Abstract: Tripterygium hypoglaucum (Levl.) Hutch, a traditional Chinese medicinal herb with a long history of use, is widely distributed in China. One of its main active components, celastrol, has great potential to be developed into anti-cancer and anti-obesity drugs. Although it exhibits strong pharmacological activities, there is a lack of sustainable sources of celastrol and its derivatives, making it crucial to develop novel sources of these drugs through synthetic biology. The key step in the biosynthesis of celastrol is considered to be the cyclization of 2,3-oxidosqualene into friedelin under the catalysis of 2,3-oxidosqualene cyclases. Friedelin was speculated to be oxidized into celastrol by cytochrome P450 oxidases (CYP450s). Here, we reported a cytochrome P450 ThCYP712K1 from Tripterygium hypoglaucum (Levl.) Hutch that catalyzed the oxidation of friedelin into polpuonic acid when heterologously expressed in yeast. Through substrate supplementation and in vitro enzyme analysis, ThCYP712K1 was further proven to catalyze the oxidation of friedelin at the C-29 position to produce polpunonic acid, which is considered a vital step in the biosynthesis of celastrol, and will lay a foundation for further analysis of its biosynthetic pathway.