Targeted Gene Disruption of the Cyclo (L-Phe, L-Pro) Biosynthetic Pathway in Streptomyces sp. US24 Strain

We have previously isolated a new actinomycete strain from Tunisian soil called Streptomyces sp. US24, and have shown that it produces two bioactive molecules including a Cyclo (L-Phe, L-Pro) diketopiperazine (DKP). To identify the structural genes responsible for the synthesis of this DKP derivative, a PCR amplification (696 bp) was carried out using the Streptomyces sp. US24 genomic DNA as template and two degenerate oligonucleotides designed by analogy with genes encoding peptide synthetases (NRPS). The detection of DKP derivative biosynthetic pathway of the Streptomyces sp. US24 strain was then achieved by gene disruption via homologous recombination using a suicide vector derived from the conjugative plasmid pSET152 and containing the PCR product. Chromatography analysis, biological tests and spectroscopic studies of supernatant cultures of the wild-type Streptomyces sp. US24 strain and three mutants obtained by this gene targeting disruption approach showed that the amplified DNA fragment is required for Cyclo (L-Phe, L-Pro) biosynthesis in Streptomyces sp. US24 strain. This DKP derivative seems to be produced either directly via a nonribosomal pathway or as a side product in the course of nonribosomal synthesis of a longer peptide

Integrative Gene Cloning and Expression System for Streptomyces sp. US 24 and Streptomyces sp. TN 58 Bioactive Molecule Producing Strains

Streptomyces sp. US 24 and Streptomyces sp. TN 58, two strains producing interesting bioactive molecules, were successfully transformed using E. coli ET12567 (pUZ8002), as a conjugal donor, carrying the integrative plasmid pSET152. For the Streptomyces sp. US 24 strain, two copies of this plasmid were tandemly integrated in the chromosome, whereas for Streptomyces sp. TN 58, the integration was in single copy at the attB site. Plasmid pSET152 was inherited every time for all analysed Streptomyces sp. US 24 and Streptomyces sp. TN 58 exconjugants under nonselective conditions. The growth, morphological differentiation, and active molecules production of all studied pSET152 integrated exconjugants were identical to those of wild type strains. Consequently, conjugal transfer using pSET152 integration system is a suitable means of genes transfer and expression for both studied strains. To validate the above gene transfer system, the glucose isomerase gene (xylA) from Streptomyces sp. SK was expressed in strain Streptomyces sp. TN 58. Obtained results indicated that heterologous glucose isomerase could be expressed and folded effectively. Glucose isomerase activity of the constructed TN 58 recombinant strain is of about eighteenfold higher than that of the Streptomyces sp. SK strain. Such results are certainly of importance due to the potential use of improved strains in biotechnological process for the production of high-fructose syrup from starch

Modeling-based optimization approaches for the development of Anti- Agrobacterium tumefaciens activity using Streptomyces sp TN71

A new aerobic bacterium TN71 was isolated from Tunisian Saharan soil and has been selected for its antimicrobial activity against phytopathogenic bacteria. Based on cellular morphology, physiological characterization and phylogenetic analysis, this isolate has been assigned as Streptomyces sp. TN71 strain. In an attempt to increase its anti-Agrobacterium tumefaciens activity, GYM + S (glucose, yeast extract, malt extract and starch) medium was selected out of five different production media and the medium composition was optimized. Plackett-Burman design (PBD) was used to select starch, malt extract and glucose as parameters having significant effects on antibacterial activity and a Box-Behnken design was applied for further optimization. The analysis revealed that the optimum concentrations for anti-A. tumefaciens activity of the tested variables were 19.49 g/L for starch, 5.06 g/L for malt extract and 2.07 g/L for glucose. Several Artificial Neural Networks (ANN): the Multilayer perceptron (MLP) and the Radial basis function (RBF) were also constructed to predict anti-A. tumefaciens activity. The comparison between experimental with predicted outputs from ANN and Response Surface Methodology (RSM) were studied. ANN model presents an improvement of 12.36% in terms of determination coefficients of anti A. tumefaciens activity. To our knowledge, this is the first work reporting the statistical versus artificial intelligence based modeling for optimization of bioactive molecules against phytopathogen

Bioactive secondary metabolites from new endophytic fungus Curvularia. sp isolated from Rauwolfia macrophylla.

Kaaniche F, Hamed A, Abdel-Razek AS, et al. Bioactive secondary metabolites from new endophytic fungus Curvularia. sp isolated from Rauwolfia macrophylla. PLoS ONE. 2019;14(6): E0217627.Over the last decades, endophytic fungi represent a new source of pharmacologically active secondary metabolites based on the underlying assumption that they live symbiotically within their plant host. In the present study, a new endophytic fungus was isolated from Rauwolfia macrophylla, a medicinal plant from Cameroon. The fungus showed a highest homology to Curvularia sp. based on complete nucleotide sequence data generated from the internal transcribed spacer (ITS) of ribosomal DNA region. Large scale fermentation, working-up and separation of the strain extract using different chromatographic techniques afforded three bioactive compounds: 2'-deoxyribolactone (1), hexylitaconic acid (2) and ergosterol (3). The chemical structures of compounds 1-3 were confirmed by 1 and 2D NMR spectroscopy and mass spectrometry, and comparison with corresponding literature data. Biologically, the antimicrobial, antioxidant activities and the acetylcholinesterase inhibitory of the isolated compounds were studied

Inhibition of Fungi and Gram-Negative Bacteria by Bacteriocin BacTN635 Produced by Lactobacillus plantarum sp. TN635

The aim of this study was to evaluate 54 lactic acid bacteria (LAB) strains isolated from meat, fermented vegetables and dairy products for their capacity to produce antimicrobial activities against several bacteria and fungi. The strain designed TN635 has been selected for advanced studies. The supernatant culture of this strain inhibits the growth of all tested pathogenic including the four Gram-negative bacteria (Salmonella enterica ATCC43972, Pseudomonas aeruginosa ATCC 49189, Hafnia sp. and Serratia sp.) and the pathogenic fungus Candida tropicalis R2 CIP203. Based on the nucleotide sequence of the 16S rRNA gene of the strain TN635 (1,540 pb accession no FN252881) and the phylogenetic analysis, we propose the assignment of our new isolate bacterium as Lactobacillus plantarum sp. TN635 strain. Its antimicrobial compound was determined as a proteinaceous substance, stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 3 and 11 with an optimum at pH = 7. The BacTN635 was purified to homogeneity by a four-step protocol involving ammonium sulfate precipitation, centrifugal microconcentrators with a 10-kDa membrane cutoff, gel filtration Sephadex G-25, and C18 reverse-phase HPLC. SDS-PAGE analysis of the purified BacTN635, revealed a single band with an estimated molecular mass of approximately 4 kDa. The maximum bacteriocin production (5,000 AU/ml) was recorded after a 16-h incubation in Man, Rogosa, and Sharpe (MRS) medium at 30 °C. The mode of action of the partial purified BacTN635 was identified as bactericidal against Listeria ivanovii BUG 496 and as fungistatic against C. tropicalis R2 CIP203

Bioactive Secondary Metabolites from a New Terrestrial Streptomyces sp. TN262

During our search for Streptomyces spp. as new producers of bioactive secondary metabolites, the ethyl acetate extract of the new terrestrial Streptomyces isolate TN262 delivered eight antimicrobially active compounds. They were identified as 1-acetyl-β-carboline (1), tryptophol (2), cineromycin B (3), 2,3-dihydrocineromycin B (4), cyclo-(tyrosylprolyl) (5), 3-(hydroxyacetyl)-indole (6), brevianamide F (7), and cis-cyclo-(l-prolyl-l-leucyl) (8). Three further metabolites were detected in the unpolar fractions using GC–MS and tentatively assigned as benzophenone (9), N-butyl-benzenesulfonamide (10), and hexanedioic acid-bis-(2-ethylhexyl) ester (11). This last compound is known as plasticizer derivatives, but it has never been described from natural sources. In this article, we describe the identification of the new Streptomyces sp. isolate TN262 using its cultural characteristics, the nucleotide sequence of the corresponding 16S rRNA gene and the phylogenetic analysis, followed by optimization, large-scale fermentation, isolation of the bioactive constituents, and determination of their structures. The biological activity of compounds (2), (3), (4), and those of the unpolar fractions was addressed as well

Regulation of the expression of amy TO1 encoding a thermostable α-amylase from Streptomyces sp. TO1, in its original host and in Streptomyces lividans TK24

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Anti-oxidant, antimicrobial and anti-acetylcholinesterase activities of organic extracts from aerial parts of three Tunisian plants and correlation with polyphenols and flavonoids contents

In goal of searching new active compounds with important biological activities, a screening of several plants from salt-marsh region of Chebba-Tunisia had been realized. Three species had been selected: Calendula arvensis, Chenopodium murale and Nicotiana glauca. The organic extracts of different aerial parts of these plants (stems, leaves and flowers) displayed variable contents of total polyphenols (TPP) and total flavonoids (TF). Flowers acetone extract from N. glauca contained the higher quantity in TPP (264.8 ± 1.6 µg GA/mg), while stems dichloromethane extract exhibited the best TF content (49.8 ± 2.2 µg QE/mg). The important TPP and TF contents reflected a good anti-oxidant and antimicrobial activities. The best acetylcholinesterase inhibitory activity had been shown in the fractions obtained after extraction with low polarity solvents. Whence, a correlation of flavonoids contents with biological activities had been shown, while, there was no correlation with acetylcholinesterase inhibitory activity. Video Clip of Methodology: DPPH radical scavenging activity: 3 min   Full Screen   Alternat

A New Endophytic Fusarium Oxysporum Gibberellic Acid: Optimization of Production Using Combined Strategies of Experimental Designs and Potency on Tomato Growth under Stress Condition

This study reports the potential of the endophytic fungi identified as a Fusarium oxysporum to produce gibberellic acid (GA3). The GA3 production was confirmed by high performance liquid chromatography. To improve the production of this phytohormone under solid state fermentation (SSF), successive optimization strategies were used. Firstly, Plackett-Burman design was applied for screening medium components and culture condition. Under the optimized condition, GA3 yield (7.14 g/kg) was 2.62-fold higher than by the use of the initial condition (2.72 g/kg). The concentration of the most influential parameters and their interaction were optimized with a Box-Behnken experimental design. The optimized condition led to a 1.14-fold enhancement in GA3 production, reaching 8.16 g/kg. The GA3 crude extract obtained by SSF was then used to study its ameliorative role on adverse salinity effect on tomato plants (Solanum lycopersicum L.). The interactive effects of different GA3 concentrations were examined on morphological and physiological parameters of tomato plants. The application of GA3 (10-6 M) under salt stress condition (100 mM) was found to improve growth and physiological parameters including plant height, total chlorophyll, starch, and proline contents. The exogenous application of GA3 is a potent strategy to reverse abiotic stress that affect the agricultural productivity and limit plant growth and yield