Antifungalni potencijal streptomiceta izolovanih iz rizosfera medicinski značajnih biljaka: karakterizacija i optimizacija biosinteze staurosporina, produkta metabolizma Streptomyces sp. BV410

Autor: Mojićević, Marija
Přispěvatelé: Grahovac, Jovana, Dodić, Jelena, Cvetković, Dragoljub, Vojnović, Sandra
Rok vydání: 2019
Předmět:
Zdroj: Универзитет у Новом Саду
CRIS UNS
Popis: Zemljište predstavlja bogat izvor različitih mikroorganizama čijiprodukti metabolizma mogu biti od izuzetnog značaja za čoveka.Dosadašnja ispitivanja mikrobnog diverziteta u zemljištu suotkrila bogati biosintetski potencijal za proizvodnju novihprirodnih proizvoda kod velikog broja mikroorganizama, naročitokada je u pitanju klasa Actinobacteria. Među zemljišnim izolatima,rod Streptomyces prednjači po broju identifikovanih bioaktivnihmolekula u odnosu na sve ostale bakterije. Stoga je jedan odciljeva u okviru ove doktorske disertacije izolacija streptomiceta izrizosfera medicinski značajnih biljaka sakupljenih na teritorijiRepublike Srbije (Papaver rhoeas, Matricaria chamomilla, i Urticadioica) i ispitivanje njihovog antifungalnog potencijala na različitevrste kandida. Morfološki različiti izolati (ukupno 103) su izolovaniiz uzoraka rizosfera i okarakterisani kao streptomicete. Dverazličite podloge i dve procedure za ekstrakciju su korišćene da bise pospešila detekcija antifungalnih jedinjenja. Ispitan je uticajukupno 412 ekstrakata na rast Candida albicans disk difuzionimesejem pri čemu je utvrđeno da 42% (43/103) izolata imajusposobnost proizvodnje antifungalnih jedinjenja pri ispitivanimuslovima. Pojedini ekstrakti su inhibirali rast važnih humanihpatogena poput Candida krusei, Candida parapsilosis, i Candidaglabrata. Na osnovu stepena i spektra antifungalne aktivnostidevet izolata je odabrano za dalja istraživanja. Ispitana jesposobnost njihovih ekstrakata da inhibiraju rast kandida u tečnojkulturi i u formi biofilma, a takođe je ispitan i njihov uticaj na većformirane biofilmove kandide u koncentracijama od 8 do 250pg/ml. Hromatografski profili ovih ekstrakata i uvid u njihovumetaboličku raznolikost dobijeni su korišćenjem tečnehromatografije visokih performansi. Tri ekstrakta sa specifičnomantifungalnom aktivnošću podvrgnuta su hemijskim analizama sciljem da se detektuju i strukturno okarakterišu molekuli koji sunosioci antifungalne aktivnosti. Na osnovu rezultata nuklearnomagnetno-rezonantne spektroskopije otkriveno je da su aktivnimolekuli genistein, daidzein i staurosporin. Genistein i daidzeinkoji su poznati fitoestrogeni poreklom iz sojinog brašna za koje jepoznato da inhibiraju ključne enzime u biosintetskom putusteroida. Njihovo prisustvo je u ovom istraživanju detektovanousled korišćenja sojinog brašna u hranljivoj podlozi. Kakostreptomicete u čijim ekstraktima su detektovani ovi molekulipokazuju sposobnost oslobađanja ovih važnih jedinjenja izkompleksne hranljive podloge, mogu se uzeti u razmatranje zabiotehnološku proizvodnju fitoestrogena. Staurosporin jedetektovan kao nosilac antifungalne aktivnosti kod ekstrakta sojaStreptomyces sp. BV410. Staurosporin je inhibitor protein kinaza injegovi derivati i analozi se koriste u kao antitumorski agensi.Biosinteza ovog molekula je optimizovana do prinosa od 36,94mg/l nakon 14 dana gajenja u hranljivoj podlozi koja sadržiglukozu, skrob, manitol i sojino brašno (JS). Dalja optimizacijahranljive podloge za biosintezu staurosporina ukazala je nasledeći sastav hranljive podloge: 20 g/l glukoze, 0,36 g/l skroba,21,46 g/l manitola, 17,32 g/l sojinog brašna. Primenomdefinisanih optimalnih vrednosti i korišćenjem odgovarajućihmatematičkih modela, predviđeno je da će se na ovaj načinpostići prinosi od 46,88 mg/l staurosporina i 12,05 mg/mlbiomase. Validnost predviđenih rezultata potvrđena jeizvođenjem bioprocesa u optimizovanoj hranljivoj podlozi (JSSta).Ispitana je kinetika biosinteze staurosporina i produkcije biomase,kao i potrošnje izvora ugljenika i razvijeni su odgovarajućiprocesni modeli. Dodatna optimizacija je podrazumevala dodataksuplemenata koji prema literature stimulišu sekundarnimetabolizam streptomiceta (joni cinka, gvožđa, fosfati, metiloleat, ulje semenki grožđa). Ovi eksperimenti su izvođeni na tri pHvrednosti (6,5, 7,5 i 8,5) a uspešnost bioprocesa je procenjivana 7.,10. i 14. dana gajenja. Dodatna optimizacija je dovela do podatkada dodatak soli gvožđa značajno pospešuje biosintezustaurosporina sa povećanjem prinosa od 25%. Dobijeni rezultatipotvrđuju da su rizosfere medicinski značajnih biljaka značajanizvor streptomiceta koje proizvode komponente saantifungalnom aktivnošću. Izolacija novog proizvođačastaurosporina i optimizacija procesa njegove biosintezeomogućiće dalja istraživanja ovog jedinjenja koje može bitiosnova za razvoj novih antifungalnih i jedinjenja koja inhibirajuangiogenezu. Rezultati dobijeni u okviru ovih istraživanjapredstavljaju početni korak ka potencijalnoj industrijalizacijiproizvodnje staurosporina.
Different soils are still a source of remarkable microbial diversitywhich also reflects in the unexplored chemical diversity. Recentadvances in assessment of microbial diversity from soil haverevealed the extraordinarily rich biosynthetic potential for theproduction of new natural products among different microbialstrains, especially within the group of Actinobacteria. Amongbacterial soil isolates, representatives of Streptomyces genus arethe most prolific producers of bioactive compounds. One of theobjectives of the present study was to isolate Streptomyces spp.from the rhizosphere soils of three ethno-medicinal plantscollected in Serbia (Papaver rhoeas, Matricaria chamomilla, andUrtica dioica) and to screen their antifungal activity againstCandida spp. Morphologically different sporulating isolates (103in total) were collected from rhizosphere soil samples anddetermined as Streptomyces spp. Two different media and twoextraction procedures were used to induce the production andfacilitate identification of antifungals. Overall, 412 crude cellextracts were tested against Candida albicans using diskdiffusion assays, with 42% (43/103) of the strains showing theability to produce antifungal agents. Also, extracts inhibitedgrowth of other important human pathogens: Candida krusei,Candida parapsilosis, and Candida glabrata. Based on theestablished degree and range of antifungal activity, nine isolateswere selected for further testing. Their ability to inhibit Candidagrowth in liquid culture, to inhibit biofilm formation, and todisperse pre-formed biofilms was assessed with activeconcentrations from 8 to 250 pg/ml. High-performance liquidchromatographic profiles of extracts derived from selectedstrains were recorded, revealing moderate metabolic diversity.The most potent extracts were subjected to comprehensiveidentification and structural characterization of antifungalcompounds. Applying a bioactivity-guided isolation approach,active compounds of three extracts were separated, and basedon NMR structure elucidation it was shown that activecompounds were genistein, daidzein and staurosporine.Genistein and daidzein, soy phytoestrogens, are known to inhibitkey enzymes in the steroid metabolism pathway and werecoming from the fermentation medium containing soy flower.Since isolated Streptomyces spp. showed good ability to extractthese molecules from complex medium, they can be furtherconsidered for biotechnological production of thesephytoestrogens. One of the isolates, Streptomyces sp. BV410,was characterized as an efficient staurosporine producer.Staurosporine is a potent inhibitor of protein kinases and isconsidered in anticancer therapy. The biotechnologicalproduction of staurosporine by strain BV410 was optimized toyield 36.94 mg/l after 14 days of incubation in soy flowerglucose-starch-mannitol based fermentation medium (JS).Further optimization of medium for biosynthesis ofstaurosporine indicated the following optimal values of theexamined factors: the content of glucose of 20 g/l, starch 0.36g/l, mannitol 21.46 g/l, soy flower 17.32 g/l. By applying thedefined optimal values and using the appropriate mathematicalmodels, the following responses were predicted: concentrationof staurosporine 46.88 mg/l and biomass yield 12.05 mg/ml. Thevalidity of the results was confirmed by performing thebiosynthesis of the staurosporine in the medium with optimalcomposition (JSSta). Kinetics of staurosporine and biomassproduction and carbon source consumption were examined andprocess models were developed. Additionally, optimization ofstaurosporine production was performed with differentsupplements which, according to literature data, had stimulativeeffect on secondary metabolism (Zn, Fe and P salts, methyloleate, grape seed oil). In order to improve the production ofstaurosporine, effects of pH (6.5, 7.5 and 8.5) and incubation time(7, 10 and 14 days) were also examined. It was found thataddition of FeS04 significantly improved the staurosporine yieldin comparison to the starting conditions (increase of 25%). Ourresults proved that rhizosphere soils of ethno-medicinal plantsare a prolific source of streptomycetes, producers of compoundswith good antifungal activity. Isolation of the new staurosporineproducing strain, allowed for its detailed bioactivity assessment.Staurosporine scaffold might serve as a lead structure for thedevelopment of new antifungal and antiangiogenic agents. Also,results obtained within this research represent the basis for thefurther scale-up and potential industrialization of the proposedproduction process.
Databáze: OpenAIRE
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