LAUSR

Abstract Celastrol with various bioactivities is a triterpenoid compound derived from Tripterygium wilfordii which is an important medicinal plant. The limited resources restrict the industrialization of celastrol. To enhance the content of celastrol, a new squalene synthase of Tripterygium wilfordii (TwSQS2) was cloned and followed by functional identification and overexpression. Recombinant TwSQS2 was obtained through prokaryotic expression and detected by SDS-PAGE and Western blot. The gas chromatograph analysis results showed that recombinant TwSQS2 converted FPP to squalene, indicating that TwSQS2 encodes a functional squalene synthase. The overexpression hairy roots were obtained by Agrobacterium rhizogenes mediated transformation and verified by β-glucuronidase histochemistry assay and genomic PCR. The enhanced expression of TwSQS2 resulted in a 2.08 fold increase of celastrol, which was up to 2.41 mg/g dry weight. These results indicated that TwSQS2 is a key enzyme in celastrol biosynthesis and its overexpression hairy roots is a promising resource to produce celastrol for replacing the natural roots. This study provides a useful genetic engineering strategy to improve the content of celastrol in Tripterygium wilfordii.