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.

Keywords: Actinomycin D; Central composite design; Electrospray mass spectrometry; Microbiology; Optimization; Plackett-Burman design; Saharan saline soil; Streptomyces sp..

Fig. 1

Actinobacteria colony number per culture media (Chitin, Gausse and Starch Casein Agar) and incubation time obtained from Saharan soil samples.

Fig. 2

Hyphae and spore chains of strain GSBNT10 grown on Gausse medium at 28 °C for 7 days; (a) A sporulated aerial mycelium, (b) Optic microscopy view (X40) and (c) Scanning electron microscopy, bar = 10 μm, (x 8500).

Fig. 3

Phylogenetic tree calculated from almost-complete 16S rRNA gene sequences (1491 nt) showing relationships between the strain GSBNT10 and the most related type species of the genus Streptomyces. The tree was constructed using Jukes and Cantor (1969) evolutionary distance methods and the neighbour-joining method of Saitou and Nei (1987). Bootstrap values above 50% (percentages of 1000 replications) are indicated at nodes. Bar, 0.02 substitutions per nucleotide position. Actinopolyspora mortivallis DSM 44261^Twas used as the out group.

Fig. 4

Online LC-MS analysis of ethyl acetate crude extract displaying actinomycin D analogue at t_R = 8.0 min from Streptomyces sp. GSBNT10: extracted–ion chromatogram (top), and corresponding MS spectra in negative (middle) and in positive ion mode (bottom).

Fig. 5

Online LC-MS analysis of ethyl acetate crude extract displaying the major metabolite actinomycin D at t_R = 9.3 min from GSBNT10: extracted–ion chromatogram (top), and corresponding MS spectra in negative (middle) and in positive ion mode (bottom).

Fig. 6

Online LC-MS analysis of ethyl acetate crude extract displaying actinomycin X2 at t_R = 10.4 min from GSBNT10 strain: extracted –ion chromatogram (top), and corresponding MS spectra in negative (middle) and in positive ion mode (bottom).

Fig. 7

Molecular structures of metabolites produced by Streptomyces sp. GSBNT10 strain: the major actinomycin D (act-D) and the minor actinomycin X₂.

Fig. 8

ESI -MS of act-D in positive ion mode: m/z 1277 corresponding to [M + Na]⁺ ion (top); MS/MS (middle) and MS³ (bottom) spectra by fragmentation experiment on m/z 1277.

Fig. 9

Pareto chart showing the effect of 6 variables on act-D production.

Fig. 10

Plot of residuals versus fitted values for act-D production.

Fig. 11

Contours plots of the predicted act-D concentration.

Fig. 12

Predicted and actual values of media composition and act-D production.

Fig. 13

(a) Relative peak areas of act-D eluted at t_R = 9.3 min in the chromatographic profile of the strain Streptomyces sp. GSBNT10, (b) Relative intensity of chromatographic peaks of act-D eluted t_R = 9.3 min by UV-DAD HPLC analysis (reversed phase C18 column, methanol-water 75:25, flow 1mLmin^-1, detection at 440 nm).