This study aims to elucidate the biosynthetic pathways of Taxol, a potent anticancer drug derived from *Taxus* plants. The biosynthesis of Taxol is believed to start from geranylgeranyl diphosphate (GGPP) and involves a complex series of reactions, including oxidation and acylation steps. Previous studies have identified 19 catalytic steps and 16 enzymes involved in this process, but the detailed stepwise reactions from the well-characterized di-oxygenated taxoids to the highly oxygenated tetracyclic core skeleton of Taxol remain unclear. This work focuses on the biosynthesis of tri-oxygenated taxoids and identifies the critical reaction order of the second and third hydroxylation steps. It also uncovers a taxoid 9α-hydroxylase that catalyzes the fourth hydroxylation and identifies CYP725A55 as the enzyme responsible for the oxetane ester formation via a cascade oxidation-concerted acyl rearrangement mechanism. Additionally, an acetyltransferase is identified that catalyzes the formation of C7-OAc, leading to the complete biosynthesis of 1β-dehydroxybaccatin VI with the Taxol tetracyclic core skeleton in engineered yeast. These findings lay the foundation for a comprehensive understanding of the biosynthetic pathway of Taxol.This study aims to elucidate the biosynthetic pathways of Taxol, a potent anticancer drug derived from *Taxus* plants. The biosynthesis of Taxol is believed to start from geranylgeranyl diphosphate (GGPP) and involves a complex series of reactions, including oxidation and acylation steps. Previous studies have identified 19 catalytic steps and 16 enzymes involved in this process, but the detailed stepwise reactions from the well-characterized di-oxygenated taxoids to the highly oxygenated tetracyclic core skeleton of Taxol remain unclear. This work focuses on the biosynthesis of tri-oxygenated taxoids and identifies the critical reaction order of the second and third hydroxylation steps. It also uncovers a taxoid 9α-hydroxylase that catalyzes the fourth hydroxylation and identifies CYP725A55 as the enzyme responsible for the oxetane ester formation via a cascade oxidation-concerted acyl rearrangement mechanism. Additionally, an acetyltransferase is identified that catalyzes the formation of C7-OAc, leading to the complete biosynthesis of 1β-dehydroxybaccatin VI with the Taxol tetracyclic core skeleton in engineered yeast. These findings lay the foundation for a comprehensive understanding of the biosynthetic pathway of Taxol.