C-2α hydroxylated triterpenoids are a large class of plant secondary metabolites. These compounds， such as maslinic， corosolic and alphitolic acid， have important biological activities against HIV， cancer and diabetes. However， the biosynthesis pathways of these compounds have not been completely elucidated. Specifically， the cytochrome P450 (CYP) enzyme responsible for C-2α hydroxylation was unknown. In this study， a novel CYP enzyme that catalyzes C-2α hydroxylation was identified in Crataegus pinnatifida (Hawthorn) using a metabolic engineering platform. It is a multifunctional enzyme with C-2α oxidase activity on oleanane-， ursane- and lupane-type pentacyclic triterpenoids. In addition， the complete biosynthesis pathways of these three triterpenoids were reconstituted in yeast， resulting in the production of 384， 141 and 23mg/L of maslinic， corosolic and alphitolic acid， respectively. This metabolic engineering platform for functional gene identification and strain engineering can serve as the basis for creating alternative pathways for the microbial production of important natural products.