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

Abstract

Zemljište predstavlja bogat izvor različitih mikroorganizama čiji produkti metabolizma mogu biti od izuzetnog značaja za čoveka. Dosadašnja ispitivanja mikrobnog diverziteta u zemljištu su otkrila bogati biosintetski potencijal za proizvodnju novih prirodnih proizvoda kod velikog broja mikroorganizama, naročito kada je u pitanju klasa Actinobacteria. Među zemljišnim izolatima, rod Streptomyces prednjači po broju identifikovanih bioaktivnih molekula u odnosu na sve ostale bakterije. Stoga je jedan od ciljeva u okviru ove doktorske disertacije izolacija streptomiceta iz rizosfera medicinski značajnih biljaka sakupljenih na teritoriji Republike Srbije (Papaver rhoeas, Matricaria chamomilla, i Urtica dioica) i ispitivanje njihovog antifungalnog potencijala na različite vrste kandida. Morfološki različiti izolati (ukupno 103) su izolovani iz uzoraka rizosfera i okarakterisani kao streptomicete. Dve različite podloge i dve procedure za ekstrakciju su korišćene da bi se pospešila detekcija antifungalnih jedinjenja. Ispitan je uticaj ukupno 412 ekstrakata na rast Candida albicans disk difuzionim esejem pri čemu je utvrđeno da 42% (43/103) izolata imaju sposobnost proizvodnje antifungalnih jedinjenja pri ispitivanim uslovima. Pojedini ekstrakti su inhibirali rast važnih humanih patogena poput Candida krusei, Candida parapsilosis, i Candida glabrata. Na osnovu stepena i spektra antifungalne aktivnosti devet izolata je odabrano za dalja istraživanja. Ispitana je sposobnost njihovih ekstrakata da inhibiraju rast kandida u tečnoj kulturi i u formi biofilma, a takođe je ispitan i njihov uticaj na već formirane biofilmove kandide u koncentracijama od 8 do 250 pg/ml. Hromatografski profili ovih ekstrakata i uvid u njihovu metaboličku raznolikost dobijeni su korišćenjem tečne hromatografije visokih performansi. Tri ekstrakta sa specifičnom antifungalnom aktivnošću podvrgnuta su hemijskim analizama s ciljem da se detektuju i strukturno okarakterišu molekuli koji su nosioci antifungalne aktivnosti. Na osnovu rezultata nuklearnomagnetno- rezonantne spektroskopije otkriveno je da su aktivni molekuli genistein, daidzein i staurosporin. Genistein i daidzein koji su poznati fitoestrogeni poreklom iz sojinog brašna za koje je poznato da inhibiraju ključne enzime u biosintetskom putu steroida. Njihovo prisustvo je u ovom istraživanju detektovano usled korišćenja sojinog brašna u hranljivoj podlozi. Kako streptomicete u čijim ekstraktima su detektovani ovi molekuli pokazuju sposobnost oslobađanja ovih važnih jedinjenja iz kompleksne hranljive podloge, mogu se uzeti u razmatranje za biotehnološku proizvodnju fitoestrogena. Staurosporin je detektovan kao nosilac antifungalne aktivnosti kod ekstrakta soja Streptomyces sp. BV410. Staurosporin je inhibitor protein kinaza i njegovi derivati i analozi se koriste u kao antitumorski agensi. Biosinteza ovog molekula je optimizovana do prinosa od 36,94 mg/l nakon 14 dana gajenja u hranljivoj podlozi koja sadrži glukozu, skrob, manitol i sojino brašno (JS). Dalja optimizacija hranljive podloge za biosintezu staurosporina ukazala je na sledeći sastav hranljive podloge: 20 g/l glukoze, 0,36 g/l skroba, 21,46 g/l manitola, 17,32 g/l sojinog brašna. Primenom definisanih optimalnih vrednosti i korišćenjem odgovarajućih matematičkih modela, predviđeno je da će se na ovaj način postići prinosi od 46,88 mg/l staurosporina i 12,05 mg/ml biomase. Validnost predviđenih rezultata potvrđena je izvođenjem bioprocesa u optimizovanoj hranljivoj podlozi (JSSta). Ispitana je kinetika biosinteze staurosporina i produkcije biomase, kao i potrošnje izvora ugljenika i razvijeni su odgovarajući procesni modeli. Dodatna optimizacija je podrazumevala dodatak suplemenata koji prema literature stimulišu sekundarni metabolizam streptomiceta (joni cinka, gvožđa, fosfati, metil oleat, ulje semenki grožđa). Ovi eksperimenti su izvođeni na tri pH vrednosti (6,5, 7,5 i 8,5) a uspešnost bioprocesa je procenjivana 7., 10. i 14. dana gajenja. Dodatna optimizacija je dovela do podatka da dodatak soli gvožđa značajno pospešuje biosintezu staurosporina sa povećanjem prinosa od 25%. Dobijeni rezultati potvrđuju da su rizosfere medicinski značajnih biljaka značajan izvor streptomiceta koje proizvode komponente sa antifungalnom aktivnošću. Izolacija novog proizvođača staurosporina i optimizacija procesa njegove biosinteze omogućiće dalja istraživanja ovog jedinjenja koje može biti osnova za razvoj novih antifungalnih i jedinjenja koja inhibiraju angiogenezu. Rezultati dobijeni u okviru ovih istraživanja predstavljaju početni korak ka potencijalnoj industrijalizaciji proizvodnje staurosporina.

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.