The main focus of the presented work concentrated on understanding and engineering the biosynthesis of three polyketides produced by filamentous fungi: squalestatin S1, strobilurin A and SCH–642305. In a combined genetic and chemical approach, the order and functions of genes and proteins involved in the three biosynthetic pathways were elucidated.Using a combination of targeted gene knockout in the native organism and heterologous expression in Aspergillus oryzae (A. oryzae) three previously unknown oxygenases were identified from SQS biosynthetic gene cluster (BGC) and their role in the biosynthesis of SQS were determined. Two non–heme iron–dependent oxygenases were shown to catalyse a series of six consecutive oxidations to form thehighly oxidised, bioactive core of squalestatins and an unusual copper–dependent oxygenase was found to introduce a hydroxyl required for later acetylation.During strobilurin biosynthesis, an FAD dependent monooxygenase was identified and shown in vitro to catalyse an unusual oxidative rearrangement to form the core beta-methoxyacrylate toxophore of this valuable class of agricultural fungicides. In vivo fungal expression studies revealed the biosynthetic pathway to bolineol from the same BGC and uncovered the role of two O–methyltransferases.In attempts to reveal each biosynthetic step in SCH biosynthesis, with special regard to formation of the 6–membered ring, the full BGC was coexpressed and re-established in A. oryzae. Only minor amounts of the final product were found due to a shunt pathway in A. oryzae. An early pathway intermediate was hydrolysed and oxidised and thus stopped the biosynthesis to SCH.