Filamentous fungi have long been recognized as prolific producers of natural products due in a considerable part to their strong track record in producing blockbuster drugs such as penicillin and lovastatin. The biosynthetic enzymes from fungi that assemble these molecules, such as polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are large highly complex multifunctional megasynthetases. Different from their well-studied bacterial counterparts, to date, the biosynthetic programming rules utilized by the fungal PKSs and NRPSs remain largely unknown. Despite the fact that a number of fungal PKSs and NPRSs genes have been discovered over the last three decades, few of them have been characterized and thus directly linked to the biosynthesis of specific fungal secondary metabolites. To fully understand the biosynthetic logic of PKSs and NRPSs and decipher the relationship between sequences and the structures of fungal polyketides and nonribosomal peptides, the thesis focus on three independent fungal biosynthetic systems involving PKSs, NRPS and PKS-NRPS.

In the first project, the biochemistry of tandem fungal PKSs for the formation of nanomolar HSP 90 inhibitor radicicol are reconstituted in vivo and in vitro and extensively investigated with the help of tool compounds acyl thioesters. Secondly, a cryptic, NRPS-like enzyme (NRPS325) mined from Aspergillus terreus was reconstituted in vitro and was shown to synthesize thiopyrroles and thiopyrazines via unprecedented mechanisms. The remarkable substrate promiscuity of NRPS325 towards different amino acids, free thiols and β-ketoacyl substrates were explored to produce hundreds of new compounds. Lastly, the genome sequence of Aspergillus clavatus was analyzed and the 30 kb cytochalasin gene cluster was identified based on the presence of the PKS-NRPS and a putative Baeyer-Villiger monooxygenase. Deletion of the central PKS-NRPS gene, ccsA, abolished the production of cytochalasin E and K, demonstrating the direct association between the natural products and the gene cluster. Overexpression of the pathway specific regulator ccsR greatly elevated the titer of cytochalasins.