Functional Characterization of Squalene Epoxidases from Siraitia grosvenorii

The medicinal plant <i>Siraitia grosvenorii</i> produces sweet-tasting cucurbitane-type mogrosides from the atypical triterpenoid precursor 2,3,22,23-dioxidosqualene (SDO), rather than the conventional 2,3-oxidosqualene (SQO). However, SDO formation in mogroside biosynthesis remains unclear. Here, we systematically characterized two squalene epoxidases (SgSQE1/2) through phylogenetic analysis, heterologous expression, subcellular localization, qRT-PCR, and alanine scanning studies. Both <i>Sg</i>SQE1 and <i>Sg</i>SQE2 exhibited squalene epoxidase activity, with <i>Sg</i>SQE2 catalyzing SDO formation in yeast. We identified two critical catalytic residues governing epoxidation efficiency through mutagenesis. Both <i>Sg</i>SQEs were localized in the ER, while expression profiling revealed a similar trend between <i>SgSQE2</i> expression and mogroside accumulation in fruits. In our study, we developed a genomically engineered strategy for heterologous SQE characterization. These results lay the foundation for the SQE catalytic reaction involved in mogroside biosynthesis, and provide gene resources and a feasible approach for triterpene metabolic engineering.