20.500.12556/RUL-176867 Razvoj inducibilnega promotorja za izražanje heterolognih genov v bakteriji Streptomyces rimosus Development of inducible promoter for expression of heterologous genes in Streptomyces rimosus Streptomyces rimosus je industrijsko pomemben mikroorganizem, ki omogoča učinkovito proizvodnjo sekundarnih metabolitov. V zadnjem desetletju pa postaja zanimiv tudi kot gostitelj za biosintezo heterolognih proteinov. Cilj magistrskega dela je bil razvoj in optimizacija na oksitetraciklin-inducibilnega promotorskega sistema Potr v sevih S. rimosus, ki ne vsebujejo biosintezne poti oksitetraciklina (OTC). Kot poročevalski gen smo uporabili mCherry, izražanje pa smo spremljali z merjenjem fluorescence. V obsegu poskusov smo testirali različne koncentracije OTC in časovne točke indukcije, ter preučevali njihov vpliv na rast sevov in količino proizvedenega heterolognega proteina. Ugotovili smo, da je optimalna koncentracija induktorja v območju 50–250 µM, pri čemer je bila najvišja stopnja izražanja poročevalskega gena dosežena, ko je bila izvedena indukcija z OTC med 2. in 12. uro kultivacije od inokulacije kulture v produkcijski fazi rasti. Pri sevih z dodatno 145 kb delecijo v genomu smo opazili povečano produkcijo heterolognega proteina, kar nakazuje na sprostitev metabolnih virov za heterologno izražanje. Največji titer heterolognega proteina je bil 22 mg/L (± 2 mg/L). Intenzivnejšo biosintezo proteina so imeli sevi, ki imajo poleg delecije biosintezne genske gruče za sintezo OTC, v genomu tudi dodatno 145 kb delecijo. Analiza izločanja proteina iz celice je pokazala, da se okoli 80 % heterolognega proteina izloči iz celic, kar nakazuje na učinkovito delovanje sekrecijskega sistema Sec. Streptomyces rimosus is an industrially important microorganism with efficient production of secondary metabolites. This strain gained increased interest as a host for heterologous protein biosynthesis. The aim of this master’s thesis was the development and optimization of an oksitetracicline (OTC)-inducible promoter system Potr in S. rimosus strains lacking the OTC biosynthetic pathway. We applyied reporter gene mCherry to monitor protein expression by measuring fluorescence. In the course of experiments, different concentrations of OTC inducer at different induction time points were tested to evaluate their impact on culture growth and the amount of heterologous protein produced. We observed that the optimal inducer concentration ranged between 50 and 250 µM, with the highest expression achieved when induction was carried between the 2. and 12. hour after the inoculation of production phase. Strains with an additional 145 kb genomic deletion exhibited increased heterologous protein production, suggesting that the reduction of metabolic resources was favourable for heterologous expression. The highest concentration of the heterologous protein reached up to 22 mg/L (± 2 mg/L). Strains without OTC biosynthetic gene cluster as well as additional 145 kb genomic deletion exhibited the highest capacity for protein secretion. Analysis of protein secretion revealed that approximately 80% of the heterologous protein was secreted, indicating efficient functioning of the Sec secretion pathway. streptomicete Streptomyces rimous heterologno izražanje oksitetraciklin mCherry fluorescenca absorbanca promotorski sistem streptomycetes Streptomyces rimous heterologous expression oxytetracycline mCherry fluorescence absorbance promoter system true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-12-12 07:15:08 2025-12-12 07:15:20 2025-12-25 03:32:43 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 272343 262791171 55526.pdf 55526.pdf 1 1C567DD2B38C62260FB6A948B1791248 7ae6f70a489af9df38065aefe9e7c25d8859bfaf444d85f724eff5b146c442c0 83a59fce-d721-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=223496 Biotehniška fakulteta 0 0 0