Open Access

Maja Klaus

• Polyketide synthases (PKSs) are large megaenzymes that occur in bacteria, fungi, and plants and produce polyketides, a class of secondary metabolites. Many polyketide natural products exhibit high biological activities e.g. as antibiotics or anti-fungal compounds. The modular architecture of assembly line PKSs makes them exciting targets for engineering approaches via the exchange of whole modules or single domains. Although many engineering attempts have been pursued over the last three decades, the resulting chimeric PKSs often exhibit decreased turnover rates or diminished product yields. In this thesis, new approaches to engineer chimeric PKSs were explored, each targeting a different aspect of the chimeric system: First the relative contribution of protein-protein and protein-substrate recognition on the turnover of chimeric PKS was assessed, revealing the importance of protein-protein interactions between the acyl carrier protein (ACP) and the ketosynthase (KS) domain in the chain translocation step. Directed evolution experiments followed to optimize the protein-protein interaction across a chimeric interface. Additionally, different junction sites for the generation of chimeric PKSs were compared, showing the ability for recombination without interfering with the chain translocation reaction, and highlighting the use of SYNZIP domains to bridge PKS modules. To optimize chimeric PKSs even further, multipoint mutagenesis of KS domains was established, with positive effects on the activity of chimeric systems. To support engineering attempts, several structure elucidation techniques were combined with in silico modeling to characterize the architecture of a PKS module and the domain-domain interactions within it. Preliminary results show a strong conformational flexibility of the PKS module and the great potential of these techniques to define the multitude of transient interactions in PKS modules.