20.500.12556/RUL-108104 Inaktivacija gena metiltransferaze oxyF iz genske skupine za biosintezo oksitetraciklina v bakteriji Streptomyces rimosus. Inactivation of methyltransferase oxyF gene from oxytetracycline biosynthesis gene cluster in Streptomyces rimous bacteria Streptomyces rimosus je pomemben industrijski mikroorganizem, ki proizvaja oksitetraciklin, antibiotik širokega spektra. V medicini in veterini se trenutno uporablja več polsinteznih tetraciklinksih antibiotikov. Zaradi širjenja bakterijske rezistence narašča interes po odkritju novih naravnih tetraciklinov in njihovih biosinteznih intermediatov, uporabnih za razvoj novih, bolj učinkovitih antibiotikov. Cilj tega dela je bila inaktivacija gena metiltransferaze oxyF znotraj genske gruče za biosintezo oksitetraciklina in identifikacija tarčnega produkta, 6-demetiloksitetraciklina. Konstruirali smo plazmid, ki vsebuje DNK fragment s homologijo regijam v okolici gena oxyF v genomu S. rimosus, vendar brez večjega dela gena oxyF. Na ta način konstruiran fragment DNK smo nato vstavili v »samomorilski« plazmid, imenovan pKC1139. pKC1139 vsebuje mesto za podvojevanje v E. coli in primerne gene za rezistenco na antibiotike, ki omogočajo selekcijo v E .coli in S. rimosus. Po transformaciji v S. rimosus smo uporabili dvostopenjski proces z integracijo v genom, ki ji je sledila delecija večjega dela gena oxyF, oboje preko homologne rekombinacije. Nato smo poiskali transformante S. rimosus z natančno inaktivacijo gena oxyF. Po kultivaciji smo analizirali metabolni profil izbranih kolonij z tankoplastno kromatografijo. Z uporabo masne spektrometrije smo potrdili produkcijo sicer nizkih količin 6-demetil oksitetraciklina. Streptomyces rimosus is an important industrial microorganism producing broad-spectrum antibiotic oxytetracycline. A number of semi-synthetic tetracyclines, are currently used in human and veterinary medicine. Due to emerging bacterial resistance, there is rising interest for new natural tetracyclines and their biosynthetic intermediates, which could be used to develop novel and more effective antibiotics . The aim of this work was to inactivate methyltransferase oxyF gene within oxytetracycline gene cluster in S. rimosus and to potentially identify target product, 6-desmethyl oxytetracycline. We consteructed a plasmid containing DNA fragment with homology to the sequence adjacent to oxyF gene from S. rimosus genome, but missing the entire oxyF gene. This way constructed DNA fragment was inserted into »suicide« plasmid designated as pKC1139, containing E. coli origin of replication and suitable resistance markers for selection in E. coli and S. rimosus. We transformed the plasmid into S.rimosus, and by applying two-stage process of chromosomal integration, followed by a double cross-over deletion, we selected for S. rimosus transformants containing precise deletion of targeted oxyF gene. We then confirmed correct double-cross over deletion of oxyF gene in a number of S. rimosus colonies. After cultivation, we analyse metabolic profile of the selected engioneered strains by thin layer chromatography. Although at low yield, we confirmed production of 6-dsemethyl oxyteracycline by mass spectroscopy. Aktinomicete Streptomyces rimosus tetraciklini inaktivacija gena metiltransferaza oksitetraciklin Actinomicetes Streptomyces rimosus tetracyclines gene inactivation methyltransferase oxytetracycline true false false [A. Pšeničnik] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2019-06-16 07:45:10 2019-06-16 07:45:16 2022-08-20 03:44:34 0000-00-00 00:00:00 2019 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2022-06-13 602.3:579.873.7:602.6:579.8(043.2) 163850 9238905 7182.pdf 7182.pdf 1 2FD56B349C140B37B0A3B5AEAB8482B7 a5607b11be4ab41abe2e596567e44d768cc2f66d0cd9f2fa5f6e6cfa113af445 933d6cc6-a1b6-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=119026 Biotehniška fakulteta 0 0 0