Friedelane‐type triterpene cyclase in celastrol biosynthesis from <i>Tripterygium wilfordii</i> and its application for triterpenes biosynthesis in yeast

Abstract

Summary Celastrol is a promising bioactive compound isolated from Tripterygium wilfordii and has been shown to possess many encouraging preclinical applications. However, the celastrol biosynthetic pathway is poorly understood, especially the key oxidosqualene cyclase ( OSC ) enzyme responsible for cyclisation of the main scaffold. Here, we report on the isolation and characterisation of three OSC s from T. wilfordii : Tw OSC 1, Tw OSC 2 and Tw OSC 3. Both Tw OSC 1 and Tw OSC 3 were multiproduct friedelin synthases, while Tw OSC 2 was a β‐amyrin synthase. We further found that Tw OSC 1 and Tw OSC 3 were involved in the biosynthesis of celastrol and that their common product, friedelin, was a precursor of celastrol. We then reconstituted the biosynthetic pathway of friedelin in engineered yeast constructed by the CRISPR /Cas9 system, with protein modification and medium optimisation, leading to heterologous production of friedelin at 37.07 mg l −1 in a shake flask culture. Our study was the first to identify the genes responsible for biosynthesis of the main scaffold of celastrol and other triterpenes in T. wilfordii . As friedelin has been found in many plants, the results and approaches described here have laid a solid foundation for further explaining the biosynthesis of celastrol and related triterpenoids. Moreover, our results provide insights for metabolic engineering of friedelane‐type triterpenes.