We cloned two squalene epoxidases and five oxidosqualene cyclases, and identified their function using CRISPR/Cas9 tool and yeast heterologous expression. Triterpenes are the main active ingredients of Tripterygium wilfordii Hook.f., a traditional Chinese medicinal plant with many encouraging preclinical applications. However, the biosynthetic pathways of triterpenes in this plant are poorly understood. Here, we report on the isolation and identification of two squalene epoxidases (SQE6 and SQE7) and five oxidosqualene cyclases (OSC4-8) from T. wilfordii. Yeast complementation assays showed that TwSQE6 and TwSQE7 can functionally complement an erg1 yeast mutant that was constructed using the CRISPR/Cas9 system. The putative OSC genes were functionally characterized by heterologous expression in yeast. GC/MS analysis of the fermentation products of the transgenic yeast showed that both TwOSC4 and TwOSC6 are cycloartenol synthases, while TwOSC8 is a β-amyrin synthase. The discovery of these genes expands our knowledge of key enzymes in triterpenoid biosynthesis, and provides additional target genes for increasing the production of triterpenes in T. wilfordii tissue cultures by disrupting competing pathways, or in chassis cells by reconstituting the triterpenoid biosynthetic pathway.

中文翻译：

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雷公藤中角鲨烯环氧酶和氧化角鲨烯环化酶的鉴定和功能表征。

我们克隆了两种角鲨烯环氧酶和五种氧化角鲨烯环化酶，并使用 CRISPR/Cas9 工具和酵母异源表达鉴定了它们的功能。三萜是雷公藤的主要活性成分，雷公藤是一种具有许多令人鼓舞的临床前应用的传统中药植物。然而，人们对这种植物中三萜的生物合成途径知之甚少。在这里，我们报告了从 T. wilfordii 中分离和鉴定两种角鲨烯环氧酶（SQE6 和 SQE7）和五种氧化角鲨烯环化酶（OSC4-8）的情况。酵母互补分析表明，TwSQE6 和 TwSQE7 可以在功能上互补使用 CRISPR/Cas9 系统构建的 erg1 酵母突变体。推定的 OSC 基因的功能特征在于酵母中的异源表达。转基因酵母发酵产物的GC/MS分析表明，TwOSC4和TwOSC6均为环蒿醇合酶，而TwOSC8为β-香树脂醇合酶。这些基因的发现扩展了我们对三萜生物合成中关键酶的认识，并提供了额外的靶基因，用​​于通过破坏竞争途径或通过重组三萜生物合成途径在底盘细胞中增加三萜生产。