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Description:
Fungal Type I polyketide synthases (PKS) are large multifunctional enzymes with a complex structure consisting of multiple functional domains. They produce complex bioactive products such as squalestatin S1 27 and byssochlamic acid 106. The main focus of the projects lies on highly reducing iterative polyketide synthases, especially on their structure and function. Previous work has shown that generating structural information from cross-linked polyketide domains can be the first step towards the elucidation of function and programming. In this thesis we have cloned and heterologously expressed the squalestatin tetraketide synthase (SQTKS) KS/AT didomain, standalone DH domain and the ACP. The SQTKS KS/AT didomain and the ACP were heterologously expressed in E. coli but were obtained as insoluble (KS/AT) or inactive (ACP) proteins. The ACP and the standalone DH domain from the Byssochlamys fulva nonadride PKS (bfPKS) were expressed for the first time. Both proteins were successfully expressed in E. coli and showed catalytic activity as measured by mass spectrometry. The bfPKS ACP was used for crosslinking attempts. Biosynthetic enzymes were expressed and used for the enzymatic conversion of chemically synthesized pantetheine linker analogues into CoA linker analogues. A phosphopantetheine transferase (PPTase, also expressed) was used to load the linkers onto the bfPKS ACP. Cross-linking between bfPKS ACP and DH domains of SQTKS, bfPKS and the Strobilurin tenacellus strobilurin PKS (stPKS) were tried but no linking was observed. In vitro investigations of the B. fulva citrate synthase (bfCS) revealed its substrate preference. The bfCS showed conversion of CoA bound substrate but no conversion for the bfPKS ACP bound one. The SQTKS standalone methyltransferase (C-MeT) domain did not convert the natural substrate which was bound to the bfPKS ACP and was inactive as in previous investigations.