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Pridobivanje proteinov minimalnega kompleksa poliketid-sintaze genske skupine za biosintezo oksitetraciklina v Streptomyces sp.
ID Kobale, Valerija (Author), ID Petković, Hrvoje (Mentor) More about this mentor... This link opens in a new window, ID Kranjc, Luka (Comentor)

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Abstract
Osnovno ogrodje molekule oksitetraciklina (OTC), ki ga sintetizira bakterija S. rimosus, sintetizirajo encimi zgodnje stopnje biosinteze, ki pripadajo kompleksu minimalnih poliketid sintaz (mPKS); to so ketosintaza-α (OxyA), ketosintaza-β (OxyB) in acil-prenašalni protein (OxyC). Ker mehanizmi začetnih reakcij v biosintezi OTC še niso docela pojasnjeni, bi bilo za razumevanje katalitične aktivnosti encimov potrebno pridobiti vse encime tega kompleksa in postaviti in vitro testni sistem, ki omogoča rekonstrukcijo in karakterizacijo aktivnosti individualnih komponent. Namen magistrske naloge je bil priprava topnih proteinov mPKS. Poskus priprave topnih proteinov mPKS je bil v E. coli neuspešen, zato smo v okviru naloge transformirali plazmide pVF s konstrukti za izražanje posameznih genov oxyA, oxyB in oxyC v sev S. rimosus, ki ne sintetizira OTC (∆OTC). Pridobljenim klonom transformant S. rimosus ∆OTC s plazmidi pVF s konstrukti posameznih genov oxyA, oxyB in oxyC ni bilo mogoče meriti aktivnosti, zato smo ustreznost klonov preverili z metodo ponovne transformacije v E. coli DH10β (»plasmid rescue«). Izkazalo se je, da so plazmidi pVF s konstrukti posameznih genov oxyA, oxyB in oxyC nestabilni, kar se je izkazalo tudi v spremenjeni morfologiji kolonij transformant S. rimosus ∆OTC. Delež klonov S. rimosus ∆OTC z ustreznimi plazmidi pVF s konstrukti posameznih genov oxyA in oxyC je bil nižji od 15 %, medtem ko ustreznih klonov s plazmidi pVF s konstrukti gena oxyB nismo uspeli potrditi. Z afinitetno kromatografijo in elektroforezo SDS-PAGE smo identificirali klone S. rimosus ∆OTC z ustreznim plazmidom pVF srT oxyA 3'HT, ki proizvajajo protein velikosti 45 kDa, ki najverjetneje ustreza proteinu ketosintazi-α. Z metodo točkovnega prenosa smo pridobili pozitiven signal, s tem smo dodatno potrdili prisotnost nekaterih ciljnih proteinov.

Language:Slovenian
Keywords:streptomicete, Streptomyces rimosus, oksitetraciklin, poliketid sintaze, izražanje proteinov, stabilnost plazmidov
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Publisher:[V. Kobale]
Year:2018
PID:20.500.12556/RUL-102828 This link opens in a new window
UDC:602.3+602.6+604.4:579.873.7
COBISS.SI-ID:4941944 This link opens in a new window
Publication date in RUL:08.09.2018
Views:1596
Downloads:470
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Secondary language

Language:English
Title:Production of polyketide synthase minimal complex proteins involved in biosynthesis of oxytetracycline by Streptomyces sp.
Abstract:
The nascent polyketide chain of the antibiotic Oxytetracyclin (OTC) produced by S. rimosus is synthesized by polyketide synthase minimal complex (mPKS) consisting of enzymes ketosynthase-α (OxyA), ketosynthase-β (OxyB) and acyl-carrier protein (OxyC), however the exact mechanism of initiation of OTC biosynthesis is not yet fully understood. Therefore, in vitro reconstitution and evaluation of its kinetics and substrate specificity could bring new information on the biosynthetic mechanism of mPKS complex. The aim of this thesis was to produce soluble proteins from mPKS complex, which are often insoluble or inactive when produced in E. coli therefore we considered using S. rimosus as an alternative production host since S. rimosus can export proteins into culture medium. Our aim was to produce the individual mPKS components in the OTC non-producing strain (∆OTC) of S. rimosus. In the scope of this master thesis we managed to transform replicative plasmid containing mPKS components into S. rimosus ∆OTC. Considering that measurement of enzymatic activity of OxyA, OxyB and OxyC was not possible, we have carried out screening of clones containing the correct version of plasmid with mPKS components by so-called plasmid rescue approach. We observed plasmid instability resulting in change of colony morphology in S. rimosus ∆OTC transformed clones. Percentage of correct S. rimosus ∆OTC clones containing pVF plasmid with genes oxyA and oxyC, was up to 15 %. We were not able to identify positive clones identified with pVF plasmid containing gene oxyB. By applying affinity chromatography and SDS-PAGE electrophoresis we have identified clones producing protein of around 45 kDa in size in the eluted protein fractions, likely corresponding to ketosynthase-α. Dot-blot analysis gave a positive signal with selected clones when targeting His-tag, thus additionally confirming production of the target proteins.

Keywords:streptomycetes, Streptomyces rimosus, oxytetracycline, polyketide synthases, protein production, plasmid stability

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