BackgroundFungi are important sources for bioactive compounds that find their applications in many important sectors like in the pharma-, food- or agricultural industries. In an environmental monitoring project for fungi involved in soil nitrogen cycling we also isolatedCephalotrichum gorgonifer(strain NG_p51). In the course of strain characterisation work we found that this strain is able to naturally produce high amounts of rasfonin, a polyketide inducing autophagy, apoptosis, necroptosis in human cell lines and showing anti-tumor activity in KRAS-dependent cancer cells.ResultsIn order to elucidate the biosynthetic pathway of rasfonin, the strain was genome sequenced, annotated, submitted to transcriptome analysis and genetic transformation was established. Biosynthetic gene cluster (BGC) prediction revealed the existence of 22 BGCs of which the majority was not expressed under our experimental conditions. In silico prediction revealed two BGCs with a suite of enzymes possibly involved in rasfonin biosynthesis. Experimental verification by gene-knock out of the key enzyme genes showed that one of the predicted BGCs is indeed responsible for rasfonin biosynthesis.ConclusionsThis study identified a biosynthetic gene cluster containing a key-gene responsible for rasfonin production. Additionally, molecular tools were established for the non-model fungusCephalotrichum gorgoniferwhich allows strain engineering and heterologous expression of the BGC for high rasfonin producing strains and the biosynthesis of rasfonin derivates for diverse applications.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40694-023-00158-x.