Diss. Nr. 6884 Naturwiss. ETH Zürich.

No description available.

The major purpose of this investigation was to determine the effect of carbon monoxide on the development of various morphological phases of a single isolate of the aquatic streptomycetes and to elucidate the physiological differences between terminal respiration in the primary and secondary stages. A secondary purpose was to repeat the work of Michael Lee Higgins involving the effect of anaerobiosis on the growth of the organism.

No description available.

<p>Zemlji&scaron;te predstavlja bogat izvor različitih mikroorganizama čiji<br />produkti metabolizma mogu biti od izuzetnog značaja za čoveka.<br />Dosada&scaron;nja ispitivanja mikrobnog diverziteta u zemlji&scaron;tu su<br />otkrila bogati biosintetski potencijal za proizvodnju novih<br />prirodnih proizvoda kod velikog broja mikroorganizama, naročito<br />kada je u pitanju klasa Actinobacteria. Među zemlji&scaron;nim izolatima,<br />rod Streptomyces prednjači po broju identifikovanih bioaktivnih<br />molekula u odnosu na sve ostale bakterije. Stoga je jedan od<br />ciljeva u okviru ove doktorske disertacije izolacija streptomiceta iz<br />rizosfera medicinski značajnih biljaka sakupljenih na teritoriji<br />Republike Srbije (Papaver rhoeas, Matricaria chamomilla, i Urtica<br />dioica) i ispitivanje njihovog antifungalnog potencijala na različite<br />vrste kandida. Morfolo&scaron;ki različiti izolati (ukupno 103) su izolovani<br />iz uzoraka rizosfera i okarakterisani kao streptomicete. Dve<br />različite podloge i dve procedure za ekstrakciju su kori&scaron;ćene da bi<br />se pospe&scaron;ila detekcija antifungalnih jedinjenja. Ispitan je uticaj<br />ukupno 412 ekstrakata na rast Candida albicans disk difuzionim<br />esejem pri čemu je utvrđeno da 42% (43/103) izolata imaju<br />sposobnost proizvodnje antifungalnih jedinjenja pri ispitivanim<br />uslovima. Pojedini ekstrakti su inhibirali rast važnih humanih<br />patogena poput Candida krusei, Candida parapsilosis, i Candida<br />glabrata. Na osnovu stepena i spektra antifungalne aktivnosti<br />devet izolata je odabrano za dalja istraživanja. Ispitana je<br />sposobnost njihovih ekstrakata da inhibiraju rast kandida u tečnoj<br />kulturi i u formi biofilma, a takođe je ispitan i njihov uticaj na već<br />formirane biofilmove kandide u koncentracijama od 8 do 250<br />pg/ml. Hromatografski profili ovih ekstrakata i uvid u njihovu<br />metaboličku raznolikost dobijeni su kori&scaron;ćenjem tečne<br />hromatografije visokih performansi. Tri ekstrakta sa specifičnom<br />antifungalnom aktivno&scaron;ću podvrgnuta su hemijskim analizama s<br />ciljem da se detektuju i strukturno okarakteri&scaron;u molekuli koji su<br />nosioci antifungalne aktivnosti. Na osnovu rezultata nuklearnomagnetno-<br />rezonantne spektroskopije otkriveno je da su aktivni<br />molekuli genistein, daidzein i staurosporin. Genistein i daidzein<br />koji su poznati fitoestrogeni poreklom iz sojinog bra&scaron;na za koje je<br />poznato da inhibiraju ključne enzime u biosintetskom putu<br />steroida. Njihovo prisustvo je u ovom istraživanju detektovano<br />usled kori&scaron;ćenja sojinog bra&scaron;na u hranljivoj podlozi. Kako<br />streptomicete u čijim ekstraktima su detektovani ovi molekuli<br />pokazuju sposobnost oslobađanja ovih važnih jedinjenja iz<br />kompleksne hranljive podloge, mogu se uzeti u razmatranje za<br />biotehnolo&scaron;ku proizvodnju fitoestrogena. Staurosporin je<br />detektovan kao nosilac antifungalne aktivnosti kod ekstrakta soja<br />Streptomyces sp. BV410. Staurosporin je inhibitor protein kinaza i<br />njegovi derivati i analozi se koriste u kao antitumorski agensi.<br />Biosinteza ovog molekula je optimizovana do prinosa od 36,94<br />mg/l nakon 14 dana gajenja u hranljivoj podlozi koja sadrži<br />glukozu, skrob, manitol i sojino bra&scaron;no (JS). Dalja optimizacija<br />hranljive podloge za biosintezu staurosporina ukazala je na<br />sledeći sastav hranljive podloge: 20 g/l glukoze, 0,36 g/l skroba,<br />21,46 g/l manitola, 17,32 g/l sojinog bra&scaron;na. Primenom<br />definisanih optimalnih vrednosti i kori&scaron;ćenjem odgovarajućih<br />matematičkih modela, predviđeno je da će se na ovaj način<br />postići prinosi od 46,88 mg/l staurosporina i 12,05 mg/ml<br />biomase. Validnost predviđenih rezultata potvrđena je<br />izvođenjem bioprocesa u optimizovanoj hranljivoj podlozi (JSSta).<br />Ispitana je kinetika biosinteze staurosporina i produkcije biomase,<br />kao i potro&scaron;nje izvora ugljenika i razvijeni su odgovarajući<br />procesni modeli. Dodatna optimizacija je podrazumevala dodatak<br />suplemenata koji prema literature stimuli&scaron;u sekundarni<br />metabolizam streptomiceta (joni cinka, gvožđa, fosfati, metil<br />oleat, ulje semenki grožđa). Ovi eksperimenti su izvođeni na tri pH<br />vrednosti (6,5, 7,5 i 8,5) a uspe&scaron;nost bioprocesa je procenjivana 7.,<br />10. i 14. dana gajenja. Dodatna optimizacija je dovela do podatka<br />da dodatak soli gvožđa značajno pospe&scaron;uje biosintezu<br />staurosporina sa povećanjem prinosa od 25%. Dobijeni rezultati<br />potvrđuju da su rizosfere medicinski značajnih biljaka značajan<br />izvor streptomiceta koje proizvode komponente sa<br />antifungalnom aktivno&scaron;ću. Izolacija novog proizvođača<br />staurosporina i optimizacija procesa njegove biosinteze<br />omogućiće dalja istraživanja ovog jedinjenja koje može biti<br />osnova za razvoj novih antifungalnih i jedinjenja koja inhibiraju<br />angiogenezu. Rezultati dobijeni u okviru ovih istraživanja<br />predstavljaju početni korak ka potencijalnoj industrijalizaciji<br />proizvodnje staurosporina.</p> / <p>Different soils are still a source of remarkable microbial diversity<br />which also reflects in the unexplored chemical diversity. Recent<br />advances in assessment of microbial diversity from soil have<br />revealed the extraordinarily rich biosynthetic potential for the<br />production of new natural products among different microbial<br />strains, especially within the group of Actinobacteria. Among<br />bacterial soil isolates, representatives of Streptomyces genus are<br />the most prolific producers of bioactive compounds. One of the<br />objectives of the present study was to isolate Streptomyces spp.<br />from the rhizosphere soils of three ethno-medicinal plants<br />collected in Serbia (Papaver rhoeas, Matricaria chamomilla, and<br />Urtica dioica) and to screen their antifungal activity against<br />Candida spp. Morphologically different sporulating isolates (103<br />in total) were collected from rhizosphere soil samples and<br />determined as Streptomyces spp. Two different media and two<br />extraction procedures were used to induce the production and<br />facilitate identification of antifungals. Overall, 412 crude cell<br />extracts were tested against Candida albicans using disk<br />diffusion assays, with 42% (43/103) of the strains showing the<br />ability to produce antifungal agents. Also, extracts inhibited<br />growth of other important human pathogens: Candida krusei,<br />Candida parapsilosis, and Candida glabrata. Based on the<br />established degree and range of antifungal activity, nine isolates<br />were selected for further testing. Their ability to inhibit Candida<br />growth in liquid culture, to inhibit biofilm formation, and to<br />disperse pre-formed biofilms was assessed with active<br />concentrations from 8 to 250 pg/ml. High-performance liquid<br />chromatographic profiles of extracts derived from selected<br />strains were recorded, revealing moderate metabolic diversity.<br />The most potent extracts were subjected to comprehensive<br />identification and structural characterization of antifungal<br />compounds. Applying a bioactivity-guided isolation approach,<br />active compounds of three extracts were separated, and based<br />on NMR structure elucidation it was shown that active<br />compounds were genistein, daidzein and staurosporine.<br />Genistein and daidzein, soy phytoestrogens, are known to inhibit<br />key enzymes in the steroid metabolism pathway and were<br />coming from the fermentation medium containing soy flower.<br />Since isolated Streptomyces spp. showed good ability to extract<br />these molecules from complex medium, they can be further<br />considered for biotechnological production of these<br />phytoestrogens. One of the isolates, Streptomyces sp. BV410,<br />was characterized as an efficient staurosporine producer.<br />Staurosporine is a potent inhibitor of protein kinases and is<br />considered in anticancer therapy. The biotechnological<br />production of staurosporine by strain BV410 was optimized to<br />yield 36.94 mg/l after 14 days of incubation in soy flowerglucose-<br />starch-mannitol based fermentation medium (JS).<br />Further optimization of medium for biosynthesis of<br />staurosporine indicated the following optimal values of the<br />examined factors: the content of glucose of 20 g/l, starch 0.36<br />g/l, mannitol 21.46 g/l, soy flower 17.32 g/l. By applying the<br />defined optimal values and using the appropriate mathematical<br />models, the following responses were predicted: concentration<br />of staurosporine 46.88 mg/l and biomass yield 12.05 mg/ml. The<br />validity of the results was confirmed by performing the<br />biosynthesis of the staurosporine in the medium with optimal<br />composition (JSSta). Kinetics of staurosporine and biomass<br />production and carbon source consumption were examined and<br />process models were developed. Additionally, optimization of<br />staurosporine production was performed with different<br />supplements which, according to literature data, had stimulative<br />effect on secondary metabolism (Zn, Fe and P salts, methyl<br />oleate, grape seed oil). In order to improve the production of<br />staurosporine, effects of pH (6.5, 7.5 and 8.5) and incubation time<br />(7, 10 and 14 days) were also examined. It was found that<br />addition of FeS04 significantly improved the staurosporine yield<br />in comparison to the starting conditions (increase of 25%). Our<br />results proved that rhizosphere soils of ethno-medicinal plants<br />are a prolific source of streptomycetes, producers of compounds<br />with good antifungal activity. Isolation of the new staurosporine<br />producing strain, allowed for its detailed bioactivity assessment.<br />Staurosporine scaffold might serve as a lead structure for the<br />development of new antifungal and antiangiogenic agents. Also,<br />results obtained within this research represent the basis for the<br />further scale-up and potential industrialization of the proposed<br />production process.</p>

1 Abstract: Secondary metabolism of Gram-positive soil bacteria from the genus Streptomyces is a inestimable source of natural products including manumycins, which belong to a polyketide group. These products possess weak antimicrobial, but important antiinflammatory, and antitumor activities. Streptomyces sp. offers broad amounts of yet undiscovered antibiotics, potentially utilizable in clinical medicine. This fact makes out of these organisms a promising solution to our present problem with rising antibiotic resistance among microorganisms. Two main ways are applied in this research: There are efforts of prepairing new derivates based on known products and creating various modifications in their structure. Next, new producers are discovered by "genome mining" methods, activation of silent gene clusters, followed by improvements of antibiotic production. One of those silent clusters was found in the Saccharothrix espanaensis DSM44229 strain. The genetic information has been transferred to a heterologous host in order to characterize its product. Cluster activation and production of novel manumycin-type metabolites occurred in the host after the transfer.

Streptomycetes, primary soil saprophytic microorganisms are at the center of interest in many research groups, mainly because of their ability to produce a wide range of biologically active substances useful in medicine, biotechnology and agriculture. The marginal, and little explored areas are the interactions that streptomycetes create with humans. Recent metagenomic studies have shown that streptomycetes colonize the skin, the respiratory and possibly the urogenital tract of humans. In addition to apparent pathogens such as S. somaliensis and S. sudanensis, the clinical impact of these streptomycetes on human health is unknown. For this reason, a unique collection of non-pathogenic streptomycetes isolated from human clinical specimens was developed. The isolates were collected by the National Reference Laboratory for Pathogenic Actinomycetes in Trutnov, Czech Republic. On the basis of pilot studies, an isolate labeled TR42 was selected from the Trutnov collection, showing a very broad spectrum of biological activities. This strain was isolated from sputum from a patient with unknown respiratory diagnosis. The TR42 strain exhibits considerable biotechnological potential and after following a thorough study, it could be a source of new biologically active substances with pharmaceutically...

Streptomyces bacteria are well-known producents of many bioactive metabolites. Its secondary metabolism is a source of many important groups of active compounds that are recently investigated by means of many new methods based on bioinformatic analyses of genome data, modern LC-MS techniques, and metabolic modeling methods. This thesis originates from the genetic screening for a specific gene (als) for cyclizing 5-aminolevulinate synthase. Based on earlier studies, we consider this gene as a genetic tag of the producers of secondary metabolites containing the C5N unit (2-amino-3-hydroxycyklopent- 2-enon). Such metabolites include several groups with variable structures and biological activities, which include manumycins as well. Manumycins are small polyketides with a weak antibiotic aktivity, especially against gram-positive bacteria. However, its cancerostatic and anti-inflammatory effects are of greater importance. Streptomyces monomycini BCCO10 1552 and Streptomyces capoamus BCCO10 1636 strains were found positive for the presence of the als gene in the targeted genetic screening. By the als phylogeny, they cluster near the producents of manumycin compounds. This thesis aimed to determine whether these new natural isolates produce any compounds containing C5N unit, and to characterize them in...

The clinically used antibiotic lincomycin consists of an amino-sugar and an amino-acid moiety. The incorporated amino-acid 4-propyl-L-prolin (PPL) is very important for the linomycin bioactivity, as evidenced by the lower activity of the related antibiotic celesticetin, which incorporates proteinogenic L-prolin instead. Gene clusters for the biosynthesis of both lincosamides are published and reflect a common basis - biosynthesis of amino-sugar precursor and condensation reactions. Additionally, in the biosynthetic gene cluster for lincomycin there is a sub-cluster of genes encoding the biosynthesis of PPL, the alkylated proline derivative (APD). PPL has a common biosynthetic origin with other APDs that are part of the structures of antitumor pyrrolobenzodiazepines and the signal molecule hormaomycin, which is also reflected in the presence of homologous genes in their gene clusters. The acquired knowledge on PPL biosynthesis thus can be applied to a larger group of natural products. The first overall concept of APD biosynthesis was published forty years ago. The milestone was the year 1995 when the gene cluster for lincomycin biosynthesis was published and specific gene products have been proposed for individual biosynthetic steps. The functional proof of proteins has been performed so far just...

Streptomyces, filamentous Gram-positive bacteria are producers of more than 70% of antibiotics used in human therapy and agriculture. They are remarkable because of their complex life cycle (morphological differentiation) which leads to a formation of dormant spores able to survive unfavorable living conditions and allowing long-term survival of the organism. Soil represents their mostly natural living environment. In laboratory conditions they are cultivated in liquid media or on agar. We have developed in our laboratory two phase cultivation system which allows quantitative and reproducible preparation of samples for proteomic, transcriptomic and metabolomic analyses of Streptomycetes differentiation. The system is composed of inert micro- beads submerged in liquid medium. We used two types of micro-beads in our studies, glass and zirconia/silica beads. We followed the surface growth and differentiation of Streptomycetes on both types of beads using optical and electron microscopy (SEM) techniques. We observed major growth and higher antibiotic production on glass beads. Another difference we observed was in size and shape of colonies. In further research, using comparative proteomics, we attempted to identify proteins which might be responsible for recognition and adhesion of Streptomycetes to...