This dissertation describes the discovery by genome mining and the characterization of thebiosynthetic pathway for sambutoxin, a 4-hydroxy-2-pyridone alkaloid Fusarium mycotoxin. The 4-hydroxy-2-pyridone alkaloid family of fungal natural products has been the subject of numerous biosynthetic studies due to their structurally diverse scaffolds that arise from a common class of biosynthetic precursors, which are transformed by various redox tailoring enzymes and cyclases that have been uncovered by genome mining. Sambutoxin is the first 2-pyridone alkaloid with a p-hydroxyphenyl moiety that is derived from L-phenylalanine via a late-stage oxidation of the Phe side chain phenyl group.