Production of the chemotherapeutic substance paclitaxel using biotechnological methods

Paklitaksel je visoko oksigeniran diterpenoid in je ena najuspešnejših učinkovin za zdravljenje raka. Zaradi velike svetovne potrebe po kemoterapevtikih, redkosti paklitaksela v naravi in trajnostno nevzdržnega načina ekstrakcije iz naravnih virov (lubja in iglic dreves Taxus spp.) se za produkcijo uporabljajo biotehnološki postopki. Ker gre za tržno pomemben proizvod z visoko dodano vrednostjo, je optimizacija obstoječih postopkov velikega pomena, prav tako pa se išče alternativne vire paklitaksela in prekurzorjevkot obetavne so se izkazale endofitske glive. Odkritje paklitaksela v navadni leski (Corylus avellana L.) nakazuje možnost izrabe odpada prehrambne industrije, kakor tudi obstoj še neznanih rastlinskih producentov paklitaksela, katerih lastnosti bi bile ugodnejše za industrijsko uporabo. Dobra proučenost, praktičnost in enostavnost uporabe mikrobnih organizmov za proizvodnjo kompleksnih molekul botruje poskusom prenosa biosintezne poti paklitaksela v E. coli in S. cerevisiae, kar pa se je sicer izkazalo za težavno. V tem delu smo napravili krajši pregled biotehnoloških postopkov proizvodnje paklitaksela, vključno z načini izboljšave donosa celičnih kultur, proizvodnjo prekurzorjev v ustaljenih industrijskih mikororganizmih in z endofitskimi glivami.Paclitaxel, a highly oxygenated diterpenoid, is one of the most successful therapeutics used in the treatment of various cancers. Due to the high global demand for the substance, its relative scarcity in nature and most of all, because the extraction from natural materials is either unsustainable or impractical a variety of biotechnological methods are employed for its industrial production. The substantial market for the substance allows for the production of a high added-value product, rationalising the ongoing optimization of the production processes as well as the search for alternative sources of paclitaxel. One of the most promising alternative sources are endophytic fungi, albeit their industrial use is yet to be realized. The discovery of paclitaxel in the common hazel (Corylus avellana L.) indicates the possible existence of yet unknown taxoid producing plant species, the intrinsic properties of which might make them more suitable for industrial use. A tentative option of utilizing the waste products of the food industry for paclitaxel extraction also emerges. Our understanding of commonly used microorganisms, their practicality and simplicity result in attempts to transfer the biosynthetic pathway for paclitaxel production away from the sometimes challenging plant hosts, although the progress is slow and troublesome. In this work we give a condensed review of the common paclitaxel production methods, the ways of improving said methods and the production of paclitaxel precursors in common industrial microorganisms and the endophytic fungi

Vpliv nadmorske višine na pojavljanje arbuskularne mikorize pri navadni ajdi (Fagopyrum esculentum)

Želeli smo ugotoviti vpliv nadmorske višine na pojavljanje arbuskularne mikorize pri navadni adji (Fagopyrum esculentum). Spremljali smo pogostost mikorize in mikoriznih struktur. Rastline so bile nabrane na treh različnih lokacijah z različno nadmorsko višino in sicer na Biotehniški fakulteti (295 m n.m) Javorovici (550 m n.m.), in Javorniku (1140 m n.m.). S tripan modrim smo pobarvali korenine, ter z metodo hitrega skeniranja s svetlobno mikroskopijo ocenili stopnjo kolonizacije, prisotnost arbuskulov, veziklov, svitkov in mikrosklerocijev. Frekvenca in intenziteta mikorize sta bili najvišji na Javorovici. V vzorcih so prevladovali mikrosklerociji. Prevladovali so temni septirani endofiti. Na podlagi danih rezultatov ne moremo sklepati na vpliv nadmorske višine na pogostost mikorize. Na to lahko vpliva še več parametrov (osončenost, tip prsti, vlažnost, padavine, temperatura)

Vpliv gnojenja z organskim gnojilom na stres zaradi slanosti pri fižolu in ječmenu

Namen: preveriti vpliv organskega gnojila na rast fižola (Phaseolus vulgaris) in ječmena (Hordeum vulgare) v zasoljenih tleh, določiti vsebnost pigmentov in mineralno sestavo v odvisnosti od načina izpostavitve rastlin. Metode: merjenje sveže in suhe mase poganjkov ter korenin, določanje vsebnosti pigmentov, analiza mineralne sestave poganjkov (XRF), kvalitativno in kvantitativno določanje arbuskularne mikorize (arbuskuli, mikrosklerociji, vezikli, svitki). Poskus smo vzporedno izvedli na primeru enokaličnic (ječmen) in dvokaličnic (fižol). Rastline smo gojili pri različnih izpostavitvah (dodatek gnojila ali brez ter zalivali z navadno vodo ali 0,1 M NaCl). Naredili smo po pet ponovitev vsake izpostavitve in na koncu podatke statistično obdelali. Rezultati: gnojilo je negativno vplivalo na rast rastlin, saj je bila ob njegovi uporabi suha masa poganjkov in korenin rastlin manjša. Vsebnost pigmentov (klorofil a in b) v poganjkih se je ob uporabi gnojila povečala pri obeh vrstah. Gnojilo je pri fižolu vplivalo na večji privzem žvepla ter zmanjšan privzem klora na slanih tleh. Ječmen je ob gnojilu bolje privzel fosfor, mangan, cink in žveplo. Tudi pri ječmenu se je ob uporabi gnojila na slanih tleh zmanjšal privzem klora. V koreninah nismo potrdili prisotnosti arbuskularno mikoriznih gliv. Zaključki: gnojilo je negativno vplivalo na rast rastlin že pri izpostavitvah brez soli. Negativen učinek gnojila se je še bolj izrazil pri izpostavitvi NaCl. Gnojilo je imelo sicer pozitiven učinek na vsebnost klorofilov in zmanjšan privzem klora rastlin v primeru prekomerne slanosti. Uporabljeno organsko gnojilo ni učinkovito pri odpravljanju simptomov prekomerne slanosti

The first record of two non-native ambrosia beetles in Slovenia: Ambrosiodmus rubricollis (Eichhoff, 1875) and Ambrosiophilus atratus (Eichhoff, 1875) (Coleoptera: Curculionidae, Scolytinae)

In September 2017, during the monitoring of the non-native ambrosia beetle Xylosandrus germanus (Blandford, 1894), one specimen of an unknown ambrosia bark beetle species was collected in Slovenia. The specimen was trapped in an ethanol-baited trap located in Klavže (46 09 39 N, 13 48 7 E), in the western part of Slovenia. The most characteristic feature distinguishing the specimen from other known ambrosia beetle species occurring in Slovenia was the asperities that covered the entire surface of the pronotum. Based on the scientific literature concerning the non-native bark and ambrosia beetles in Europe (Kirkendall & Faccoli 2010) and illustrated identification keys (Rabaglia et al. 2006Faccoli et al. 2009), we identified the beetle by its morphological characteristics as Ambrosiodmus rubricollis (Eichhoff, 1875). As a result of this find, a specific monitoring was set up in 2018 in Slovenia with the aim to improve the knowledge about occurrence and distribution of A. rubricollis in this country

Cutting corners

The purpose of our study was to investigate methods of short-term storage that allow preservation, transport and retrieval of genetic information contained in honeybee queen’s spermatheca. Genotyping of the honeybee colony requires well ahead planned sample collection, depending on the type of data to be acquired. Sampling and genotyping of spermatheca’s content instead of individual offspring is timesaving, allowing answers to the questions related to patriline composition immediately after mating. Such procedure is also cheaper and less error prone. For preservation either Allprotect Tissue Reagent (Qiagen) or absolute ethanol were used. Conditions during transportation were simulated by keeping samples 6–8 days at room temperature. Six different storing conditions of spermathecas were tested, complemented with two DNA extraction methods. We have analysed the concentration of DNA, RNA, and proteins in DNA extracts. We also analysed how strongly the DNA is subjected to fragmentation (through amplification of genetic markers ANT2 and tRNA$^{leu}$-COX2) and whether the quality of the extracted DNA is suitable for microsatellite (MS) analysis. Then, we tested the usage of spermatheca as a source of patriline composition in an experiment with three instrumentally inseminated virgin queens and performed MS analysis of the extracted DNA from each spermatheca, as well as queens’ and drones’ tissue. Our results show that median DNA concentration from spermathecas excised prior the storage, regardless of the storing condition and DNA extraction method, were generally lower than median DNA concentration obtained from spermathecas dissected from the whole queens after the storage. Despite the differences in DNA yield from the samples subjected to different storing conditions there was no significant effect of storage method or the DNA extraction method on the amplification success, although fewer samples stored in EtOH amplified successfully in comparison to ATR storing reagent. However, we recommend EtOH as a storing reagent due to its availability, low price, simplicity in usage in the field and in the laboratory, and capability of good preservation of the samples for DNA analysis during transport at room temperature