The project focused on understanding the biosynthesis of sporothriolides, sporochartines and trienylfuranol A. Gene cluster identification, gene knock out, heterologous expression and protein in vitro assays were used during the investigation.Alkyl citrate biosynthetic gene clusters of the antifungal metabolite sporothriolide 1 were identified from the genomes of the ascomycetes: Hypomontagnella monticulosa MUCL 54604, H. spongiphila CLL 205 and H. submonticulosa DAOMC 242471. A transformation protocol was established, and genes encoding a fatty acid synthase subunit and a citrate synthase were simultaneously knocked out which led to the loss of sporothriolide and sporochartine production. Heterologous expression of the spo genes in Aspergillus oryzae then led to the production of intermediates and shunts and delineation of a new fungal biosynthetic pathway originating in fattyacid biosynthesis. Finally, a hydrolase was revealed by in vitro studies likely contributing towards self-resistance of the producer organism. In vitro reactions showed that the sporochartines are derived from non-enzymatic Diels-Alder cycloaddition of 1 and trienylfuranol A 2 during the fermentation and extraction process.Several hrPKS gene clusters were identified as the potential polyene BGC for trienylfuranol A 2 through multiple bioinformatic analysis, however metabolites produced from the PKS in heterologous expression belong to either different polyene type compounds or pyrone derivatives. Based on these results, a highly unusual epoxidation/decarboxylation mechanism was proposed to be involved during trienylfuranol A 2 biosynthesis, and a new pyrone BGC likely to encodethe biosynthesis of a large class of bioactive compounds related to islandic acid 161 was identified.