The genome analysis of Tripterygium wilfordii reveals TwCYP712K1 and TwCYP712K2 responsible for oxidation of friedelin in celastrol biosynthesis pathway

Tripterygium wilfordii is a Traditional Chinese Medicine (TCM) from family Celastraceae and celastrol is one of the strongest active ingredients belonging to friedelane-type pentacyclic triterpenoid, which has a large clinical application value of anti-tumor, immunosuppression, and obesity treatment. The first committed biosynthesis step of celastrol is the cyclization of 2, 3-oxidosqualene to friedelin, catalyzed by the oxidosqualene cyclase, while the rest of this pathway is still unclear. In this study, we reported a reference genome assembly of T. wilfordii with high-quality annotation by using a hybrid sequencing strategy (Nanopore, Bionano, Illumina HiSeq, and Pacbio), which obtained a 340.12 Mb total size and contig N50 reached 3.09 Mb. In addition, we successfully anchored 91.02% sequences into 23 pseudochromosomes using Hi-C technology and the super-scaffold N50 reached 13.03 Mb. Based on integration genome, transcriptom and metabolite analyses, as well as in vivo and in vitro enzyme assays, two CYP450 genes, TwCYP712K1 and TwCYP712K2 have been proven for C-29 position oxidation of friedelin to produce polpunonic acid, which clarifies the second biosynthesis step of celastrol. Syntenic analysis revealed that TwCYP712K1 and TwCYP712K2 derived from the common ancestor. These results have provided insight into illustrating pathways for both celastrol and other bioactive compounds found in this plant.