Functional validation of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing

Background: Squalene epoxidase is one of the rate-limiting enzymes in the biosynthetic pathway of membrane sterols and triterpenoids. The enzyme catalyzes the formation of oxidized squalene, which is a common precursor of sterols and triterpenoids. In recent years, CRISPR/Cas9 gene editing technology has emerged and other functional genes in the Poria cocos triterpene synthesis pathway have been studied; PcSE has not been reported. Results In this study, the squalene epoxidase gene ( PcSE ) was evaluated in Poria cocos . Molecular docking between PcSE and squalene was performed and the active amino acids were identified. sgRNA sequences were designed based on the active site residues. In vivo verification of PcSE function was performed using a PEG-mediated protoplast transformation approach. The effect on triterpene synthesis in P. cocos was consistent with the results from ultra-high-performance liquid chromatography-quadruplex time-of-flight-double mass spectrometry (UHPLC-QTOF-MS/MS) analysis. Which showed that deletion of PcSE inhibited triterpene synthesis. Conclusions A gene editing system based on molecular docking was successfully constructed to demonstrate that PcSE functions as a house squalene cyclooxygenase, which provides a basis for further studies on the heterologous biosynthesis of P. cocos secondary metabolites.