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.
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