Pyrichalasin H 65 belongs to the cytochalasan family of fungal natural products, and is the major phytotoxic metabolite produced by the fungal plant pathogen Magnaporthe grisea NI980. In a combined chemical and genetic approach, the biosynthesis and chemical modification of 65 were investigated in this work.Using a bipartite gene knockout strategy targeting the pyi biosynthetic gene cluster, two fundamental genes pyiS and pyiC were disrupted, resulting in the complete abolition of 65. Five tailoring genes (two P450 encoding genes, one O-AcT, one O-MeT and one OXR encoding gene) were also inactivated individually; thirteen novel cytochalasans were isolated and identified by HRMS and NMR. For the first time, we discovered that O-methyltyrosine 175 was the initial precursor of 65 from the results of pyiA KO.Six cryptic P450 genes were investigated in two pyrichalasin H P450 disruption strains by heterologous expression. HffD and CYP1 were confirmed to catalyse a C-18 hydroxylation reaction, HffG catalyses a C-7 hydroxylation reaction and CYP3 catalyses an epoxidation reaction between C-6 and C-7. Three novel epoxide cytochalasans were obtained.Four 4’- halogenated phenylalanines, together with 4’-allyl, 4’-propargyl and 4’-azido phenylalanine were fed to a ∆pyiA KO strain. This resulted in production of seven new 4’-functionalised pyrichalasins with titres around 30 - 60 mg/L. Based on seven 4’-substituted pyrichalasins generated by mutasynthesis, many functionalised pyrichalasins were created by various chemical modifications. Triazole pyrichalasins were generated by click chemistry, using azide- 258 and alkyne pyrichalasin 252 as starting materials. Bromo- pyrichalasin 256 is the substrate for the synthesis of dimeric pyrichalasin 318 via Suzuki cross coupling, while Iodo- pyrichalasin 257 is a substrate for Sonogashira coupling reactions. Moreover, dye linked pyrichalasins 308 - 316 were generated for actin visualization in cell biology. In total, eighteen novel functional pyrichalasins were synthesized.