Optimizacija gensko spremenjenih sevov Streptomyces rimosus za proizvodnjo heterolognih proteinov s pristopi naključne mutageneze in selekcije

Žitnik, Katja (avtor), Petković, Hrvoje (mentor), Avbelj, Martina (komentor)

V obsegu magistrskega dela smo optimizirali produkcijski sev Streptomyces rimosus z uvajanjem naključne mutageneze in vzpostavitvijo presejalnega testa za hitro izbiro visoko-produktivnih klonov. S pomočjo elektroporacije smo v sev S. rimosus ATCC deltaOTC soft vnesli plazmidni vektor pAB04_Per, ki nosi zapis za proteazo pernizin in na osnovi azokazeinskega testa izbrali najboljše transformante. Seve S. rimosus ATCC deltaOTC soft, ki nosijo zapis za proteazo pernizin ali za fuzijski proteinski kompleks mCherry-kaminizin, smo testirali v različnih produkcijskih gojiščih. V nadaljevanju smo izvedli mutagenezo s pomočjo UV-svetlobe in etil metansulfonata ter vzpostavili knjižnice mutant sevov S. rimosus ATCC deltaOTC soft, postopek gojenja in presejanja pa smo prenesli v manjše merilo (mikrotitrske plošče), kar je omogočilo hitrejše testiranje. Encimsko aktivnost mutant smo vrednotili z azokazeinskim testom in fluorescenco proteinskega kompleksa mCherry-kaminizin. Najvišje proteolitične aktivnosti v kulturi sevov smo dosegli pri izolatih, ki nosijo zapis za biosintezo pernizina, pridobljenih po izpostavljenosti UV-svetlobi in sicer je aktivnost znašala v povprečju približno 1750 U/mL, kar je 2x več v primerjavi s kontrolnim sevom. Pri izolatih, ki nosijo zapis za proteinski kompleks mCherry-kaminizin, izboljšav v proteolitični aktivnosti nismo zaznali. Opazili smo tudi, da merjenje fluorescence mCherry ni zanesljiva metoda za hitro kvantifikacijo proteolitične aktivnosti v manjšem merilu. Podrobnejša analiza encimskih aktivnosti v kulturah 191 klonov izbranega seva, izoliranih po UV-mutagenezi, je pokazala veliko variabilnost v aktivnosti pernizina, vendar najboljši kloni statistično v proteolitični aktivnosti niso presegli kontrolnega seva, ki ni bil tretiran z mutagenom. Naši rezultati potrjujejo uporabnost naključne mutageneze v kombinaciji z optimizacijo gojišč in presejanjem kot strategije za povečanje aktivnosti produkta pri Streptomyces rimosus. Hkrati pa naši rezultati kažejo, da so potrebne nadaljnje izboljšave v postopku gojenja sevov za stabilnejšo selekcijo visoko-produktivnih sevov za zagotovitev višjih aktivnosti proteaz pernizin in kaminizin.

• Jezik: Slovenski jezik
• Ključne besede: Streptomyces rimosus, heterologno izražanje proteinov, pernizin, kaminizin, naključna mutageneza, presejalno testiranje, azokazeinski test, proteolitična aktivnost
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: BF - Biotehniška fakulteta
• Založnik: [K. Žitnik]
• Leto izida: 2026
• PID: 20.500.12556/RUL-180061
• UDK: 602.3:579.873.7:601.4:577.21
• COBISS.SI-ID: 270510339
• Datum objave v RUL: 01.03.2026

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Optimisation of genetically engineered strains of Streptomyces rimosus for heterologous protein production by random mutagenesis and selection approaches

Izvleček

In this study, we optimized the production strain Streptomyces rimosus encoding proteases pernisine and caminisine by random mutagenesis and screening for the rapid selection of high-producing clones. Using electroporation, we introduced the plasmid vector pAB04_Per, carrying the gene for the protease pernisine, into the S. rimosus ATCC deltaOTC soft strain, and selected the best protease-producing transformants with azocasein assay. The S. rimosus ATCC deltaOTC soft strains, containing the plasmid construct encoding the protease pernisine or the fusion protein complex mCherry-caminisine, were tested in various production media. Subsequently, mutagenesis was performed by applying UV light and ethyl methanesulfonate, and this way, libraries of mutant S. rimosus ATCC deltaOTC soft strains were collected. The cultivation and screening process was transferred to a smaller scale (microtiter plates), which enabled faster testing. The activity of mutant isolates was evaluated using the azocasein assay and fluorescence of the mCherry-caminisine protein complex. The highest proteolytic activity was observed in isolates containing the plasmid construct with pernisine, obtained after UV exposure, reaching approximately 1750 U/mL, which is two times higher compared to the control strain. In isolates containing the plasmid construct with mCherry-caminisine, no improvement in proteolytic activity was observed. We also found that measuring mCherry fluorescence is not a reliable method for rapid quantification of protease activity at a small scale. Detailed analysis of 191 clones of the selected strain, obtained after UV mutagenesis, revealed high variability in pernisine activity; however, the best clones did not statistically exceed the proteolytic activity of the control strain not treated with the mutagen. These results confirm the applicability of random mutagenesis in combination with medium optimization and screening as a strategy to enhance product activity in Streptomyces rimosus. At the same time, our results indicate that further improvements in the cultivation procedure are required to achieve a more stable selection of high-producing strains and ensure higher activities of the proteases pernisine and caminisine.

• Ključne besede: Streptomyces rimosus, heterologous protein expression, pernisine, caminisine, random mutagenesis, screening assays, azocasein assay, proteolytic activity