Abstract：
Triterpenoids from Camellia species comprise a diverse class of bioactive compounds with great therapeutic potential. However, triterpene biosynthesis in tea plants (Camellia sinensis) remains elusive. Here, we identified eight putative 2,3-oxidosqualene cyclase (OSC) genes (CsOSC1-8) from the tea genome and characterized the functions of five through heterologous expression in yeast and tobacco and transient overexpression in tea plants. CsOSC1 was found to be a β-amyrin synthase, whereas CsOSC4, 5, and 6 exhibited multifunctional α-amyrin synthase activity. Molecular docking and site-directed mutagenesis showed that the CsOSC6M259T/W260L double mutant yielded >40% lupeol, while the CsOSC1 W259L single mutant alone was sufficient for lupeol production. The V732F mutation in CsOSC5 altered product formation from friedelin to taraxasterol and ψ-taraxasterol. The L254 M mutation in the cycloartenol synthase CsOSC8 enhanced the catalytic activity. Our findings shed light on the molecular basis governing triterpene diversity in tea plants and offer potential avenues for OSC engineering.
摘要：
来自山茶属物种的三萜类包括具有巨大治疗潜力的多种生物活性化合物。然而,茶树（茶树）中的三萜生物合成仍然难以捉摸。这里,我们从茶基因组中鉴定了八个推定的2,3-氧化角鲨烯环化酶（OSC）基因（CsOSC1-8），并通过在酵母和烟草中的异源表达以及在茶树中的瞬时过表达来表征五个基因的功能。CsOSC1被发现是一种β-淀粉酶合成酶，而CsOSC4、5和6表现出多功能α-amyrin合酶活性。分子对接和定点诱变表明，CsOSC6M259T/W260L双突变体产量>40%羽扇豆醇，而单独的CsOSC1W259L单突变体足以生产羽扇豆醇。CsOSC5中的V732F突变改变了产物从friedelin到taraxasterol和Φ-taraxasterol的形成。环阿替烯醇合酶CsOSC8中的L254M突变增强了催化活性。我们的发现揭示了控制茶树中三萜多样性的分子基础，并为OSC工程提供了潜在的途径。
