involved in the production of antimicrobial compounds. Metabolic profiling of the EtAc-PBR11 was performed by thin-layer chromatography and flash chromatography resulted in the extraction of two bioactive fractions, namely, PBR11Fr-1 and PBR11Fr-2. Liquid chromatography-tandem mass spectrometry analysis of both the fractions demonstrated the presence of significant antimicrobial compounds, including ethambutol. This is the first report on the detection of antituberculosis drug in the bioactive fractions of Streptomyces sp. PBR11. EtAc-PBR11 and PBR11Fr-1 showed the lowest MIC values (>0.097 and >0.048 μg/mL, respectively) against Candida albicans MTCC 227, whereas they showed the highest MIC values (>0.390 and >0.195 μg/mL, respectively) against Escherichia coli ATCC BAA-2469. The effects of PBR11Fr-1 were investigated on the pathogens by using a scanning electron microscope. The results indicated major morphological alterations in the cytoplasmic membrane. PBR11Fr-1 exhibited low cytotoxicity on normal hepatocyte cell line (CC-1) and the percent cell viability started to decline as the concentration increased from 50 μg/mL (87.07% ± 3.22%) to 100 μg/mL (81.26% ± 2.99%). IMPORTANCE Novel antibiotic breakthroughs are urgently required to combat antimicrobial resistance. Actinomycetia are the principal producers of antibiotics. The present study demonstrated the broad-spectrum antimicrobial potential of an Actinomycetia strain Streptomyces sp. strain PBR11 isolated from the PWS of Assam, India, which represents diverse, poorly screened habitats for novel microorganisms. The strain displayed 92.4% sequence similarity with genes of the closest type strain, indicating that the strain may represent a novel taxon within the phylum Actinomycetota. The metabolomics studies of EtAc-PBR11 revealed structurally diverse antimicrobial agents, including the detection of the antituberculosis drug ethambutol, in the bioactive fraction of Streptomyces sp. PBR11 for the first time. The PBR11 strain also yielded positive results for the antibiotic synthesis gene and the chitinase gene, both of which are responsible for broad-spectrum antimicrobial activity. This suggests that the untouched forest ecosystems have a tremendous potential to harbor potent actinomycetia for future drug discovery.

Project description:Sequencing the actinomycin (acm) biosynthetic gene cluster of Streptomyces antibioticus IMRU 3720, which produces actinomycin X (Acm X), revealed 20 genes organized into a highly similar framework as in the bi-armed acm C biosynthetic gene cluster of Streptomyces chrysomallus but without an attached additional extra arm of orthologues as in the latter. Curiously, the extra arm of the S. chrysomallus gene cluster turned out to perfectly match the single arm of the S. antibioticus gene cluster in the same order of orthologues including the the presence of two pseudogenes, scacmM and scacmN, encoding a cytochrome P450 and its ferredoxin, respectively. Orthologues of the latter genes were both missing in the principal arm of the S. chrysomallus acm C gene cluster. All orthologues of the extra arm showed a G +C-contents different from that of their counterparts in the principal arm. Moreover, the similarities of translation products from the extra arm were all higher to the corresponding translation products of orthologue genes from the S. antibioticus acm X gene cluster than to those encoded by the principal arm of their own gene cluster. This suggests that the duplicated structure of the S. chrysomallus acm C biosynthetic gene cluster evolved from previous fusion between two one-armed acm gene clusters each from a different genetic background. However, while scacmM and scacmN in the extra arm of the S. chrysomallus acm C gene cluster are mutated and therefore are non-functional, their orthologues saacmM and saacmN in the S. antibioticus acm C gene cluster show no defects seemingly encoding active enzymes with functions specific for Acm X biosynthesis. Both acm biosynthetic gene clusters lack a kynurenine-3-monooxygenase gene necessary for biosynthesis of 3-hydroxy-4-methylanthranilic acid, the building block of the Acm chromophore, which suggests participation of a genome-encoded relevant monooxygenase during Acm biosynthesis in both S. chrysomallus and S. antibioticus.

Dataset Information

Modeling improved production of the chemotherapeutic polypeptide actinomycin D by a novel Streptomyces sp. strain from a Saharan soil.

Publications

Modeling improved production of the chemotherapeutic polypeptide actinomycin D by a novel <i>Streptomyces</i> sp. strain from a Saharan soil.

Similar Datasets

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