Antifungal potential of streptomycetes isolated from ethnomedicinal plants' rhizospheres: characterization and optimization of staurosporine biosynthesis by Streptomyces sp. BV410 strain | Antifungalni potencijal streptomiceta izolovanih iz rizosfera medicinski značajnih biljaka : karakterizacija i optimizacija biosinteze staurosporina, produkta metabolizma Streptomyces sp. BV410

Different soils are still a source of remarkable microbial diversity which also reflects in the unexplored chemical diversity. Recent advances in assessment of microbial diversity from soil have revealed the extraordinarily rich biosynthetic potential for the production of new natural products among different microbial strains, especially within the group of Actinobacteria. Among bacterial soil isolates, representatives of Streptomyces genus are the most prolific producers of bioactive compounds. One of the objectives of the present study was to isolate Streptomyces spp. from the rhizosphere soils of three ethno-medicinal plants collected in Serbia (Papaver rhoeas, Matricaria chamomilla, and Urtica dioica) and to screen their antifungal activity against Candida spp. Morphologically different sporulating isolates (103 in total) were collected from rhizosphere soil samples and determined as Streptomyces spp. Two different media and two extraction procedures were used to induce the production and facilitate identification of antifungals. Overall, 412 crude cell extracts were tested against Candida albicans using disk diffusion assays, with 42% (43/103) of the strains showing the ability to produce antifungal agents. Also, extracts inhibited growth of other important human pathogens: Candida krusei, Candida parapsilosis, and Candida glabrata. Based on the established degree and range of antifungal activity, nine isolates were selected for further testing. Their ability to inhibit Candida growth in liquid culture, to inhibit biofilm formation, and to disperse pre-formed biofilms was assessed with active concentrations from 8 to 250 pg/ml. High-performance liquid chromatographic profiles of extracts derived from selected strains were recorded, revealing moderate metabolic diversity. The most potent extracts were subjected to comprehensive identification and structural characterization of antifungal compounds. Applying a bioactivity-guided isolation approach, active compounds of three extracts were separated, and based on NMR structure elucidation it was shown that active compounds were genistein, daidzein and staurosporine. Genistein and daidzein, soy phytoestrogens, are known to inhibit key enzymes in the steroid metabolism pathway and were coming from the fermentation medium containing soy flower. Since isolated Streptomyces spp. showed good ability to extract these molecules from complex medium, they can be further considered for biotechnological production of these phytoestrogens. One of the isolates, Streptomyces sp. BV410, was characterized as an efficient staurosporine producer. Staurosporine is a potent inhibitor of protein kinases and is considered in anticancer therapy. The biotechnological production of staurosporine by strain BV410 was optimized to yield 36.94 mg/l after 14 days of incubation in soy flowerglucose- starch-mannitol based fermentation medium (JS). Further optimization of medium for biosynthesis of staurosporine indicated the following optimal values of the examined factors: the content of glucose of 20 g/l, starch 0.36 g/l, mannitol 21.46 g/l, soy flower 17.32 g/l. By applying the defined optimal values and using the appropriate mathematical models, the following responses were predicted: concentration of staurosporine 46.88 mg/l and biomass yield 12.05 mg/ml. The validity of the results was confirmed by performing the biosynthesis of the staurosporine in the medium with optimal composition (JSSta). Kinetics of staurosporine and biomass production and carbon source consumption were examined and process models were developed. Additionally, optimization of staurosporine production was performed with different supplements which, according to literature data, had stimulative effect on secondary metabolism (Zn, Fe and P salts, methyl oleate, grape seed oil). In order to improve the production of staurosporine, effects of pH (6.5, 7.5 and 8.5) and incubation time (7, 10 and 14 days) were also examined. It was found that addition of FeS04 significantly improved the staurosporine yield in comparison to the starting conditions (increase of 25%). Our results proved that rhizosphere soils of ethno-medicinal plants are a prolific source of streptomycetes, producers of compounds with good antifungal activity. Isolation of the new staurosporine producing strain, allowed for its detailed bioactivity assessment. Staurosporine scaffold might serve as a lead structure for the development of new antifungal and antiangiogenic agents. Also, results obtained within this research represent the basis for the further scale-up and potential industrialization of the proposed production process.