20.500.12556/RUL-108617 Modulacija morfoloških lastnosti sevov Streptomyces clavuligerus s pristopi genskega inženiringa Morphological Modulation of Strains Streptomyces clavuligerus by Genetic Engineering Bakterije iz rodu Streptomyces so bogat vir sekundarnih metabolitov. Industrijski bioprocesi s streptomicetami so pogosto energetsko zahtevni, saj mnoge vrste tvorijo viskozne kulture. Viskoznost brozge je v veliki meri odvisna od morfologije. V zadnjih letih so identificirali številne proteine, ki so vpleteni v fiziološke procese, ki vplivajo na morfologijo streptomicet v submerznih kulturah (npr. CslA, SsgA). V magistrski nalogi smo želeli pridobiti seve s spremenjeno morfologijo z namenom zmanjšanja viskoznosti in posledično energijske zahtevnosti procesa. V ta namen smo s pomočjo bioinformatskih metod pri S. clavuligerus ATTC 27064 sprva identificirali homologne proteine SsgA in CslA. Z uporabo sistema CRISPR/Cas in integrativnih plazmidov smo genetsko modificirali seve S. clavuligerus in pridobili seve z izbitjem izbranega gena (ssgA, cslA) ali vneseno dodano kopijo. Fenotipe pridobljenih sevov smo ovrednotili na laboratorijskem in pilotnem merilu. Morfološke lastnosti smo ocenili na podlagi dolžin in razvejenosti hif (v primeru ssgA) ter velikosti in strukture peletov (v primeru cslA). Pri tem smo uporabili metodo digitalne analize mikroskopskih slik tekoče kulture. Rezultati kažejo, da je za sev s prekomerno izraženim genom ssgA značilna višja stopnja razvejanosti, večji delež krajših hif in manjša viskoznost brozge ob povečani tvorbi biomase. Z izbitjem gena cslA s sistemom CRISPR/Cas smo pridobili sev, ki tvori manjše pelete z večjim deležem biomase na robovih peleta in manjšo notranjostjo peleta. Pridobljena seva s spremenjeno morfologijo tako kažeta potencial za izboljšanje bioprocesa za produkcijo klavulanske kisline. Members of the genus Streptomyces are rich source of medically important natural products. Industrial bioprocesses with Streptomyces species often face difficulties caused by high viscosity of the cultures, thus increasing energy demands of the bio-processes. The viscosity of the culture is influenced by strain morphology. In recent years, several proteins that effect mycelial growth and morphology in liquid cultures (including CslA, SsgA) were identified. In this work, we aimed to engineer strains with specific morphological phenotypes of reduced viscosity in liquid culture. Using bioinformatics approaches we identified ssgA and cslA gene homologues in S. clavuligerus ATTC 27064. We engineered mutant strains of S. clavuligerus with deleted genes ssgA and cslA by applying the CRISPR/Cas system. We also constructed strains where ssgA and cslA genes were over-expressed by in-trans chromosome integration. The phenotypes of the genetically modified strains were evaluated in liquid medium on the laboratory and pilot plant scale. Morphological differences between the genetically modified strains and the wild-type strain were determined by image analysis of hyphal length and level of branching (for SsgA) or by analyzing the size and structure of pellets (for CslA). Our results show that the strain with ssgA over-expressing had shorter hyphae with more branches. The viscosity and energy demand in the pilot scale fermentors with the culture over-expressing ssgA was much lower compared to the wild type strain culture. The strain with the cslA deletion formed smaller pellets with longer hyphae on the periphery of the pellets and a smaller “dead” core in the pellet center. Both strains with changed morphologyzapia therefore show potential for improvement of the bioprocess for clavulanic acid production. biotehnologija filamentozne bakterije Streptomyces clavuligerus genski inženiring morfološke lastnosti slikovna analiza viskoznost klavulanska kislina biotechnology filamentous bacteria Streptomyces clavuligerus genetic engineering morphological properties image analysis viscosity clavulanic acid true false false [A. Juršič] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2019-07-10 07:45:38 2019-07-10 07:45:40 2022-08-20 03:47:32 0000-00-00 00:00:00 2019 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 602.6:579.873.7:579.23(043.2) 164035 9258873 7303.pdf 7303.pdf 1 31F1687F7EA8F753AFB7430921A72968 e2672062b0dc7c536a6134ccebb6ac29622865063761789d79b656405583acf6 ae130aaa-a1b6-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=119597 Biotehniška fakulteta 0 0 0