Mining and Functional Characterization of Four Oxidosqualene Cyclases from Cereals

Cereals serve as both essential food sources and prolific producers of diverse triterpenoids. In this study, we identified four previously uncharacterized cereal OSCs through genome mining and heterologous expression in <i>Nicotiana benthamiana</i>. Among them, maize ZmαβAS, ZmMDDS, and ZmGLS yielded β-amyrin (<b>1</b>)/α-amyrin (<b>2</b>), (3<i>S</i>,13<i>S</i>)-malabarica-17,21-dien-3β,14-diol (<b>3</b>) and glutinol (<b>4</b>), respectively. While oat AsLCS functioned as a new multifunctional OSC, predominantly generating lactucenyl (<b>5</b>). Site-directed mutagenesis of ZmMDDS, ZmGLS, and AsLCS identified critical residues governing catalytic activity. It was worth noticing that Y407 in AsLCS played a decisive role in modulating D-ring expansion versus the deprotonation process via the dammarenyl cation, which becomes a switch to produce <b>5</b> or dammara-20,24-dien-3β-ol (<b>6</b>). Evolutionary relationship analysis revealed that cereal OSC genes experienced complex diversification to yield various triterpenoids. These findings lay the foundation for further understanding the diversity of triterpenoids from cereals.